IMMUNOLOGY AND SEROLOGY

LECTURE/FINALS
1st SEM, 2022

IMMUNODEFICIENCY DISORDER ➔ But for immunodeficiency patients hindi nila

Unit Intended Outcomes: kayang labanan nagiging susceptible sila that’s
why the MOST CLINICAL SYMPTOMS OF
Identify and differentiate the different immune- IMMUNODEFICIENCY DISORDERS are
deficiency disorders in relation to their general RECURRENT INFECTIONS because of
immunologic defects, defects in the B-cell, T-cell, DYSFUNCTION OF IMMUNE SYSTEM
myeloid, or complement systems.
Immunodeficiencies can be INHERITED or ACQUIRED
OVERVIEW INHERITED - known as PID (Primary immune
Immunodeficiencies is a collective name for a LARGE deficiencies)
GROUP OF DISORDERS when the function of the ➔ PRIMARY IMMUNE DEFICIENCY DISORDER
immune system is NOT strong enough to protect the ◆ through parents basta naiinherit
organism against foreign invaders, especially microbes
and their toxic products, or against the organism’s ACQUIRED - yung mga nakukuha such as HIV which
own malignantly transformed cells or what we know causes AIDS which is an immunodeficiency disorder is
as CANCER CELLS ACQUIRED like nakukuha sa infection or
microorganism,
IMMUNE DISORDERS - are a collective disorder in MALIGNANCIES, IMMUNOSUPPRESSIVE THERAPIES
which there is a part of immune system that is missing and that known as SECONDARY IMMUNE DEFICIENCY
or dysfunctional → that’s why nagkakaroon ng mga DISORDERS
dysfunction or deficient ang ating immune system ➔ most common is AIDS

People with conditions, they have tendency to have a Categories of Primary Immunodeficiencies
decreased ability to defend themselves against
difference infections or against different Category 1: Combined Immunodeficiencies
microorganisms → that’s why the decreased ability to Category 2: Combined Immunodeficiencies with
protect themselves they are MORE SUSCEPTIBLE to associated or Syndromic Features
developing certain types of diseases or acquiring Category 3: Predominantly Antibody Deficiencies
certain types of infection as well as developing certain (pinakamarami) - the PREDOMINANTLY antibody
types of CANCER deficiencies have the MOST common
immunodeficiency representing about 50% of all the
➔ These is a RISK FACTORS that could make those PIDs
patients more SUSCEPTIBLE to develop more Category 4: Diseases of Immune Dysregulation
MALIGNANCIES/CANCER CELLS Category 5: Congenital Defects of Phagocyte Number,
Function, or Both
The clinical symptoms associated Category 6: Defects in Innate Immunity
with immunodeficiencies range from very MILD or Category 7: Auto-inflammatory Disorders
subclinical to SEVERE MANIFESTATIONS, and the MOST Category 8: Complement Deficiencies
clinical symptom of patients having immunodeficiency Category 9: Phenocopies of Primary
is having RECURRENT INFECTIONS or FAILURE TO Immunodeficiencies
THRIVE or FAILURE of eliciting an immune response or
immune mechanism against different invasion of RECURRENT INFECTION - most common hallmark of
microorganisms immunodeficiency
➔ the most common hallmark na meron talagang
➔ so usually ang pinaka SYMPTOM na meron or immunodeficiency disorder yung isang
immune deficiency disorder pag paulit ulit patient is the RECURRENCE OF REPEATED
yung INFECTION because sa mga taong may INFECTION and even by organism considered
as LOW VIRULENT
IMMUNOCOMPETENT/NORMAL IMMUNE SYSTEM -
IMMUNODEFICIENCY DISORDER or meron mga organism that DO NOT cause severe
IMMUNOCOMPROMISED PEOPLE yung mga simpleng infections to those people who have normal immune
sakit na NORMALLY na nalalabanan ng katawan. response
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 IMMUNOLOGY AND SEROLOGY
LECTURE/FINALS
1st SEM, 2022

→ a SEVERE DEFECT on T Cells function will also have

LOW VIRULENT ORGANISM - these organisms DO NOT effects on the IMMUNOGLOBULIN LEVELS as well
normally cause severe infections in human These categories involve defects on
→ B Cells and T Cells (kasi interrelated sila pareho)
BUT in patient with IMMUNODEFICIENCY DISORDER ➔ T helper is necessary for antigen precitation
nagkaroon sila ng REPEATED RECURRENT with that because B cells can also be activated using
LOW VIRULENT ORGANISMS and nagiging severe pa through the action of T helper cells
➔ T helper cells is necessary for normal activation
In the PAST IMMUNODEFICIENCY DISORDER have been of B cells by secreting cytokines by necessary
classified as defects in: to activate and to secrete antibody
➔ T cells
➔ B cells DEFECT ON B CELLS AND T CELLS - SA CATEGORY 1
➔ Phagocytes
➔ Complement and another component of Diseases include:
INNATE ○ Severe Combined Immunodeficiency (SCID)
➔ will have an effect on immunoglobulin levels
BUT in 2014 the international union of immunological ○ Purine-Nucleoside Phosphorylase (PNP) Deficiency
societies have updated their classification of the
PRIMARY IMMUNODEFICIENCIES by grouping them
into 9 different categories based on their Severe Combined Immunodeficiency Disease (SCID)
characteristics, clinical features, immunologic defects
and genetic abnormalities kung ano ba yung na ● MOST SERIOUS of the among congenital
inherent mutation or AUTOSOMAL or RECESSIVE immunodeficiencies meaning there is
GENES ASSOCIATION or there is an INVOLVEMENT OF
➔ that would cause the immune deficiency A MUTATION in the GENETIC COMPOSITION /
disorder INHERITED GENES

Although they are separate in different categories, ➔ POTENTIALLY FATAL/MOST SERIOUS

some of them listed in more than one category ➔ PRIMARY IMMUNODEFICIENCY
because of overlapping features like a disease that ➔ Combined absence of T Cells and B cells
cause a defect in T cells and defect in phagocytic
functions. ● A group of related diseases that all affect T-
and B-cell function but with differing causes
Category 1: Combined Immunodeficiencies
CATEGORY 1 - known as the COMBINED causing a SEVERE COMBINED IMMUNODEFICIENCY
IMMUNODEFICIENCY because deficiency or disorder DISEASE wherein parehong T Cells and B cells
under category 1 involves defect in both HUMORAL DEPLETED FUNCTION or DECREASE FUNCTION
and those MEDIATED BY B CELLS as well as defects in T
CELLS functions. Interleukin-2 receptor gamma (IL2RG)

● Defects in both humoral (B cell) and cell- Usually, the MUTATION occurs with the frequency of
mediated (T cell) immunity about 1 in 50,000 births - NOT common, VERY RARE
● Result from MUTATION that affect immunodeficiency and usually cause in mutation
development of both types of lymphocytes INTERLEUKIN-2 RECEPTOR GAMMA GENES (IL2RG)
(which are B CELLS and T CELLS may defect
located on the X-chromosome which is the most
pareho and T Cells and B Cells function that’s
common form of the disease
why DECREASED and DYSFUNCTIONAL) or
cause defective interaction between the two INTERLEUKIN-2 RECEPTOR GAMMA GENES - is a gene
antigen-specific limbs of the adaptive immune that CODES for a PROTEIN CHAIN called the COMMON
system. GAMMA CHAIN that is common to receptor for

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 IMMUNOLOGY AND SEROLOGY
LECTURE/FINALS
1st SEM, 2022

INTERLEUKIN 2,7,9,15,21 or those INTERLEUKINS that LYMPHOCYTE number - this is SIMILAR to

are secreted by T CELLS another form APAID deficiency which is the
PNP
NORMAL SIGNALING CANNOT occur in cells with the
effective receptors and thus halting the maturation of In patients with only MILDLY REDUCED ADA ACTIVITY
T cells and B cells. so meron lang SLIGHT IMPAIRMENT of immune
function so it DEPENDS upon the SEVERITY of the ADA
If meron MUTATION INTERLEUKIN-2 RECEPTOR activity
GAMMA GENES (kung wala or if meron mutation sa
INTERLEUKIN-2 RECEPTOR GAMMA GENES - RAG-1 or RAG-2 GENES
nagkakaroon ng DEFECTIVE SIGNALING and HALTING
now the natural maturation of T cells and B cells that's ○ A MUTATION in a recombinase activating
why nagkakaroon ng DEPLETED or DECREASED T Cells gene (RAG-1 or RAG-2) - important because
and B Cells function. they are activating genes that are very
important in DIFFERENTIATION of PRO-B Cells
or IMMATURE B cells
JAK3 gene
Patient with SCID were found to have a MUTATION in
○ An additional defect in the JAK3 gene RAG-1 or RAG-2 → they LACK both T Cells and B Cells
BUT they have functioning NK cells
JACK3 GENE - is required for processing an NK cells are NOT affected
INTERLEUKIN BINDING SIGNAL from the cell’s
membrane will enter the nucleus and ACTIVATE
RAG-1 or RAG-2 GENES
NORMAL SIGNALING and DIFFERENTIATION of T Cells
and B Cells - are important in differentiation of the EARLY
CELLS or EARLY PRO-B Cells and PRE-B Cells
● kailangan sya dahil sa binding ng interleukin
stages
cell membrane will enter in nucleus activate
- very important in the REARRANGEMENT of
normal signalling and differentiation of cells
the light chains and heavy chains genes
● it could also result for the decrease,
- aslo for the REARRANGEMENT of the
dysfunction or depletion of T cells and B cells
DEOXYRIBONUCLEIC ACID (DNA) inside of B
Adenosine deaminase (ADA) deficiency Cells
- necessary to PRODUCE to FUNCTIONAL
○ About 15% to 20% of the patients with SCID IMMUNOGLOBULINS in the differentiation of
have an adenosine deaminase (ADA) B cells during the coding
deficiency, leading to a NEGATIVE T-B-NK- - functional T Cells receptor or TCR
phenotype - meaning nagkakaroon ng SEVERE
- important in the normal maturation and
DEPLETION or PROGRESSIVE decrease in
differentiation of T Cells and B Cells
lymphocyte numbers.
→ that’s why a DEFECT or MUTATION of these genes
ADA DEFICIENCY - affects an enzyme that is could also result in SEVERE COMBINED DEFICIENCY of
involved in the METABOLISM of PURINES (toxic T Cells and B Cells function
metabolites of purines accumulate in
LYMPHOID CELLS that’s why because of these → usually with patients with RAG-1 or RAG-2
toxic metabolites of purines due to the DEFICIENCY have DECREASED CLASS 2 MHC (antigen
deficiency of ADA - nagkakaroon ngayon ng presentation) MOLECULE adding up to the deficiency
IMPARED PROLIFERATION of T Cells and B or to the disorder of the immune system.
Cells) that’s why can also result in SCID leading CD45
to the progressive DECREASE LYMPHOCYTE
number also leading to a PHENOTYPE which ○ A molecular defect in the gene encoding a
causes REDUCE or progressive DECREASE common leukocyte protein called CD45
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 IMMUNOLOGY AND SEROLOGY
LECTURE/FINALS
1st SEM, 2022

involved in the metabolism of purines (same

CD45 - transmembrane phosphatase which regulate as ADA)
the SIGNAL TRANSDUCTION of T Cells and B Cells
receptors. ● Produces a MODERATE to SEVERE defect in cell
➔ that’s why a DEFECT of these PROTEIN can also mediated immunity that’s why
IMPAIR of immune response → The number of T cells progressively
decreases
→ all of these CONFORMATIONAL or GENETIC ● The levels of immunoglobulins are generally
ABNORMALITIES can induce an immune deficiency normal or increased BUT the T cells
disorder, such as this SCID progressively decreases because of the
accumulation of deoxyguanosine
● Patients with SCID generally present early in triphosphate, a toxic purine metabolite.
infancy with infection by nearly any type of
organism. PNP deficiency - lacking an enzyme that is involved in
● Oral candida yeast infections, pneumonia, purine metabolism led to TOXIC METABOLITE OF
and diarrhea are the most common PURINE known as deoxyguanosine triphosphate
MANIFESTATION/SYMPTOMS. ➔ TOXIC METABOLITE OF PURINE - That’s why
● The administration of live vaccines can cause accumulate it to lymphoid organs causing
severe illness. severe decrease on T cells
● Unless immune reconstitution can be achieved
by bone marrow transplantation or by ● About two thirds of PNP-deficient patients also
specifically replacing a deficient enzyme, have neurological disorders (MENTAL
patients with SCID die before they are 2 years DISABILITIES), but NO characteristic physical
old abnormalities
PNP deficiency can be confused with neonatal HIV
successful reconstitution by using STEM CELLS so that infection. The two conditions can usually be
mag increase yung number ng T Cells and B Cells distinguished by specific tests for HIV and by assays for
PNP activity.
VERY FATAL & VERY SERIOUS PID AMONG CHILDREN →
the severe combine kasi parehong T Cells and B Cells Category 2: Combined Immunodeficiencies with
are severely DECREASED that’s why they are very prone Associated or Syndromic Features
and usually ang kinamamatay nila is SECONDARY Category 2 are characterized by non-IMMUNOLOGIC
INFECTIONS kasi hindi malabanan ang kanilang features in addition to the combined
katawan since bata pa sila and common is immunodeficiency.
PNEUMONIA
→ known as combined immunodeficiencies with
Purine-Nucleoside Phosphorylase (PNP) Deficiency associated or SYNDROMIC FEATURES
also, a CATEGORY 1 in which there is combined category 2 is characterized by nonimmunologic
immunodeficiency of T Cells and B Cells and the associated
condition present in INFANCY as well with RECURRENT indirect din sya
● Diseases in this category are typically caused
● CATEGORY 1 in which there is combined by DEFECTS in CELL-MEDIATED IMMUNITY
immunodeficiency of T Cells and B Cells The ○ majority of the disease is caused by the
condition presents in INFANCY with cell defects
RECURRENT or chronic pulmonary infections, ● Often these diseases can result from
oral or cutaneous candidiasis, diarrhea, skin abnormalities at different stages of T-cell
infections, urinary tract infections, and development LEADS to other problems with
FAILURE to thrive or failure to produce other branches of immune response such as
immune response. HUMORAL IMMUNITY
● PNP deficiency is also a RARE AUTOSOMAL Examples of specific immunodeficiencies in this
RECESSIVE TRAIT and affects an enzyme category are:

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 IMMUNOLOGY AND SEROLOGY
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1st SEM, 2022

○ Wiskott-Aldrich syndrome (WAS) CD43 - which is involved in the regulation of protein

○ DiGeorge anomaly glycosylation
○ Ataxia-Telangiectasia (AT) Abnormalities cause defective actin polymerization
and affect its signal transduction in lymphocytes and
Wiskott-Aldrich Syndrome (WAS) platelets
AUTOSOMAL DISORDER & NON-IMMUNOLOGIC ➔ that’s why nagkakaroon ng both decrease ng
● A rare X-linked recessive syndrome that is level ang lymphocytes and platelets
defined by the triad of immunodeficiency,
eczema, and thrombocytopenia ● Platelets have a shortened half-life and T
WAS is usually lethal in childhood because of lymphocytes are also affected, although B
RECURRENT infection, hemorrhage because of lymphocytes appear to function normally
thrombocytopenia, or malignancy/formations of ○ if there’s a defect in t cells, it can affect
cancer. other different branches of immune
system as a humoral immune response
The laboratory features of WAS include a decrease in
platelet number and size with a prolonged bleeding Current treatment: Transplantation of bone marrow
time because of the decrease in platelet function. or cord blood stem cells from an HLA identical sibling.
Splenectomy can be very valuable in controlling the
The bone marrow contains a normal or somewhat thrombocytopenia.
increased number of megakaryocytes.
Non-immunologic disorders or features appear in
● Milder variants have also been described such Category 2
as an X-linked form of thrombocytopenia ● thrombocytopenia
● eczema
● There are abnormalities in both the ● other non-immunologic features as well as
cellular (t cells) and humoral (b cells) branches immunodeficiency or combined
of the immune system related to a general immunodeficiency
defect in antigen processing
● generally, there is a defect in antigen DiGeorge Anomaly
processing as a result of this defect known as CONGENITAL THYMIC HYPOPLASIA
● As a result, patients display a severe ● A developmental abnormality of the third and
deficiency of the naturally occurring fourth pharyngeal pouches that affects thymus
IgM antibodies to ABO blood group development in the embryo
antigens known as ○ defect in certain part during
ISOHEMAGGLUTININS embryogenesis

● Absence of isohemagglutinins is the most → All organs derived from these embryonic structures
consistent laboratory finding in WAS and is can be affected
often used diagnostically
- Primary Immunodeficiency disease caused by
● Patients also have persistently increased levels ABNORMAL MIGRATION and development of
of serum alpha fetoprotein certain cells and tissues during the fetal
development
Patients with WAS can have a variety of different
patterns of immunoglobulin levels, but they usually
have low levels of IgM, normal levels of IgA and IgG, Defects of the third and fourth pharyngeal pouches
and increased levels of IgE. during the embryogenesis and therefore affects the
development of the THYMUS
● The primary molecular defect in the syndrome
appears to be an abnormality of the integral These children tend to have severe, recurrent viral and
membrane protein CD43 fungal infections.
.

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● A disorder associated by a defect in Telangiectasias - capillary swelling resulting in red

chromosome 22 blotches on the skin especially on the earlobes and
● A severe and persistent DECREASE in T-cell conjunctiva.
numbers and other branches of immune
response will also be INDIRECTLY AFFECTED → Blood vessels in the sclera of the eyes may be dilated
such as HUMORAL IMMUNE RESPONSE and there may also be a reddish butterfly area on the
● Patients tend to have severe, recurrent viral face and ears.
and fungal infections. ● 95% of patients exhibit increased levels of
○ because of decrease T cells there may serum alpha-fetoprotein
have ○ The incidence of this disease is
● Severely affected children usually present in between 1:10,000 to 1:100,000,
the neonatal period with TETANY (caused by although as much as 1% of the
hypocalcemia resulting from population is heterozygous for the
hypoparathyroidism) or manifestations of gene
cardiac defects ○ Antibody response to antigens,
especially polysaccharides, are usually
Non-Immunologic features are slow/blunted.
→ MENTAL RETARDATION (mental disorder)
➔ mental retardation ● Abnormal genes produce a combined defect of
➔ absence of ossification of the hyoid bone both humoral and cellular immunity
➔ cardiac anomalies ● There is a defect in the AT gene is located on
chromosome 11, region q22
➔ abnormal facial development (FISH SHAPE
● The only effective therapy for AT is allogeneic
MOUTH & tendency to have a LOW EARS)
bone marrow transplantation
➔ thymic hypoplasia - kasi yung problem is may
defect sa THYMUS DEVELOPMENT which
IMPAIRS the T cells number)
Usually the cause of Ataxia-Telangiectasia (AT) is a
AUTOSOMAL RECESSIVE DEFECT IN THE AT GENE
Many patients with a partial DiGeorge anomaly have
results in a defective kinase involved in DNA repair and
only a minimal thymic defect and, thus, near normal
in cell cycle control
immune function. However, about 20% of children
with a defect of the third and fourth pharyngeal
Abnormal rearrangement of TCR and immunoglobulin
pouches have a severe and persistent decrease in T-cell
genes does not occur normally. Patients’ lymphocytes
numbers.
often exhibit chromosomal breaks and other
abnormalities involving the TCR genes in T cells and
The immunodeficiency associated with the DiGeorge
immunoglobulin genes in B cells.
anomaly is a QUANTITATIVE DEFECT in thymocytes.
→ number yung cause, there is SEVERE or PERSISTENT
The levels of IgG2, IgA, and IgE are often low or absent,
DECREASE in T cells number.
although the pattern can be quite variable.
NOT enough to become activated.
In addition, the number of circulating T cells is often
decreased. Death usually occurs in early adult life from
Treatment: Fetal thymus transplantation, Bone
either pulmonary disease or malignancy
marrow transplantation, administration of thymic
● causing of the death is having a low immune
hormones
response or they cannot protect their body
○ it is important to have a strong
Ataxia-Telangiectasia (AT)
immune response
● A RARE autosomal recessive syndrome
characterized by cerebellar ataxia and
telangiectasias

Cerebellar ataxia - involuntary muscle movements

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 IMMUNOLOGY AND SEROLOGY
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1st SEM, 2022

The blood level of IgG at birth is about the SAME as the

adult level, reflecting transfer of maternal IgG across
the placenta.
● nattransfer ang IgG from mother that’s why
increase ang IgG ng fetus

→ The IgG level declines over the first 6 months of life

as maternal antibody is catabolized.
→ Levels of IgA and IgM are very low at birth. The
concentrations of all immunoglobulins gradually RISE
when the infant begins to produce antibodies at a few
months of age in response to environmental stimuli.
IgM reaches normal adult levels first, around 1 year of
age, followed by IgG at about 5 to 6 years of
age.(pagnaactivate na)

Predominantly Antibody Deficiencies include:

○ Transient hypogammaglobulinemia of infancy
Category 3: Predominantly Antibody Deficiencies ○ Selective IgA deficiency
● Include genetic defects in B-cell maturation or ○ Btk deficiency
mutations leading to defective interactions ○ Common variable immunodeficiency
between B and T cells ○ Isolated IgG subclass deficiency
○ CD154 deficiency
This category encompasses conditions in which the
MAIN characteristic is low levels of serum Transient Hypogammaglobulinemia of Infancy with
immunoglobulins. Normal Numbers of B Cells
All infants experience low levels of immunoglobulins
Low levels of serum immunoglobulins means at approximately 5 to 6 months of age. However, in
deficiency in immunoglobulin is some babies the low levels persist for a longer time.
AGAMMAGLOBULINEMIA or (meron deficiency in the
concentration of antibodies) ● A relatively common primary
immunodeficiency disease that affects infants
gammaglobulins - region of globulins and young children.
● Infants experience low levels of
The mechanisms of the AGAMMAGLOBULINEMIA immunoglobulins persisting for a longer
include genetic defects in B-cell maturation or period of time.
mutations leading to defective interactions between B → Because these children do NOT begin synthesizing
and T cells. immunoglobulins promptly, they can experience
severe pyogenic sinopulmonary and skin infections as
Wide range of immunoglobulin deficiency states protective maternal IgG is cleared.
have been reported and involve virtually all
combinations of immunoglobulins and all degrees of ● Cell-mediated immunity is NORMAL and there
severity. In some cases, only a single isotype of one may be normal levels of IgA and IgM
immunoglobulin class is deficient, whereas all of the
other isotypes are NORMAL. ● IgG appears to be the → MOST AFFECTED
dropping to at least 2 standard deviations (SDs)
In evaluating immunoglobulin deficiency states, it is below the age-adjusted mean with or without
important to remember that blood levels of a depression of IgM and IgA
immunoglobulins change with age.
● Immunoglobulin levels in infants with this
condition usually NORMALIZE spontaneously,
often by 9 to 15 months of age.

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Selective IgA Deficiency

The cause may be related to a delayed maturation ● It is hypothesized that lack of IgA is caused by
of one or more components of the immune system, impaired differentiation of lymphocytes to
possibly T helper cells. become → IgA-producing plasma cells
agammaglobimna
Selective IgA deficiency is the most common congenital
X-Linked Bruton’s Tyrosine Kinase (Btk) Deficiency immunodeficiency, occurring in about 1 in 500
→ first described in 1952, is X chromosome linked, so persons. Most patients with a deficiency of IgA are
this syndrome affects MALES almost exclusively ASYMPTOMATIC. Those with symptoms usually have
● A hereditary immunodeficiency due to the infections of the respiratory and gastrointestinal tract
failure of B lymphocytes to mature and and an increased tendency to develop autoimmune
rearrangement failure of Ig Heavy Chain diseases such as:
● Patients with X-linked agammaglobulinemia ➔ systemic lupus erythematosus (SLE),
lack of circulating mature CD19+ B cells and ➔ rheumatoid arthritis (RA)
exhibit a deficiency or lack of ➔ celiac disease
immunoglobulins of all classes
➔ thyroiditis
→ They have NO plasma cells in their lymphoid tissues. About 20% of the IgA-deficient patients who develop
The patients do, however, have pre-B cells in their infections also have an IgG2 subclass deficiency.
bone marrow. Because of the lack of B cells, the tonsils
and adenoids are →small or entirely ABSENT and
● IgE antibodies specifically directed against IgA
lymph nodes lack normal germinal centers.
are produced by 30% to 40% of patients with
severe IgA deficiency, causing anaphylactic
They develop recurrent bacterial infections beginning
reactions when blood products containing IgA
in INFANCY as maternal antibody is cleared. The
are transfused
patients most commonly develop
Patients with severe IgA deficiency have no other
→ sinopulmonary infections caused by encapsulated symptoms, the IgA deficiency may not be detected
organisms such as streptococci, meningococci, and until the patient experiences a transfusion reaction,
Haemophilus influenzae. resulting in the production of anti-IgA antibodies.
Therefore, products for transfusion to known IgA-
→ Other infections seen include bacterial otitis media,
deficient patients should be collected from
bronchitis, pneumonia, meningitis, and dermatitis.
IgAdeficient donors or cellular products should be
washed to remove as much donor plasma as possible.
X-linked hypogammaglobulinemia results from
arrested differentiation at the pre–B-cell stage,
DEPLETED is IgA type of immunoglobulins.
leading to a → complete absence of B cells and plasma
→ Usually the reaction is ANAPHYLACTIC during
cells. BLOOD TRANSFUSION
The underlying genetic mechanism is a deficiency of an
Common Variable Immunodeficiency (CVI)
enzyme called the Btk in B-cell progenitor cells. ● A low incidence, but the most common PID
with a severe clinical syndrome
Lack of the enzyme apparently causes a failure of
● The disorder can be congenital or acquired, or
immunoglobulin VH gene rearrangement.
familial or sporadic, and it occurs with equal
The syndrome can be differentiated from transient
frequency in men and women.
hypogammaglobulinemia of infancy by the absence of
CVI - heterogeneous group of disorders with a
CD19+ B cells in the peripheral blood, the abnormal
prevalence of about 1 in 25,000.
histology of lymphoid tissues, and its persistence
→ Patients usually begin to have symptoms in their 20s
beyond 2 years of age. and 30s, but age at onset ranges from 7 to 71 years of
age.
● T cells are normal in number and function.
TREATMENT: administration of intramuscular or
CVI is characterized by hypogammaglobulinemia that
intravenous immunoglobulin preparations and
leads to recurrent bacterial infections, particularly
vigorous antimicrobial treatment of infections
sinusitis and pneumonia.

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● There is usually a deficiency of both IgA and Isolated IgG Subclass Deficiency
IgG, but selective IgG deficiency may occur This may result in a LOW LEVEL of IgG
● It is also associated with an increased risk of ● These are conditions where the level(s) of one
lymphoproliferative disorders, gastric or more of the four IgG subclasses is (are) more
carcinomas, and autoimmune disorders than two SDs below the mean age-appropriate
● Diagnosed by demonstrating a low serum IgG level
level in patients with recurrent bacterial Normally, about 70% of the total IgG is IgG1, 20% IgG2,
infections 6% IgG3, and 4% IgG4.
up to 20% of CVI patients develop HERPES ZOSTER
(shingles), a much higher incidence than in In patients with recurrent infections, levels of the
immunologically normal young adults. different subclasses should be measured if the total IgG
CVI is often associated with a spruelike syndrome level is normal kasi pwede ang baba ni IgG1 and IgG3
characterized by malabsorption and diarrhea. pero NORMAL padin yung total IgG → because the LOW
NUMBER of IgG1 AND IgG3 is being COMPENSATED by
The most common autoimmune manifestations of CVI the HIGH AMOUNTS kasi normal naman si IgG2 and
are immune thrombocytopenia and autoimmune IgG4
hemolytic anemia.
Other symptoms may include: Most IgG antibodies directed against protein antigens
➔ lymphadenopathy are of the IgG1 and IgG3 sub-classes, whereas most
➔ splenomegaly - IgG antibodies against carbohydrate antigens
➔ intestinal hyperplasia are IgG2 or IgG4.
- Thus, deficiencies involving IgG1 or IgG3 lead
Blood group isohemagglutinins, or the so-called to a reduced capability of responding to
naturally occurring antibodies, are typically absent or protein antigens such as toxins, whereas
low. In contrast to X-linked agammaglobulinemia, most selective deficiencies of IgG2 can result in
patients with CVI have normal numbers of mature B impaired responses to polysaccharide
cells. However, these B cells DO NOT differentiate antigens, which cause recurrent infections
normally into immunoglobulin-producing with polysaccharide-encapsulated bacteria
plasma cells. such as:
➔ Streptococcus pneumoniae
➔ H influenzae.

IgG4 - The most common subclass deficiency, although

IgG4 subclass deficiency may have the least clinical
Three major types of cellular defects have been significance (NOT SEVERE )
found in CVI patients:
➔ T cells or their products appear to suppress IgG1 & IgG3 - TOXIN, bacteria produce ENTEROTOXIN
differentiation of B cells into plasma cells. IgG2- can result in impaired polysaccharide-
➔ T cells may FAIL to provide adequate help to encapsulated bacteria such as Streptococcus
support terminal differentiation of B cells. pneumoniae & H influenzae.
➔ Primary defect in the B-cell line in some
IgG1 deficiency - being the least common
patients.
Hindi siya AGAMMAGLOBULINEMIA meron plasma
● IgG1 and IgG3 sub-classes: protein antigens
cells and B cells BUT hindi siya mag differentiate
● IgG2 or IgG4 sub-classes: carbohydrate
normally into PLASMA CELLS → that’s why IMPAIRED
antigens
ang ADAPTIVE IMMUNITY with patients with CVI
● The most common subclass deficiency is IgG4,
● CVI can usually be effectively treated with with IgG1 deficiency being the least common
intramuscular or intravenous immunoglobulin
preparations

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Category 5: Congenital Defects of Phagocyte

Number, Function, or Both
DEPLETED neutrophils and phagocytic cells or
NORMAL naman yung count pero DEFECTIVE naman
sila → hindi kaya yung MICROBIAL KILLING that’s why
impaired din ang immune response

➔ Defective ang number of phagocytes

➔ or NORMAL ang NUMBER but DEFECTIVE yung
function or combination of BOTH
➔ ABNORMAL FUNCTION and LOW NUMBER of
PHAGOCYTES

Characterized by abnormalities in phagocytic cells

○ Chronic Granulomatous Disease (CGD)
○ Other Microbiocidal Defects
Category 4: Diseases of Immune Dysregulation
○ Leukocyte Adhesion Deficiency (LAD)
MAJORITY AUTOIMMUNE DISEASE
● Many of the diseases may involve normal
Neutrophils play a crucial role in the immediate and
numbers of T or B cells but with reduced
nonspecific response to invading organisms by
control over their functions
responding before specific antibody and cell-mediated
The autoimmune lymphoproliferative syndrome (ALPS)
immune responses can be mounted.
→ for example, may involve mutations in genes coding
- Neutrophils are even more effective at
for caspase enzymes involved in APOPTOSIS of B Cells
ingesting and killing organisms coated with
specific antibody and thus continue to play an
● May also have features of autoimmunity
important role in host defense even after an
● Autoimmune lymphoproliferative syndrome
adaptive immune response is established.
(ALPS)
- To destroy invading organisms, neutrophils
● CD25 deficiency
must adhere to vascular endothelial lining
CD25 deficiency - is manifested by a lack of T regulatory
cells, migrate through the capillary wall to a
(Treg) cells, which leads to lymphoproliferation and
site of infection, and ingest and kill the
autoimmunity
microbes
Defects affecting each of these steps can lead to an
● Mutations in the FoxP3 gene
increased susceptibility to pyogenic infections.
Mutations in the FoxP3 gene - which is required for
Treg differentiation, may show a similar clinical
Chronic Granulomatous Disease (CGD)
presentation
● A group of disorders involving inheritance of
either an X-linked or autosomal recessive gene
● Chediak-Higashi syndrome: caused by a
that affects neutrophil microbiocidal function
mutation in the LYST gene
Chediak-Higashi syndrome - an immunodeficiency
CGD - is the one of the most common and best
with hypopigmentation (loss of skin color) caused by a
characterized of neutrophil abnormalities
mutation in the LYST gene, is characterized by a
reduced number of natural killer (NK) cells and
● Result in the inability of the patient’s
neutrophils, as well as an increased production of
neutrophils to produce the reactive forms of
inflammatory proteins.
oxygen necessary for normal bacterial killing
Patients with Chediak-Higashi syndrome show
➔ Normally, neutrophil stimulation leads to the
granulocytic inclusions attributed to enlarged
production of reactive oxygen molecules, such
lysosomes
as hydrogen peroxide (H2O2), by NADPH
oxidase reactivity on the plasma membrane.

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➔ The plasma membrane enfolds an organism as ➔ The neutrophils are then activated using
it is phagocytized and hydrogen peroxide is phorbol myristate acetate (PMA), which is
generated in close proximity to the target mitogenic for neutrophils.
microbe. ➔ The resultant oxidative burst will reduce the
➔ Neutrophil granules fuse with and release the DHR, resulting in fluorescence that may be
enzyme myeloperoxidase into the forming quantitated on a flow cytometer.
phagosome. The myeloperoxidase uses the
➔ Neutrophils from CGD patients will be unable
hydrogen peroxide to generate the potent
microbicidal agent, hypochlorous acid to undergo the oxidative burst and show less
fluorescence than normal neutrophils.
Hexose monophosphate shunt - the process of ➔ Although therapy with granulocyte
generating partially reduced forms of oxygen by transfusions may allow resolution of an acute
stimulated neutrophils was first detected as an infectious episode, it is impossible to provide
increase in oxygen consumption, known as enough granulocytes to treat the chronic
→ RESPIRATORY BURST / OXIDATIVE BURST condition.
➔ Administration of cytokines, such as
NADPH oxidase system can result in the CGD
interferon, may increase the oxidative burst
phenotype by making the neutrophil incapable of
activity in some patients. Continuous use of
generating an oxidative burst
antibiotics can greatly reduce the occurrence
of severe infections.
● Symptoms include: recurrent suppurative ➔ Bone marrow transplantation or use of
infections, pneumonia, osteomyelitis, peripheral blood stem cells may result in a
draining adenopathy, liver abscesses, permanent cure
dermatitis, and hypergammaglobulinemia
Diagnosis of Phagocytic Cells
● Staphylococcus aureus, Burk olderia cepacia,
and Chromobacterium violaceum; In patient
with CGD in additional to FUNGI such as ● The nitroblue tetrazolium test (NBT) usually is
Aspergillus and Nocardia and INFECTION used to assay the phagocytic function of
usually begins before 1 year of age and the neutrophils.
syndrome is usually often FATAL in ● This assay for metabolism and generation of
CHILDHOOD for CGD toxic molecules determines whether
UNABLE to produce NORMAL BACTERIAL KILLING phagocytic cells are using hexose
because they are INCAPABLE of producing ROS or monophosphate shunt (HMP) and generating
activate form reactive oxygen species which are toxic materials to kill microorganism.
necessary for normal bacterial killing kasi nga ● It is used in the diagnosis of chronic
→ ABNORMAL or INCAPABLE of generating an granulomatous disease (CGD).
OXIDATIVE BURST there is GENETIC DEFECT in the ● The neutrophils of CGD patients fail to reduce
several NADPH OXIDASE SYSTEM the NBT dye
● More recently, a flow cytometric assay has
➔ CGD was historically diagnosed by measuring been used. In this assay, neutrophils are
labeled with dihydrorhodamine (DHR).
the ability of a patient’s neutrophils to reduce
● DHR will fluoresce when it is reduced.
the dye nitroblue tetrazolium (NBT).
● Uses Phorbol myristate acetate (PMA), which
➔ NBT reduction is caused by the production of is mitogenic for neutrophils.
hydrogen peroxide and other reactive forms ● Neutrophils from CGD patients will be unable
of oxygen. to undergo the oxidative burst and will show
➔ Reduction converts the nearly colorless NBT less fluorescence than normal neutrophils.
into a blue precipitate that can be assessed ● This technique is more objective and
visually on a microscope slide. quantitative than the traditional NBT
➔ DHR will fluoresce when it is reduced. technique
OTHER MICROBICIDAL DEFECTS
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Neutrophil glucose-6-phosphate dehydrogenase Category 6: Defects in Innate Immunity

deficiency Category 6 DEFECTS in the innate immune system. At
○ Aerobic system of neutrophils are impaired. least one disease, chronic mucocutaneous candidiasis,
Neutrophil glucose-6-phosphate dehydrogenase which was previously classified as a T-cell defect, is now
deficiency included in this classification
- leads to an inability to generate enough
NADPH to supply reducing equivalents to the Other rare entities classified under this heading
NADPH oxidase system include mutations in Toll-like receptors (TLRs)
- There is a HEMOLYTIC ANEMIA that is present
.because G6PD also causes POIKILOCYTOSIS or ● Chronic mucocutaneous candidiasis
ANOMALY in RBCs wherein nagkakaroon ng ● Other rare entities classified under this
HEINZ BODIES or PITTED GOLF BALL heading include mutations in Toll-like
appearance ang rbcs that's why they are being receptors (TLRs)
destroyed in SPLEEN and nagkakaroon ng ● Few clinical laboratory assays are currently
HEMOLYTIC ANEMIA in G6PD available for assessing innate immune system
functional capabilities
Myeloperoxidase deficiency - defects can lead to bacterial infections
- These are SPECIFIC GRANULES in
NEUTROPHILS that are needed for bacterial Category 7: Autoinflammatory Disorders
killing. Autoinflammatory disorders are subdivided into two
Myeloperoxidase deficiency is relatively common, classifications:
occurring in about 1 in 3,000 persons in the United ➔ Those involving the inflammasome
States. ➔ non inflammasome conditions
○ Deficient patients may have recurrent Inflammasome - is a protein oligomer that contains
candidal infections caspase enzymes and other proteins associated with
○ Defects of neutrophil secondary granules apoptosis.
- Defects of neutrophil secondary granules have - The inflammasome is located primarily in
been described also. myeloid cells and may be activated by various
However, the molecular nature of the defects is microbial substances. Once activated, the
unknown inflammasome stimulates the production of
the proinflammatory cytokines IL-1 and IL-18
Leukocyte Adhesion Deficiency (LAD)
● There is a deficiency in a protein called CD18, ● Genetic defects involving the inflammasome
which is a component of adhesion receptors on include the Hyper IgD syndrome, also referred
neutrophils and monocytes (CD11b or CD11c) to as periodic fever syndrome (mevalonate
and on T cells (CD11a) kinase), and Muckle-Wells syndrome
● Abnormal adhesion, motility, aggregation, (cryopyrin)
chemotaxis, and endocytosis by the affected Muckle-Wells syndrome is caused by a mutation in the
leukocytes. CIAS1 gene coding for cryopyrin, a component of the
Even if microbiocidal activity is normal, neutrophils inflammasome.
cannot perform their functions properly if they fail to
leave the vasculature and migrate to a site of incipient ● Defects not involving the inflammasome
infection. include tumor necrosis factor (TNF) receptor-
associated periodic syndrome (TRAPS)
Adhesion receptors on leukocytes and their counter (TNFRSF1A gene) -
receptors on endothelial cells and the extracellular TRAPS is caused by a mutation in the TNFRSF1A gene,
matrix play important roles in these activities. which codes for a TNF receptor and may result in
recurrent fevers, as well as ocular and joint
● Manifestations: delayed wound healing, inflammation.
chronic skin infections, intestinal and
respiratory tract infections, and periodontitis

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early-onset inflammatory bowel disease (IBD) ● Chronic mucocutaneous candidiasis

(IL-10 or its receptor)
Early-onset IBD - is caused by mutations in genes Acquired Immunodeficiency syndrome (AIDS)
coding for IL-10 or its receptor
● Etiologic agent: Human immunodeficiency
Category 8: Complement Deficiencies virus (HIV) - The virus attacks the immune
● Deficiencies in the early complement system and leaves the body vulnerable to a
components, C1q, C4, and C2, are usually variety of life-threatening illnesses and prone
associated with a lupuslike syndrome cancers
Complement - consists of a SERIES of proteins that
work in a cascade to assist in antibody destruction of HIV - causative agent
cells AIDS - disease known as <-
The complement system is also part of the innate BUT hindi porket meron HIV yung isang patients meron
immune system and can work as part of the na kaagad AIDS → nagkaroon lang ng AIDS if CD4 T Cells
inflammatory system to directly eliminate a potential count falls BELOW 200 (NORMAL → 1500 up to 3000)
pathogen. meaning hindi na kaya ng katawan,
- Deficiencies in each of the major complement immunocompromised na and have now AIDS, and the
components have been described, leading to patient are now VULNERABLE to different LIFE-
various clinical symptoms THREATENING ILLNESS
Deficiency of C2 - is believed to be the most common
complement component deficiency. Human immunodeficiency virus (HIV) - is the etiologic
C3 deficiency - may also have a lupuslike clinical agent of acquired immunodeficiency syndrome, or
presentation, but is more likely to involve recurrent AIDS, a disease that has posed one of the greatest
infections with encapsulated organisms. medical challenges worldwide - because wala pang
cure and treatment is usually management
A deficiency of C1 esterase inhibitor - has been found (pinapalakas ang immune system)
in patients with hereditary neuroangioedema.
Transmission
● Deficiencies of the later components of ➔ intimate sexual contact
complement (C5–C9) - are often associated ➔ contact with blood or other body fluids, or
with recurrent Neisseria meningitidis ➔ perinatally (from infected mother to infant)
infections.
- The majority of cases of HIV infection have
Category 9: Phenocopies of Primary occurred through sexual transmission
Immunodeficiencies involving either vaginal or anal intercourse
This category comprises a new classification of PIDs.
Disorders that fall into this category have an inherited - transmission is perinatal, from infected
genetic component but also include an acquired mother to her fetus or infant. Transmission
component, such as: through this route can occur during pregnancy,
➔ somatic mutations by transfer of blood at the time of delivery, or
➔ autoantibody production through breastfeeding
Chronic mucocutaneous candidiasis, a disease that
was once classified as a cell-mediated deficiency, is
now included in Category 9.
- This disease is induced by a genetic mutation
in the AIRE gene, but also involves an antibody
to either (or both) IL-17 and IL-22.

● Disorders that fall into this category have an

inherited genetic component but also include
an acquired component, such as somatic
mutations or autoantibody production
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HIV belongs to the genus Lentivirinae of the virus

family Retroviridae.
- It is classified as a retrovirus because it
contains ribonucleic acid (RNA) as its nucleic
acid and a unique enzyme, called reverse
transcriptase, which transcribes the viral RNA
into DNA, a necessary step in the virus’s life
cycle.
- HIV is a spherical particle, 100 to 120 nm in
diameter, which contains an inner core with
two copies of single-stranded RNA surrounded
by a protein coat or CAPSID and an outer
envelope of glycoproteins embedded in a lipid
bilayer
The genome of HIV includes three main structural
genes:
➔ gag,
➔ env
➔ pol
The gag gene codes for p55, a precursor protein with a
molecular weight of 55 kd, from which four core
structural proteins are formed:
➔ p6
➔ p9
➔ p17
➔ p24
All four are located in the nucleocapsid of the virus.

The env gene codes - for the glycoproteins gp160,

gp120, and gp41, which are found in the viral Viral Replication
envelope.→ important for the ATTACHMENT of HIV to
CD4 cells

The third structural gene, pol, codes for enzymes

necessary for HIV replication

These include reverse transcriptase (p51); ribonuclease

(RNase H; p66) - an enzyme involved in the
degradation of the original HIV RNA

integrase (p31) - an enzyme which mediates the

integration of viral DNA into the genome of infected
host cells

protease (p10) - that cleaves precursor proteins into

smaller active units used to make the mature virions.

- These proteins are located in the core of the

virus in association with HIV RNA.
The first step in the reproductive cycle of HIV occurs
when the virus attaches to a susceptible host cell. This

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interaction is mediated through the host-cell CD4 - Double-stranded DNA is synthesized and, with
antigen, which serves as a receptor for the virus by the help of the HIV integrase enzyme, becomes
binding the gp120 glycoprotein on the outer envelope integrated into the host cell’s genome as a
of HIV. provirus
- The provirus can remain in a latent state for a
T helper (Th) cells - are the MAIN TARGET for HIV long time, during which viral replication does
infection because they express high numbers of CD4 not occur. Eventually, expression of the viral
molecules on their surface and bind the virus with high genes is induced when the infected host cell is
affinity activated by binding to antigen or by exposure
to cytokines.
Other cells such as macrophages, monocytes, dendritic - Viral DNA within the cell nucleus is then
cells, Langerhans cells, and microglial brain cells can transcribed into genomic RNA and messenger
also be infected with HIV because they have some RNA (mRNA), which are transported to the
surface CD4. cytoplasm.
- Translation of mRNA occurs, with production
HIV viruses that preferentially infect T cells are known of viral precursor proteins and assembly of
as T-tropic or X4 strains, whereas those strains that can viral particles.
infect both macrophages and T cells are called M-tropic - The intact virions bud out from the host cell
or R5 strains. membrane and acquire their envelope during
the process.
TROPIC/ TROPISM - means they only affect a SINGLE - The precursor proteins are cleaved by the viral
TYPE of cell protease enzyme in the mature virus particles.
- These viruses can proceed to infect additional
host cells.
- Viral replication occurs to the greatest extent
in antigen-activated Th cells. Because viral
replication occurs very rapidly and the reverse
→ Entry of HIV into the host cells to which it has transcriptase enzyme lacks proofreading
attached requires an additional binding step, involving activity, genetic mutations occur at a high rate,
co-receptors that promote fusion of the HIV envelope producing distinct isolates that exhibit an
with the plasma cell membrane. extraordinary level of antigenic variation.
- These co-receptors belong to a family of - In fact, the level of HIV diversity in a single
proteins known as chemokine receptors, individual is greater than the diversity of all the
whose main function is to direct white blood influenza virus isolates throughout the world in
cells (WBCs) to sites of inflammation. a given year!
- This tremendous genetic diversity of HIV
CXCR4 - required for HIV to enter T lymphocytes hinders the ability of the host to mount an
CCR5 - is required for entry into macrophages. effective immune response.

- In fact, individuals who have a genetic Immunologic Manifestations

mutationin the CCR5 gene have been found to ● Initial viral replication: presence of increased
be resistant to HIV infection. levels of p24 antigen
- Binding of the co-receptors allows for HIV ● As the virus replicates, some of the viral
entry by inducing a conformational change in proteins produced within host cells form
the gp41 glycoprotein, which mediates fusion complexes with class I major histocompatibility
of the virus to the cell membrane. complex (MHC) antigens and are transported
- After fusion occurs, the viral particle is taken to the cell surface, where they stimulate
into the cell and uncoating of the particle lymphocyte responses.
exposes the viral genome ● HIV-specific CD4+ Th cells are generated and
- Action of the enzyme reverse transcriptase assist both humoral and cell-mediated immune
produces complementary DNA from the viral responses against the virus.
RNA.

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B lymphocytes are stimulated to produce antibodies to IgA def: selective IgA deficiency
HIV, which can usually be detected in the host’s serum IgG def: IgG subclass deficiency
by 6 weeks after primary infection. JAK3: Janus kinase-3 deficiency
LAD: leukocyte adhesion deficiency
● The first antibodies to be detected are directed PNP: purine nucleoside phosphorylase deficiency
against the gp41 transmembrane glycoprotein WAS: Wiskott-Aldrich syndrome
followed by production of antibodies to the xSCID: X-linked severe combined
gag proteins such as p24, and finally immunodeficiency disease
production of antibodies to the env, pol, and KEY TO CELLS
regulatory proteins. PS: pluripotent stem cell
CMP: common myeloid precursor
● T-cell–mediated immunity is thought to play an CLP: common lymphoid precursor
important role in the immune response to HIV NK: natural killer cell
IB: immature B cell
● CTL and antibody responses to HIV are IT: immature T cell
hindered by the virus’s ability to undergo rapid Th: helper T cell
genetic mutations, generating ESCAPE Tc: cytotoxic T cell
mutants with altered antigens toward which PC: plasma cell
the host’s initial immune responses are
ineffective PRINCIPLES OF IMMUNOLOGIC AND
SEROLOGIC PROCEDURES
● HIV Pro-viral state: HIV is protected from Learning Intended Outcomes
attack by the immune system until cell ● Explain the principles and apply the procedures of the
different serologic tests in the detection of antigens and
activation stimulates the virus to multiply and
antibodies.
display its viral antigens ● Distinguish between precipitation and agglutination
- SILENCE PRO VIRUS for LONG PERIOD OF TIME reaction correctly.
● The ability of HIV to evade the immune ● Compare and describe the different agglutination
response results in a persistent infection that reactions and give examples of each appropriately.
can destroy the immune system ● Compare and contrast single and double
SUMMARY immunodiffusion discuss the role of each in
measurement of precipitation reactions.
● Compare flocculation and agglutination
● Explain the principles of complement fixation and
neutralization test accurately.

Antigen–Antibody Binding
In serology the focus is to → determine the absence or
presence of an antibody or an antigen to help in the
diagnosis of certain infections and diseases
The combination of an antigen with a specific antibody
plays an important role in the laboratory in diagnosing
many different diseases and there are a lot of
SEROLOGICAL TEST or IMMUNOASSAYS.
IMMUNOASSAYS - we're using ANTIBODIES in the
KEY TO IMMUNODEFICIENCIES diagnosis of different diseases.
ADA: adenosine deaminase deficiency
AT: ataxia-telangiectasia There are several immunoassays that have been
developed to detect (sa isang immunoassays we’re
BTK: Bruton’s tyrosine kinase deficiency
detecting either the ANTIGEN or ANTIBODY of a
CGD: chronic granulomatous disease
particular INFECTION, and kung ano yung denedetect ang
C-H: Chediak-Higashi syndrome
reagent is yung OPPOSITE niya)
CVI: common variable immunodeficiency
DiGeorge: DiGeorge anomaly

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If there is an ANTIGEN ANTIBODY BINDING there would One antibody may initially attract numerous different
be a particular reaction to help diagnose a particular antigen but the epitopes shape and the way it fits
infection. together with binding site of an antibody determines
whether the bonding will be stable or not.
If you are detecting patients antigen the reagent is consist
Antibodies are capable of reacting with antigens
of the KNOWN ANTIBODY for it (para mag positive at ma
resembling the original antigen that induced antibody
detect yung certain infection)
production, which is known as → cross-reactivity.
● Either you are detecting the patient antigen or
antibody and → the reagent will consist of either The more the cross-reacting antigen resembles the
the antigen or antibody. original antigen, the stronger the bond will be between
the antigen and the binding site. However, if the epitope
Antigen–Antibody Binding and the binding site have a perfect lock-and-key fit, as is
The primary union of binding sites on an antibody with the case with the original antigen, the affinity will be
specific epitopes on an antigen depends on two maximal.
characteristics of antibody known as AFFINITY and
When the affinity is higher, the assay reaction is more
AVIDITY.
sensitive because more antigen–antibody complexes will
For such reactions to occur or for an antigen antibody be formed and visualized more easily.
reaction to occur both antigen and antibody must have ● When the affinity is higher, the assay reaction is
MULTIPLE BINDING SITE for them to form a LATTICE more sensitive because more antigen–antibody
FORMATION of CROSSLINK → and the binding complexes will be formed and visualized more
characteristics now of antibodies are termed as AFFINITY easily.
and AVIDITY.
AFFINITY and AVIDITY - plays MAJOR role in antigen and
antibody binding or reaction,

AFFINITY
● INITIAL FORCE OF ATTRACTION that exists
between a single Fab site on an antibody
molecule and a single epitope or determinant FIGURE 10–1 Affinity is determined by the three-dimensional fit
and molecular attractions between one antigenic determinant and
site on the corresponding antigen
one antibody-binding site. The antigenic determinant on the left
● The strength of attraction DEPENDS on the has a better fit and charge distribution than the epitope on the
specificity of antibody for a particular antigen right and hence will have a higher affinity.
An antigen has different antigenic determinants.
Antigenic determinants or epitope - is the binding site of
AVIDITY
the antibody molecule.
● SUM of all attractive forces between an antigen
Cross reaction mechanism - wherein an antigen can have
and an antibody
several antigenic determinants and antibodies can react
● This involves the strength with which a
to this.
multivalent antibody binds a multivalent antigen,
eg: antibody of smallpox pwedeng mag react sa antigenic Avidity → represents the overall strength of antigen–
determinant ng cowpox and that is a cross reaction antibody binding and is the sum of the affinities of all the
mechanism but the affinity increases if the particular individual antibody–antigen combining sites.
antibody is for the particular antigen (so mas malakas ang Avidity → refers to the strength with which a multivalent
affinity ni cowpox antibody to the cowpox antigen kesa antibody binds a multivalent antigen and is a measure of
cowpox antibody to the smallpox antigen) the overall stability of an antigen–antibody complex.
The more cross-reacting antigen resembles the original In other words, once binding has occurred, it is the force
antigen the STRONGER the bond will be, between the that keeps the molecules together
antigen and the binding site of an antibody. ● A measure of the overall stability of an antigen–
antibody complex.
So if the epitope or the antigenic determinants and the
● A high avidity can actually compensate for a low
binding site of an antibody exhibits a perfect lock and key
affinity.
that is → the case of of an original antigen and the affinity
The more bonds that form between antigen and antibody,
will be at its maximum or maximal capacity
the higher the avidity is. IgM → for instance, has a higher
avidity than IgG because
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IgM - has the potential to bind 10 different antigens

Both affinity and avidity → contribute to the stability of
the antigen–antibody complexes,which is essential to HIGHER AVIDITY = HIGHER ASSOCIATION or BINDING
detecting the presence of an unknown, whether it is HIGHER AVIDITY = LOW DISSOCIATION
antigen or antibody. The ideal conditions in the clinical laboratory would
● Stability of the antigen–antibody complex is be to have an antibody with a high affinity, or initial force
essential in serology or to detecting the presence of attraction, and a high avidity, or strength of binding.
of an unknown, whether it is antigen or antibody The higher the values are for both of these and the more
antigen–antibody complexes that are formed, the more
sensitive the test.
Precipitation and Agglutination
Precipitation and Agglutination are the visible expression
of the aggregation of antigens and antibodies through the
formation of a framework in which antigen particles or
molecules alternate with antibody molecules.
PRECIPITATION - this involves combination of a soluble
antigen with a soluble antibody (so pareho dapat soluble
antigen when it comes to precipitation reactions)

PRECIPITATION - this involves a combination of a soluble

antigen and then the soluble antibody to → produce an
insoluble complex which are visible
Avidity is the sum of the forces binding multivalent
AGGLUTINATION - this is the process by which the
antigens to multivalent antibodies. In a comparison
between IgG and IgM, IgM has the most potential antigen is PARTICULATE such as particles → such as for
binding sites for antigen and thus the higher avidity. example cells which aggregate to form a large complex
Note that the monomers in IgM can swing up or down in when a specific antibody is present or if the antibody is
order to bind more effectively. bound to the particulate antigen.
Law of Mass Action Precipitation and agglutination - these are the visible
All antigen–antibody binding is REVERSIBLE and is expression of the aggregation of antigen and antibodies
governed by the → LAW OF MASS ACTION through the formation of a framework
● This law states that free reactants are in
equilibrium with bound reactants So for a visible reaction or expression of an antibody
● The equilibrium constant K represents the complex to occur or the antigen and antibodies should
difference in the rates of the forward and reverse produce a LATTICE FORMATION or a FRAMEWORK in
reactions according to the following equation: which the particles or the molecules alternate with the
▹ K = [AgAb]/[Ab][Ag] antigen rather alternate with the antibody molecules.
(Equilibrium constant is equals to antigen-antibody
association divided by antibody and antigen PRECIPITATION REACTIONS
dissociation complex) Precipitation Curve
PRECIPITATION - this involves a combination of a soluble
■ where [AgAb] = concentration of the antigen-antibody antigen and then the soluble antibody to → produce an
complex (mol/L) insoluble complex which are visible
■ [Ab] = concentration of free antibody (mol/L)
■ [Ag] = concentration of free antigen (mol/L) Precipitation - depends on the relative proportions of
antigen and antibody present in the reaction.
As the strength of binding, or avidity, increases,
the tendency of the antigen–antibody complexes to Optimum precipitation should be in the zone of
dissociate decreases, which increases the value of K. equivalence.
When the value of K is higher, the amount of antigen–
antibody complex is larger and the assay reaction is more ZONE OF EQUIVALENCE
visible or easily detectable. ● In this zone, precipitation is the result of random,
reversible reactions whereby each antibody

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binds to more than one antigen and vice versa, A false-negative reaction may take place in the prozone
forming a stable network or lattice because of high antibody concentration.
In the zone of equivalence, the number of multivalent If it is suspected that the reaction is a false negative,
sites of antigen and antibody are approximately equal diluting out antibody and performing the test again may
(para magkaroon ng stable network or LATTICE produce a positive result. In the postzone,excess antigen
FORMATION); maximum may obscure the presence of a small amount of antibody.
Typically, such a test is repeated with an additional
The lattice hypothesis, as formulated by Marrack, is patient specimen taken about a week later.
based on the assumptions that each antibody molecule The extra time would allow for the further production of
must have at least two binding sites and the antigen must antibody. If the repeated test is negative, it is unlikely that
be multivalent. the patient has that particular antibody.
As they combine, this arrangement results in a
multimolecular lattice that increases in size until it 3 test for precipitation
precipitates out of solution ● Precipitation by Light Scattering
● Precipitation by Immunodiffusion/ diffusion into
Prozone phenomenon: antibody excess the gel
The prozone phenomenon occurs, in which antigen ● Precipitation by using electric current /
combines with only one or two antibody molecules and electrophoresis
no cross-linkages are formed.
A. Precipitation by Light Scattering
In the prozone, usually only one site on an antibody 1. Turbidimetry
molecule is used and many free antibody molecules - directly through the solution
remain in solution. ● a MEASURE of the turbidity or cloudiness of a
● Antigen combines with only one or two antibody solution
molecules and no cross-linkages are formed. because most antigens are multivalent and thus capable
● This is because usually only one site on an of → forming aggregates in the presence of the
antibody molecule is used, and many free corresponding antibody. When antigen and antibody
antibody molecules remain in solution solutions are mixed, the antigen cross-links with
Postzone phenomenon numerous antibody molecules and the lattice networks
● At the other side of the zone, where there is become so large that they precipitate out of solution.
antigen excess, the postzone phenomenon Precipitates in fluids can be measured by means of
occurs in which small aggregates are surrounded → turbidimetry or nephelometry.
by excess antigen, and again no lattice network ● A detection device is placed in direct line with the
is formed. incident light, collecting light after it has passed
● In this case, every available antibody site is through the solution.
bound to a single antigen, and no cross-links are ● Scattering of light occurs in proportion to the
formed. size, shape, and concentration of molecules
● Thus, for precipitation reactions to be present in solution.
detectable, they must be run in the zone of ● Measurements are made using a
equivalence spectrophotometer or an automated clinical
chemistry analyzer
2. Nephelometry
- gumagamit ng certain angle
- Nephelometers typically measure light scatter at
angles ranging from 10 degrees to about 90
degrees.
FIGURE 10–3 Precipitin curve. The precipitin curve shows how the ● measures the light that is scattered at a
amount of precipitation varies with varying antigen concentrations particular angle from the incident beam as it
when the amount of antibody is kept constant. Excess antibody is passes through a suspension
called the prozone and excess antigen concentration is called the
postzone.
● If a solution has excess antibody, adding
increasing amounts of antigen results in an
The prozone and postzone phenomenon must be increase in antigen–antibody complexes and thus
considered in the clinical setting because negative an increase in light scattering.
reactions occur in both. ● Nephelometry can be used to detect either
antigen or antibody, but typically it is run with
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antibody as the reagent and patient antigen as gel matrix and if there is a reaction or combination visible
the unknown lines of precipitation will form in the gel → PASSIVE
● Although the sensitivity of turbidity has DIFFUSION
increased, nephelometry is more sensitive, with - When no electrical current is used to speed up
a lower limit of detection of 1 to 10 mg/L for this process, it is known as passive
serum proteins immunodiffusion. The rate of diffusion is affected
● The amount of light scattered is an index of the by the size of the particles, the temperature, the
solution’s concentration gel viscosity, and the amount of hydration.
1. Radial Immunodiffusion (RID)
● James Oudin
● A single-diffusion technique (meaning
isang antigen isang antibody) detect is
the ANTIGEN OF THE PATIENT
In this technique, antibody is uniformly distributed in the
support gel and antigen is applied to a well cut into the
FIGURE 10–4 Principles of nephelometry. The light detection device gel. As the antigen diffuses out from the well, antigen –
is at an angle to the incident light, in contrast to turbidity, which antibody combination occurs in changing proportions
measures light rays passing directly through the solution. until the zone of equivalence is reached and a stable
lattice network is formed in the gel. The area of the ring
B. Precipitation by Passive Immunodiffusion obtained is a measure of antigen concentration that can
PRECIPITATION - always dealing with SOLUBLE ANTIGEN be compared with a standard curve obtained by using
and SOLUBLE ANTIBODY that precipitates out of the antigens of known concentration, depicts some typical
serum forming an → INSOLUBLE COMPLEX results.
When the number of antigen and antibody is EQUAL the ● Antibody is uniformly distributed in
ZONE OF EQUIVALENCE will occur resulting to a VISIBLE support gel, and antigen is applied to a
REACTION well cut into the gel
● Antigen and antibody are added to wells in the ● The area of the ring obtained is a
gel (AGAROSE) an antigen– antibody measure of antigen concentration, and
combination occurs by means of diffusion this can be compared to a standard
➔ Agarose, a purified high-molecular-weight curve obtained by using antigens of
complex polysaccharide derived from seaweed, known concentration
is used for this purpose. Radial Immunodiffusion (RID)
➔ When antigen and antibody diffuse toward one
another in a gel matrix, visible lines of
precipitation will form.
➔ Agarose - helps stabilize the diffusion process
and allow better visualization of the precipitin
bands (bonds?) (that’s why yun yung ginagamit
na support medium in immunodiffusion)
● No electrical current is used to speed up the
process → that's why passive diffusion
● The rate of diffusion is affected by the size of the
particles, the temperature, the gel viscosity, and
the amount of hydration.
The agar concentration that is used as a support medium
in this reaction ranges from → 0.3% - 1.5% of agar and Antibody is incorporated into the agar and then layer on
concentration allows for diffusion of most reactants top the antigen as the antigen moves down towards the
TECHNIQUES: gel if present siya into particular antibody a precipitation
○ Radial Immunodiffusion reaction will occur at the zone of equivalence forming
- single immunodiffusion lattice formation and because of this magkakaroon
○ Ouchterlony Double Diffusion precipitation bond that resembles a RING that’s why this
- double immunodiffusion is called → Radial Immunodiffusion and the diameter of
same gumagamit ng agarose gel and then hahayaan mag the ring will be compared to a standard concentration and
diffuse ang antigen at antibody towards one another in a

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the proportions of the ring is DIRECTLY PROPORTIONAL to ● Both antigen and antibody diffuse
the antigen concentration independently through a semisolid medium in
(so, compare yung diameter nung RING to the STANDARD two dimensions
of KNOWN antigen concentration for us to known the In this technique, both antigen and antibody diffuse
ANTIGEN CONCENTRATION OF UNKNOWN) independently through a semisolid medium in two
dimensions, horizontally and vertically.
● Precipitin lines form where the moving front of
RID have 2 types of measurements antigen meets that of antibody.
● A. Mancini/ Endpoint Method ● The density of the lines reflects the amount of
● Fahey and McKelvey/ Kinetic Method immune complex formed
same lang na ginagamit na RID pero ang difference is the Wells are cut in a gel and reactants are added to the wells.
TIME that is required to measure the ring or the - Antibody that is multispecific/ cross-reactive is
precipitating ring placed in the central well and different antigens
are placed in the surrounding wells to determine
MEASURING RADIAL IMMUNODIFFUSION if the antigens share identical epitopes.
A. Mancini/ Endpoint Method - Diffusion takes place radially from the wells.
- PASSIVE IMMUNODIFFUSION TECHNIQUE After an incubation period of between 12 and 48
- measure at the END hours in a moist chamber, precipitin lines form
One technique for the measurement of radial where the moving front of antigen meets that of
immunodiffusion was developed by Mancini and is antibody and the point of equivalence is reached.
known as the endpoint method.
Ouchterlony Double Diffusion
Equivalence occurs between 24 and 72 hours.
● IgM - 50-72 hours
● IgG - 24 hours for the zone of equivalence to
occur
● Antigen is allowed to diffuse to completion
● Once equivalence is reached, no further change
in ring diameter takes place
● In this technique, antigen is allowed to diffuse to
completion, and when equivalence is reached,
there is no further change in the ring diameter.
● The square of the diameter is then directly
FIGURE 10–6
proportional to the concentration of the antigen. Ouchterlony diffusion patterns. An antibody mixture is placed in
● The major drawback (disadvantage) to this the central well. Unknown antigens are placed in the outside wells.
method is the time it takes to obtain results The antibodies and antigens all diffuse radially out of the wells.
B. Fahey and McKelvey/ Kinetic Method
(A) Serological identity. If the antigens are identical, they will react
- measure while the test is being conducted with the same antibody and the precipitate line forms a
● Uses measurements taken before the point of continuous arc.
equivalence is reached
the kinetic or Fahey method, uses ring diameter readings (B) Serological Nonidentity. If the antigens share no identical
determinants, they will react with different antibodies and two
taken at about 19 hours before the zone of equivalence is
crossed lines are formed.
reached
● The diameter is proportional to the log of the (C) If antigen 3 has a determinant in common with antigen 1, one
concentration of the antibodies reacts with both antigens.
RID - Radial immunodiffusion has been used to measure Another antibody that reacts with different determinants on
antigen 1 (absent on antigen 3) passes through one precipitation
IgG and IgA subclasses as well as complement line and forms the spur on the other line. The spur formed always
components. points to the simpler antigen with fewer antigenic determinants.

The position of the precipitin bands between wells

B. Precipitation by Passive Immunodiffusion
allows for the antigens to be compared with one
2. Ouchterlony Double Diffusion
another. Several patterns are possible:
- Ouchterlony method
(1) Fusion of the lines at their junction to form an arc
One of the older, classic immunochemical techniques is
represents serological identity or the presence of a
Ouchterlony double diffusion
common epitope,

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(2) a pattern of crossed lines demonstrates two ● Incubated time: 18 to 24 hours.

separate reactions and indicates that the compared double-diffusion occurs at right angles and
antigens share no common epitopes, and electrophoresis will be used to separate the reactions if
(3) fusion of two lines with a spur indicates partial there is presence of antigens towards the antibodies in
identity. the trough precipitin lines or bond will be formed, where
the specific antigen antibody combination took place.
C. Precipitation by Electrophoretic Techniques - However, interpretation is difficult for
● Diffusion can be combined with electrical immunoelectrophoresis therefore it has largely
current to speed up reaction and sharpen been replaced with immunofixation
results electrophoresis give faster and more reliable
● Electrophoresis separates molecules according results and is easier to interpret
to differences in their electric charge when they 3. Countercurrent Immunoelectrophoresis
are placed in an electric field. same as immunoelectrophoresis but instead of a trough
● A direct current is forced through the gel, ● Ag and ab placed on a well directly opposite
causing antigen, antibody, or both to migrate. each other
As diffusion takes place, distinct precipitin use to facilitate migration towards the center (magkikita
bands are formed. sa gitna)
TECHNIQUES Precipitin line is formed it will indicate that there has
○ Rocket Immunoelectrophoresis been a reaction, so precipitin line closer to the antigen
○ Immunoelectrophoresis well it means that there is a higher concentration
○ Immunofixation Electrophoresis antibody
1. Rocket immunoelectrophoresis: single- diffusion 4. Immunofixation Electrophoresis
technique immunoelectrophoresis this was describe by ALPER and
is a one dimension electroimmunodiffusion, this is a JOHNSON
adaptation of the regional immunodiffusion or RID which ● Similar to immunoelectrophoresis except that
is developed by Laurell, same principle but this is after electrophoresis takes place, antiserum is
combined with electrical current to speed up reaction applied directly to the gel’s surface rather than
and sharpen results placed in a trough
- it just RID + electrophoresis ● Immunodiffusion takes place in a shorter time
● Antibody is distributed in the gel, and antigen is (less than 1 hour) and results in a higher
placed in wells cut in the gel, just as in RID. resolution
● Electrophoresis is used to facilitate migration of ● Agarose or cellulose acetate is used.
the antigen into the agar ● An antibody of known specificity is used to
● When the antigen diffuses out of the well, determine whether patient antigen is present.
precipitation begins.
● The end result is a precipitin line that is conical
in shape, resembling a rocket, hence the name
rocket immunoelectrophoresis.

FIGURE 10–7 Immunofixation electrophoresis. A complex antigen

mixture such as serum proteins is separated by electrophoresis. An
antiserum template is aligned over the gel. Then protein fixative
and monospecific antisera, IgG, IgA, IgM, κ, and λ are applied to
the gel. After incubating for 30 minutes, the gel is stained and
examined for the presence of paraproteins. Precipitates form
2. Immunoelectrophoresis where specific antigen–antibody combination has taken place. In
introduced by GRAY BAR and WILLIAMS GRAY this case, the patient has an IgG monoclonal antibody with λ
chains. (Courtesy of Helena Laboratories, Beaumont, TX.)
● A double-diffusion technique that incorporates
electrophoresis current to enhance results
● A trough (parang butas na pa-curve) is cut in
the gel parallel to the line of separation
● Antiserum is placed in the trough

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have antigens on its surface and combining with

antibody molecules that form bridges between the
antigenic determinants of the antigens
SUMMARY
Agglutination, like precipitation, is a two-step process
that results in the formation of a stable lattice network.
➔ The first reaction, called sensitization, involves
antigen–antibody combination through single
antigenic determinants on the particle and is
rapid and reversible.
➔ The second step, or lattice formation, is the
formation of cross-links that form the visible
aggregates. This represents the stabilization of
antigen–antibody complexes with the binding
together of multiple antigenic determinants
Sensitization
AGGLUTINATION REACTIONS (PART 2) ● involves antigen–antibody combination through
Agglutination single antigenic determinants on the particle
Agglutination - it needs that the antigen is a surface
PARTICULATE MATTER kasi ang SOLUBLE ANTIGEN kahit Lattice formation
na may combination sila with an antibody hindi sila mag ● Formation of cross-links that form the visible
formed ng visible agglutination aggregates.
● Agglutination (clumping) is the term used to ● This represents the stabilization of antigen–
describe the aggregation of particulate test antibody complexes with the binding together
antigens. of multiple antigenic determinants
● Process by which particulate antigens such as
cells aggregate to form large complexes when a Phases of Agglutination
specific antibody is present.
● Agglutination occurs only if the antigen is in the
form of particles such as bacteria, red blood
cells, latex particles, white blood cells or any
substance that appears cloudy when suspended
in saline. Lifted from: Stevens
In 1896, Gruber and Durham published the first report FIGURE 10–8 Phases of agglutination. Sensitization: Antigen and
about the ability of antibody to clump cells, based on antibody unite through antigenic determinant sites. Lattice formation:
observations of agglutination of bacterial cells by serum. Rearrangement of antigen and antibody bonds to form a stable lattice.

This finding gave rise to the use of serology as a tool in Sensitization is affected by the nature of the antigens on
the diagnosis of disease and also led to the discovery of the agglutinating particles. If epitopes are sparse or if
the ABO blood groups. they are obscured by other surface molecules, they are
Widal and Sicard developed one of the earliest less likely to interact with antibody.
diagnostic tests in 1896 for the detection of antibodies
occurring in typhoid fever, brucellosis, and tularemia. Additionally, red blood cells (RBCs) and
➔ Direct agglutination bacterial cells have a slight negative surface charge;
➔ Passive Agglutination because like charges tend to repel one another, it is
sometimes difficult to bring such cells together into a
➔ Reverse Passive Agglutination
lattice formation.
➔ Agglutination Inhibition
➔ CO agglutination - The class of immunoglobulin is also important; IgM
➔ Antiglobulin mediated immune agglutination with a potential valence of 10 is over 700 times more
Mechanism of particle agglutination efficient in agglutination than is IgG with a valence of
Agglutination is the clumping aggregation of particles Antibodies of the IgG class often cannot bridge the
that's why it involves particulate antigens distance between particles because their small size and
restricted flexibility at the hinge region may prohibit
It involves clumping or aggregation of particles that
multivalent binding.
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IgM antibodies, on the other hand, are strong Hemagglutination Reactions

agglutinins because of their larger size. Achieving visible
reactions with IgG often requires the use of
enhancement techniques that vary physicochemical
conditions such as the ionic strength of the solution, the
pH, and the temperature.

Antibodies belonging to the IgG class agglutinate

best at 30°C to 37°C, whereas
IgM antibodies react best at temperatures between 4°C
and 27°C.
Because naturally occurring antibodies against the ABO FIGURE 10–11 Grading of agglutination reactions:
A. tube method. If tubes are centrifuged and shaken to resuspend the
blood groups belong to the IgM class, these reactions
button, reactions can be graded from negative to 4+, depending on
are best run at room temperature. the size of clumps observed. B. Rapid slide method.
Antibodies to other human blood groups usually belong
to the IgG class; reactions involving these must be run at
37°C

In addition to temperature considerations, detection of

IgG antibodies often requires the use of a second
antibody, antihuman immunoglobulin, to visualize a
reaction

TYPES OF AGGLUTINATION REACTONS

1. DIRECT AGGLUTINATION FIGURE 10–10 Red blood cell agglutination. The tube on the left is a
● Occurs when antigens are found naturally on positive test for RBC agglutination, whereas the tube on the right is a
the surface of a particle. negative test showing that the RBCs have remained in a smooth
● One of the best examples of direct agglutination suspension.
testing involves known bacterial antigens used
2. PASSIVE AGGLUTINATION
to test for the presence of unknown antibodies
gumagamit here ng carrier
in the patient.
A variety of particles, including erythrocytes, latex, and
● Detection of antibodies is primarily used in
gelatin, are used for passive agglutination.
diagnosis of diseases for which the bacterial
● Passive, or indirect, agglutination employs
agents are extremely difficult to cultivate.
particles that are coated with antigens not
One such example is the Widal test, a rapid screening
normally found on their surfaces.
test used to help determine the possibility of typhoid
● Particle sizes vary from 7 μm for RBCs down to
fever.
0.8 μm for fine latex particles
A significant finding is a fourfold increase in antibody
● Used to detect rheumatoid factor; antinuclear
titer over time when paired dilutions of serum samples
antibody occurring in the disease lupus
are tested with any of these antigens.
erythematosus; antibodies to group A
streptococcus antigens; antibodies to Trichinella
Hemagglutination:
spiralis; antibodies to Treponema pallidum;
● Antigen is found on surface of RBCs
and antibodies to viruses such as
If an agglutination reaction involves RBCs, then it is
cytomegalovirus, rubella, varicella-zoster, and
called hemagglutination.
HIV-1/ HIV-2
- The best example of this occurs in ABO blood
Different carrier particles such as:
group typing of human RBCs, one of the world’s
➔ commonly use is LATEX
most frequently used immunoassays.
- Patient RBCs mixed with antisera of the IgM ➔ RBCs
type can be used to determine the presence or ➔ Gelatine
absence of the A and B antigens; this reaction is ➔ Silicates
usually performed at room temperature ➔ Bentonite
without the need for any enhancement ➔ Charcoal
techniques.
PASSIVE AGGLUTINATION
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Streptococcus, Staphylococcus aureus, streptococcal

groups A and B, rotavirus, and Cryptococcus
neoformans.

In 1955, Singer and Plotz found by happenstance that

IgG was naturally adsorbed to the surface of polystyrene Passive and Reverse Passive Agglutination
latex particles.
- Latex particles are inexpensive, are relatively
stable, and are not subject to cross-reactivity
with other antibodies. A large number of
antibody molecules can be bound to the surface
of latex particles, so the number of antigen-
binding sites is large
Additionally, the large particle size facilitates reading of
the test. Latex agglutination tests have been used to
detect rheumatoid factor, antibodies to Group A
Streptococcus antigens, and antibodies to viruses such
as rotavirus, cytomegalovirus, rubella, and varicella-
zoster

3. REVERSE PASSIVE AGGLUTINATION FIGURE 10–12 Passive and reverse passive agglutination. (A)
Same as passive agglutination meaning it also use a Passive
agglutination. Antigen is attached to the carrier particle;
carrier particles → the antigens used in this reaction agglutination occurs if patient antibody is present. (B) Reverse
cannot form visible reaction that's why it needs or have passive agglutination. Antibody is attached to the carrier particle;
a carrier molecule but the difference of passive to agglutination occurs if patient antigen is present.
reverse passive agglutination -
4. COAGGLUTINATION
Reverse passive - uses antibody rather than antigen so
This is just the same with reverse passive agglutination
the antibody is the one attached to the carrier to the
but the difference the particle or the carrier is bacteria,
latex so the carrier molecule that's why if agglutination
antibody ang naka attached
occur patient antigen is present
Because staphylococcus aureus has a protein on its
(Kung anong reagent mo yung kabaliktaran non yun
surface that we call protein A which naturally absorbs
yung detect)
like hinihigop niya naturally fc portion na antibody
So dahil reagent is carrier particles mixed with the molecules that's why staphylococcus aureus serve at the
reagent antibody so carrier + antibody if there is carriers and observe the fc portion of antibodies
agglutination → it means there is presence of patient
● Uses bacteria as the inert particles to which
antigen antibodies are attached
● Antibody rather than antigen is attached to a ● Staphylococcus aureus is most frequently used
carrier particle. ● These particles exhibit particles and are more
● The antibody must still be reactive and is joined refractory to changes in ionic strength.
in such a manner that the active sites are facing ● Coagglutination reagents have been used in
outward (so that presence and available parin identification of streptococci, Neisseria
yung binding site) meningitidis, Neisseria gonorrhoeae, Vibrio
● This type of testing is often used to detect cholera 0139, and Haemophilus influenzae
microbial antigens Protein A absorbs the fc portion of IgG except for IgG3.
Numerous kits are available for the rapid identification IgG3 - fails to attached to S.aureus Protein A
of antigens from such infectious agents as Group B
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illicit drugs such as cocaine or heroin are

examples of agglutination inhibition tests.

TYPES OF AGGLUTINATION REACTONS

HEMAGGLUTINATION INHIBITION
used RBCs
● Reactions use the same principle, except red
blood cells are the indicator particles
5. AGGLUTINATION INHIBITION
● Presence of patient antibody inhibits the
● Based on competition between particulate and
agglutination reaction
soluble antigens for limited antibodycombining
Agglutination inhibition reactions are based on
sites
competition between particulate and soluble antigens
● Lack of agglutination is an indicator of a positive
for limited antibody combining sites.
reaction
● Classic example is the human chorionic
Hemagglutination inhibition reactions use the same
gonadotropin (HCG)
principle, except RBCs are the indicator particles.
Typically, this type of reaction involves haptens
that are complexed to proteins; the hapten–protein
This type of testing has been used to detect antibodies
conjugate is then attached to a carrier particle.
to certain viruses, such as rubella, influenza, and
respiratory syncytial virus (RSV)

6. ANTIGLOBULIN-MEDIATED AGGLUTINATION
● Detects non-agglutinating antibody which is IgG
by means of coupling with a second antibody
- The patient sample is first reacted with a limited which is known → antibody to human globulin
amount of reagent antibody that is specific for or anti-human globulin
the hapten being tested.
- Indicator particles that contain the same hapten ● The key component of the test is antibody to
one wishes to measure in the patient are then human globulin that is made in animals or by
added. means of hybridoma techniques.
- If the patient sample has no free hapten, the IgG cannot bridge the gap of two antigen that's why
reagent antibody is able to combine with the cannot form a stable lattice formation that's why kahit
carrier particles and produce a visible na IgG nakabond sa kanyang antigen it cannot produce a
agglutination. In this case, however, visible agglutination → IgG needs a second antibody for
agglutination is a negative reaction, indicating an agglutination to occur and that antibody is known as
that the patient did not have sufficient hapten anti-human globulin
to inhibit the secondary reaction
- Either antigen or antibody can be attached to Two different types of Coombs test:
the particles.
- The sensitivity of the reaction is governed by ● Direct antiglobulin test
the avidity of the antibody itself. ● Indirect antiglobulin test
- It can be a sensitive assay capable of detecting
Coombs test - checks your blood for antibodies that
small quantities of antigen. Tests used to detect
attack red blood cells.

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A. DIRECT ANTIGLOBULINTEST - This use to validate a negative reaction kung

● Used to demonstrate in vivo (in the body) talagang negative ba o nag negative lang kasi na
attachment of antibody or complement to an neutralize yung AHG
individual’s red blood cells.
● This test serves as an indicator of autoimmune ● Group O RBCs sensitized with IgG
hemolytic anemia, hemolytic disease of the ● After addition of O check cells, agglutination
newborn, sensitization of red blood cells caused indicates:
by the presence of drugs, or a transfusion ■ AHG reagent was added
reaction ■ AHG reagent was not neutralized
● Lack of agglutination, invalidates results

Causes of False-Positive Reactions in Agglutination

Testing

B. INDIRECT ANTIGLOBULIN TEST

- cross-matching
● Detectin vitro sensitization
● Used to determine the presence of a particular
antibody in a patient, or it can be used to type
patient red blood cells for specific blood group
antigens
Compatibility test - between the donor the recipient

very important ang WASHING IN THIS TEST

Improper washing will produce false-negative reaction

because unbound antibody mga antibody na di naka
attach sa red cells can neutralized AHG

After that mag aadd na ng AHG kung present ang patient

antibody for this red cells magkakaroon ng agglutination
reaction ibig sabihin ng patient serum may antibody INTRODUCTION
that's why hindi pwede transfuse kasi sisirain lang din LABELED IMMUNOASSAY
niya yung red cells. ❖ Some antigen/ antibody reactions not
TYPES OF AHG REAGENT detected by precipitation or agglutination
a. Polyspecific AHG contains o However, they are relatively
● Anti IgG and anti C3d insensitive because those reaction
b. Monospecific AHG contains rely on a high enough concentration
● Contain anti-IgG or anti-C3d of the unknown to visualize the
O Check Cells reaction such as precipitation or
agglutination

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o In contrast, labelled immunoassay are ● hormones, drugs

designed for antigens or antibodies ● tumor markers
that may be small in size or present in ● specific immunoglobulins
very low concentrations which are ● and many other substances.
NOT detected by unlabeled
immunoassays such as precipitation ● Gumagamit tayo ng label to detect small
or agglutination because mainly those sample concentrations
ANALYTES do not have a high enough ● LABELLED can be an enzyme an → pag
concentration of the unknown to ENZYME ang gamit there in color change
visualize a precipitation or ● ANALYTES ARE BOUND TO MOLECULES
agglutination reaction that’s why THAT REACT SPECIFICALLY WITH THEM.
these labelled immunoassays are ENZYME - the reactions is color change
designed for those instances. RADIOISOTOPES - the antigen antibody combines
❖ Measured indirectly using labelled reactants there would be a radioactivity
such as → if the label use is a FLUOROCHROME there is binding
o enzymes there will be fluorescence
o radio isotopes
o and it can be fluorochromes Unlabeled immunoassays, → such as the precipitation
- to detect whether or not and agglutination reactions
specific binding has taken
place Labeled immunoassays - are designed for antigens and
❖ Referred to as receptor → ligand assays antibodies that may be small in size or present in very
Ligands → is the substance to be measured and is low concentrations.
defined as a molecule that binds to another molecule
of a complementary configuration. The presence of such antigens or antibodies is
determined indirectly by using a labeled reactant to
LABELED - usually binds to the substance that the test detect whether or not specific binding has taken place
is trying to detect so that is the receptor, the receptor
is what binds to the specific target molecules. Examples of ANALYTE can include:
● bacterial antigens
eg: labeled analyte or yung labeled mo is enzymes ● hormones
tapos naka attach yung enzymes sa antibody so dahil ● drugs
yun yung reagent/ligand yung idedetect ni labelled ● tumor markers
analyte which is antibody is the → ANTIGEN ● specific immunoglobulins and
● many other substances
If that immunoassay uses a labelled antibody the one .
that is being detected is patient antigen, if that
immunoassay using a labelled antigen the test is
detecting patient antibody ANALYTES - can be antibodies but it can also be antigen
● One reactant, either the antigen or the
LABELED IMMUNOASSAY antibody, is labelled with a marker so that the
❖ Labelled immunoassays are designed for amount of binding can be monitored.
antigens and antibodies that may be small in kung ano naka marker or naka labelled it will bind kung
size present in very low concentrations. ano ang kanyang dinedetect.
❖ The substance to be measured, often called
the typically is a protein. CONSTITUENTS OF LABELED ASSAYS
LIGANDS - the one that binds ● Fluorescent
LABELED - so that ma detect yung SMALL ● Radioactive
● Chemiluminescent
CONCENTRATION
● Enzyme labels.
Examples include:
The assays includes the use of:
● bacterial antigens
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● The concentration of the labeled analyte

Labeled (known) and is in excess, so all binding sites on the
non labeled (patient sample) ligands antibody will be occupied.

● Specific antibody ● After the separation, the amount of

● Standards and calibrators - this are also bound label is measured and used to
unlabelled analytes are made up in known determine the amount of patient antigen
concentrations of the substance to be present.
measured that it could be compared the result
of the action ● If patient antigen is present, some of the
● Means of separating bound from free binding sites will be filled with unlabelled
components analyte, thus decreasing the amount of
● Means of label detection. It could be bound label.
scintillating counters, it could be fluorescence
microscope and other detection equipments. Therefore, the amount of bound label is inversely
proportional to the concentration of the labeled
FORMATS OF LABELED ASSAYS antigen, which means that more labeled antigen that is
2 FORMATS detected, the less there is of patient antigen.
● Competitive Immunoassays - the both labeled
analytes and unlabeled analytes COMPETES Enzyme-labeled antigen competes with unlabeled
with the limited number of antibody binding patient antigen for a limited number of binding sites on
site that's why and the relationship is inversely antibody molecules that are attached to a solid phase.
proportional
● Non-competitive Immunoassays Enzyme activity is inversely proportional to the
concentration of the test substance.
COMPETITIVE IMMUNOASSAY

Competitive Immunoassays - labeled antigen

competes with unlabeled patient antigen for a limited
number of antibody which is also known which is (on
the sample) and the relationship is inversely
proportional because they are completing to the
LIMITED NUMBER of antibody which is also known.

labeled antigen - KNOWN/REAGENT

unlabeled patient antigen - from PATIENT’S SAMPLE
UNKNOWN - PATIENT ANTIGEN

● All reactants are mixed together

simultaneously;
● Labeled antigen competes with
unlabelled patient antigen for a limited
number of antibody-binding sites.
● The amount of bound label is inversely
proportional to the concentration of the
labeled antigen, which means that the
more labeled antigen is detected, the less
there is of patient antigen.

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❖ In this case, the amount of label measured is

directly proportional to the amount of the
patient antigen.
● This type of assay is more sensitive than
competitive immunoassay.
● In both types assays, the label must not
alter the reactivity of the molecule and
should remain stable for the reagent’s
1. Unknown concentration of analyte in patient shelf life.
sample (red dots) competes with labeled
analyte (yellow stars) for binding sites on
immobilized antibody. Sample A is a negative
control.
2. Wash to remove unbound materials.
3. After substrate is added, a colored product
(signal) is generated with an intensity
proportional to the amount of enzyme-labeled
analyte bound to the antibody.
4. The signal strength is usually inversely related
to the analyte concentration

NONCOMPETITIVE IMMUNOASSAY
The amount of labeled detect is DIRECTLY
PROPORTIONAL to the amount of the patient that is
being detected or patient antigen.
(hindi sila nag aagawan)

Label usually a marker for a specific reaction or

combination of a patient antigen and antibody.

Labeled usually attached to an antibody molecule CAPTURED ANTIBODY - these are antibodies that are
attached in the medium or solid phase antibodies
Antibody, is first passively absorbed to a solid phase
such as a MICROTITER PLATES, NITROCELLULOSE 1. For Sample B, immobilized reagent antibodies
MEMBRANES OR PLASTIC BEADS. capture analyte in patient sample (red dots)
while in the Ab capture technique, immobilized
● It is also used labeled antibody or reagent antigen captures patient antibodies.
captured technique. Sample A is a negative control.
2. Wash to remove unbound materials.
Unknown patient antigen is then allowed to react with 3. Add enzyme-labeled (detection) antibody that
and be captured by the SOLID PHASE antibody. binds to a different epitope on analyte for
Sample B (or binds Fc region on human Ig for
also called a CAPTURED ANTIBODY - it capture the Antibody capture).
specific antigen 4. Wash to remove unbound materials.
5. Add substrate and measure color change.
A second antibody with a label is added to the reaction. Color intensity is directly related to analyte
(Sample B) or human antibody (Antibody
● After washing to remove unbound capture).
antigen, a second antibody with a label is ● Noncompetitive immuassay also requires
added to the reaction. that antigen should be MULTIVALENT. So
that it would be able to attach to two

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antigen binding sites of antibody

molecule.
LABELED ANTIBODY - will serves as a DIRECT MARKER
of patient antigen that’s why the relationship is directly
proportional

The amount of color, fluorescence, or luminescence

detected is directly proportional to the amount of
antibody in the specimen.

SEPARATION TECHNIQUES
HETEROGENOUS ENZYME IMMUNOASSAYS Homogeneous immunoassay. Reagent antibody is in
o Require a step to physically solution. Patient antigen and enzyme-labeled antigen
separate free from bound are added to the test tube. Patient antigen and
analyte. (requires washing) enzyme-labeled antigen compete for a limited
● Antigen or antibody is attached by number of binding sites on the antibodies. When
physical adsorption; when specific patient antigen is present, the enzyme label on the
binding takes place, complexes remain reagent antigen is not blocked, so color development
attached to the solid phase medium. is observed. Sample A has a low concentration of
● This step provides a simple way to patient antigen, whereas Sample B contains more
separate bound and free reactants. patient antigen and has stronger color development.

HOMOGENOUS ENZYME IMMUNOASSAY

o Do not need separation step. RADIOIMMUNOASSAY
o Simpler to perform. ❖ The First type of immunoassay developed was
● The activity of the labeled attached to the radioimmunoassay (RIA), pioneered by
antigen is diminished when binding of YALOW and BERSON in the late 1950’s.
antibody and antigen occurs. ● It was used to determined the level of
● Typically, homogenous assays involve as insulin-anti-insulin complexes in diabetic
enzyme label, chosen so that the enzyme patients.
is inactivated when bound to an antibody. ● The technique proved to be valuable in
● The sample containing patient antigen is measuring a number of substances such
incubated with the labeled antigen and as hormones, serum proteins and
the antibody; the amount of activity then vitamins that either occur at very low
can be measured directly. levels that’s why we used labeled analytes
● Homogenous assay are less sensitive than to mark or to detect those very low
heterogenous assay. concentrations that cannot be detected
by agglutination and precipitation
reaction in blood plasma.

❖ The assay uses a radioactive substance as a

label.
❖ Radioactive elements have nuclei that decay
spontaneously, emitting matter and energy.
❖ Radioactivity is measured by a scintillation
counter.
❖ 131 Iodine; 125 iodine → is the most popular )
and tritiated hydrogen, or 3H have been used.

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● RIA is an extremely sensitive and precise ● Enzymes used as labels for immunoassay
technique for determining trace amounts are typically chosen according to the
of analytes that are small in size. number of substrate molecules converted
per molecule of enzyme, ease and speed
● However, the chief disadvantage is the of detection and stability
health hazard involved in working with
radioactive substances. ● Enzyme assay are classified as either
heterogenous or homogenous on the
● Disposal problems, short shelf life, and basis of whether a separation step is
the need for expensive equipment has necessary.
caused laboratorians to utilize other
techniques for identifying analytes in low
concentration. TYPICAL ENZYMES USED INCLUDE:
● Horseradish peroxidase (has the highest turn
RADIOIMMUNOASSAY over and sensitivity)
COMPETITIVE BINDING SITES ● Glucose oxidase
o The analyte being detected competes ● Glucose-6-phosphate dehydrogenase
with a radiolabeled analyte for a limited ● Alkaline phosphatase (has the highest turn
number of binding sites on a high-affinity over and sensitivity)
antibody. ● Beta- galactosidase
o Amount of label in the bound phase is
INVERSELY PROPORTIONAL to the HETEROGENOUS ENZYME IMMUNOASSAY
amount of patient antigen present. COMPETITIVE ENZYME LINKED IMMUNOABSORBENT
ASSAY (ELISA)
● If little or no antigen is present means ● Based on the principles of RIA.
there would be a strong positive reactions ● ENZYME- LABELED antigen competes
radioactivity. with unlabelled patient antigen for
limited number of binding sites on
NON-COMPETITIVE BINDING ASSAYS (IRMA) antibody molecules that are attached to a
o Amount of bound labeled antibody solid phase.
is DIRECTLY proportional to the ● Enzyme activity is inversely proportional
amount of patient serum. to the concentration of the test
substance.
ENZYME IMMUNOASSAY This method is typically used for measuring small
As labels for immunoassay, enzymes are cheap and antigens that are relatively pure, such as drugs and
readily available, have a long shelf life, are easily hormones.
adapted to automation and cause changes that can be
measured using inexpensive equipment. The higher color change is detected meaning the lesser
- WIDELY USE IMMUNOASSAY patient antigen is detected vice versa.
Enzyme labels can either be used qualitatively to
determine the presence of an antigen or antibody or NON-COMPETITIVE ENZYME LINKED
quantitatively to determine the actual concentration of IMMUNOABSORBENT ASSAY (ELISA)
an analyte in an unknown specimen.
● Often referred to as indirect (ELISA) because
● Enzymes are naturally occurring the enzyme-labeled reagent does not
molecules that catalyses certain participate in the initial antigen- antibody
biochemical reactions. binding reaction.
● Amount of enzyme label is DIRECTLY
● They react with suitable substrates to proportional to the amount of the test
produce breakdown products that may be substance.
chromogenic, fluorogenic or luminescent.

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Used a solid phase antigen ● The amount of color, fluorescence or

and then detect patient luminescence detected is directly
antibody. proportional the amount of antibody in
the specimen.
Afte
r a wash step,
an enzyme-
labeled
antiglobulin, or
secondary
antibody was CAPTURE ASSAYS (SANDWICH)
added. ● Antibody is bound to the solid phase
● Used with antigens HAVING MULTIPLE
EPITOPES
● After incubation, enzyme labelled antibody is
added
● The second antibody recognizes a different
epitope or binding site than the solid-phase
antibody and completes the “sandwich”.
● Enzyme activity directly related to
concentration of antigen.
● Detect antigens present in low concentration
and suited to antigens with multiple epitopes.

● If antibody, rather than antigen, is bound

to the solid phase, these assays are often
called sandwich immunoassays or
capture assays
● The enzymatic activity is directly
proportional to the amount of antigen it
the test sample.
● It also depends upon the particular
enzyme used either a colored or
● Antigen is typically bound to solid phase chemiluminescent reaction product is
then an unknown antibody is added and detected.
given time to react. ● Capture assays are best suited to antigens
● If antibody is present to the patient there that have multiple determinants, such as
would be a specific binding. antibodies, cytokines, proteins, tumor
● After a wash step, an enzyme-labeled markers and microorganism specially
antiglobulin, or secondary antibody was viruses.
added. ● In INDIRECT ELISA using solid-phase
● This second antibody reacts with any antigen instead of solid-phase antibody
patient antibody that is bound to solid and detecting patient antibody and use
phase, the second antibody will not be enzyme labelled anti-human globulin.
bound.
● If no patient antibody is bound to the
solid phase, the second labeled antibody
will not be bound.
● The amount of color, fluorescence, or
luminescence is measured using a
detection device and is compared with
the amount of product according to a
standard curve.
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● Design as a single use, disposable assays

in a plastic cartridge

● Membrane is usually nitrocellulose—Able

to easily immobilize proteins and nucleic
acids.

● Antigen or antibody can be coupled to the

membrane

● Reaction is read by looking for the

presence of a colored reaction product.

Design primarily for point of care testing, many of these

have been modified for increased sensitivity and can be
HOMOGENOUS ENZYME IMMUNOASSAY made semiquantitative for use in clinical laboratory.
● Less sensitive than heterogeneous assays,
but they are rapid, simple to perform, and The membrane is usually made of microporous nylon,
adapt easily to automation. which is able to easily immobilize proteins or nucleic
● Their chief use has been in the acid.
determination of low- molecular weight
analytes such as HORMONES, The rapid flow through the membrane and it’s large
THERAPEUTIC DRUGS, and DRUGS OF surface area enhance the speed and sensitivity of ELISA
ABUSE in both serum and urine. reactions.
● Homogenous assays are based on the
principle of change in enzyme activity as Some test devices require the separate addition of a
specific antigen-antibody combination patient sample, wash reagent, labelled antigen or
occurs. antibody and the substrate.
● Enzyme activity is directly proportion to
the concentration of patient antigen or It combines all the previously mentioned steps into
hapten present in the test solution. one.

Most frequent enzyme that are used for homogenous The analyte is applied at one end of the strip and
enzyme immunoassays include malate dehydrogenase migrates toward the distal end where there is an
and G-6-PD. absorbent pad to maintain a constant capillary flow
rate.
Enzyme immunoassay advantage in general have
achieved sensitivity similar to that RIA without creating The labelling and the detection zones are set between
a health hazard or causing disposal problems. the two ends.

Disadvantages include the fact that some specimen An antigen or antibody immobilized in the detection
may contain natural inhibitors, the size on enzyme zone captures the immune complex and forms a
label may be a limiting factor in the design of some colored line for a positive test, which may be in the
assays, nonspecific protein binding is another difficulty form of a plus sign.
encountered with the use of enzyme labels.
This type of test device has been used to identify
RAPID IMMUNOASSAYS microorganisms such as Streptococcus pyogenes, the
MEMBRANE-BASED CASSETTE ASSAYS cause of strep throat and has been used for pregnancy
testing as well as testing for cardiac troponin after a
● Rapid, easy to perform and give heart attack.
reproducible results

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Test results are most often qualitative rather than ❖ Involve two steps, the first of which is
quantitative. incubation of patient serum with a known
antigen attached to a solid phase.
FLOURESCENT IMMUNOASSAY ❖ The slide is washed, and then an antihuman
In 1941, Albert Coons demonstrated that antibodies immunoglobulin containing a fluorescent tag is
could be labelled with molecules that fluoresce. added
❖ This combines with the first antibody to form a
The fluorescent compunds, called flourophores or sandwich, which localizes the fluorescence.
flourochromes, can absorb energy from an incident ● Fluorescence means there is a presence
light source and convert that energy into light of a of patient antigen.
longer wavelength and lower energy as the excited ● In indirect immuno fluorescence tatlong
electrons return to the ground state. patong sila.

Two compounds used are phycobiliprotein, europium

(B-napthyl triflouroacetone), and Lucifer yellow VS

The presence of a specific antigen is determined by the

appearance of localized color against a dark
background.

● Flourophores are typically organic

molecules with a ring structure; each has
a characteristic optimum absorption
range.
● The time interval between absorption of
energy and emission of fluorescence is
very short and can be measured in
nanoseconds.
● Fluorescein absorbs maximally 490 to 495 FIGURE 11–5 Direct versus indirect immunofluorescent
nm and emits a green color at 520 nm. assays. (A) In direct fluorescent assay, the patient antigen is
● It has a high intensity, good fixed to a microscope slide and incubated directly with a
fluorescent-labeled antibody. The slide is washed to
phostostability and a high quantum yield.
remove unbound antibody. If specific antigen is present in
● While the tetramethylrhodamine absorbs the patient sample, fluorescence will be observed. (B) In
at 550 nm and emits red light at 585 nm. indirect fluorescence, well-characterized tissues or cells are
● These two has absorbance and emission fixed to slides. Specific antibody in patient serum (in red)
patterns differ, fluorescein and binds to the antigens on the slides. A wash step is
rhodamine can be used together. performed and a labeled anti-human immunoglobulin is
added. After a second wash step to remove any
DIRECT IMMUNOFLOURESCENT ASSAYS uncombined anti-immunoglobulin, fluorescence of the
● Antibody is conjugated with a fluorescent tag sample is determined. The amount of fluorescence is
● Added directly to unknown antigen that is fixed directly in proportion to the amount of patient antibody
present.
to a microscope slide
● Read using fluorescence microscope.
● It is also use to Treponema pallidum.
● Antigens are typically visualized as bright apple
green or orange-yellow objects against a dark
● Indirect assays results in increased
background.
staining because multiple molecules can
bind to each primary molecule, thus
● Antigens detected by the direct method
making this a more sensitive technique.
include Legionella pneumophila and
Chlamydia trachomatis.
FLUORESCENCE POLARIZATION IMMUNOASSAY
INDIRECT IMMUNOFLOURESCENT ASSAYS

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❖ Based on the change in polarization of If sample analyte concentration is low, the antibody
fluorescent light emitted from a labelled will bind most of the labelled analyte, restricting it’s
molecule when it is bound by antibody. rotation. When excited by polarized light, the emitted
❖ If a molecule is small and rotates quickly fluorescence will remain polarized (the light waves all
enough, the emitted light is unpolarised after oscillate with the same orientation).
it is excited by polarized light
❖ If the labelled molecule is bound to antibody, If the patient sample has a high concentration of
the molecule in unable to tumble as rapidly analyte, this unlabelled analyte will occupy most of the
and it emits an increased amount of polarized antibody- binding sites, leaving the labelled analyte
light. free to rotate. The light waves emitted by the rotating
labels will not be uniformly oriented (less polarization).
❖ The degree of fluorescence polarization is
inversely proportional to concentration of the
Less polarization means higher antigen or analyte
analyte.
concentration, and higher polarization means lesser
patient antigen concentration.

CHEMILUMINESCENT IMMUNOASSAY

● Emission of the light caused by chemical

reaction, typically an oxidation reaction,
producing a molecule that decays back to it’s
original ground state
● Molecules capable of chemiluminescence:
○ Luminal
○ Acridinium esters
○ Ruthenium derivatives
○ Nitrophenyl oxalates
● When these substances are oxidized (typically
using H2O2), intermediates are produced that
are of a higher energy state
● These intermediates spontaneously return to
their original state, giving off energy in the
form of light.
● Light emissions range from a rapid flash of
FIGURE 11–6 Fluorescence polarization immunoassay.
Reagent antibody and fluorescently labeled analyte are light to a more continuous glow that can
added to the patient sample. (A) If sample analyte last for hours.
concentration is low, the antibody will bind most of the ● For example, when acridinium esters
labeled analyte, restricting its rotation. When excited by oxidized by hydrogen peroxide under
polarized light, the emitted fluorescence will remain alkaline conditions, they emit a quick
polarized (the light waves all oscillate with the same burst or flash of light.
orientation). (B) If the patient sample has a high ● The light remains for a longer time with
concentration of analyte, this unlabeled analyte will occupy luminol. Different types of
most of the antibody-binding sites, leaving the labeled instrumentation are necessary for each
analyte free to rotate. The light waves emitted by the
kind of emission.
rotating labels will not be uniformly oriented (less
polarization).
IMMUNOLOGY AND SEROLOGY OF BACTERIAL &
VIRAL INFECTIONS
Fluorescence polarization immunoassay. Reagent
antibody and fluorescently labelled analyte are added IMMUNOLOGY AND SEROLOGY OF INFECTIOUS
to the patient sample. DISEASE
(Bacterial, Viral, Fungal and Parasitic)

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Serological and Molecular Detection of Bacterial ● Symbiotic bacteria that reside on and colonize
Infections these host surfaces
The bacterial populations that exist on the body are
INTRODUCTION not homogenous; they vary in composition and
Human Microbiome numbers, depending on the area of the body or the
part of the body that is invaded by those
● Collection of microorganisms that exists on
microorganisms
the body, such as bacteria, viruses, and
single-celled prokaryotic organisms (yeast and For a microorganism to survive, the organism needs
fungi) to colonize the host and acquire nutrients.
keeps us healthy in many ways and there commonly Importantly, it must not stimulate the host’s immune
known as NORMAL FLORA response (in the case of the indigenous microbiota) or
it must avoid or circumvent the immune responses.
They reside on our body. They get nutrients, vitamins
for our body but at the same time some of these Once established, it needs to be able to replicate and
human microbiomes also protect us against disease disseminate to a preferred site in the body for
causing bacteria. survival and eventually be transmitted to a new
susceptible host.
In digestion of our food they produce also certain
vitamins and they stimulate INNATE and ADAPTIVE Three types of symbiotic relationships can exist
IMMUNE SYSTEMS between humans and bacteria;
Infectious disease Commensalistic - No benefits, No harm
● When a microbe causes damage to host cells - In commensalistic relationships, there is no
or altered physiology that results in clinical apparent benefit or harm to either organism.
signs and 3 symptoms of disease, the phrase “ - An example of a commensalistic organism is
Staphylococcus epidermidis, which colonizes
There are also microbes that causes damages to host and inhabits the human skin
cells or altered physiology that can result in clinical
- Staphylococcus epidermidis do not harm the
signs and symptoms of disease and that is known as
host pero wala din naman binibigay na benefit
→ INFECTIOUS ORGANISM to the host, so neutral lang ang organism to
The establishment of an organism that leads to host the host
injury is referred to as an → “infection.” / infectious Neutral; No harm or benefit to either organism (EX.
disease Staphylococcus epidermidis and skin
Human–Microbe Relationships neutral ang benefit to the host
When an individual is born, a dynamic relationship
begins between the human host and the bacteria in
the environment.
Various bacteria establish themselves on the surfaces
of an individual, including the gastrointestinal tract,
creating a symbiotic relationship.

Symbiotic relationship - this is a living together of 2 Mutualistic - normal flora which helps us
unlike organism, in that case the bacteria or the
human microbiome and human body also known as - In a mutualistic relationship, both humans
Indigenous microbiota (known as normal flora) and the bacteria benefit.
- One example is the Lactobacillus species that
Indigenous microbiota colonizes the epithelial surfaces of the vaginal
canal.

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- The human host provides conditions Pathogenicity refers to the inherent capacity of an
(temperature, atmosphere, nutrients) that organism to cause disease. This is a qualitative trait of
allow the bacteria to grow and multiply; in the organism determined by its genetic makeup.
exchange, the bacteria produce lactic acid to
help innate immune response, which prevents Always symptomatic (APPARENT ILLNESS)
colonization of bacteria and yeast that may can produce clinically illness
cause disease
Although an organism may be pathogenic in nature, it
both organisms benefit with each other (Ex. may not always cause disease.
Lactobacillus and epithelial surfaces of vaginal canal at
intestinal surfaces) The outcome of the host–pathogen interaction is
determined by several factors, including the host’s
● Parasitic immunologic status. Some microorganisms may only
- the encounter with specific organism cause disease or infection in individuals who have
or viruses or bacteria are occasionally compromised immune systems because of factors
results in harm to the host such as chemotherapy, radiation therapy, or various
- Parasite/bacteria or other organism chronic diseases. These organisms are referred to as
could get nutrients to the host “opportunistic pathogens.”
provide condition to allow grow and
multiplication Virulence
- while the parasite or the organism ● A quantitative trait that refers to the extent of
causes harm to the host 5 damage, or pathology, caused by the
- No benefits, nahaharm pa niya yung organism
host
Severity of the reaction of the pathology produced
In this case, a parasitic relationship exists between the and measured in terms of FATALITY
other organisms and the host. Establishment of an
organism that leads to host injury is referred to as an degree of damage was determined by specific
infection. virulence factors and virulent factors may increase
an organisms pathogenicity as well by contributing
Viruses, bacteria, or any parasite gets benefits and to the organisms ability to establish itself in the host
causes harm 4 to the host invade or damage the host tissue and evade your
Infecting organism and host Interactions host immune response
Infectivity For example,
● An organism’s ability to establish an infection Yersinia pestis, the causative agent of bubonic and
Infectivity - describes the proportion of individuals pneumonic plague, is considered to be extremely
exposed to a pathogen through horizontal virulent and is likely to cause severe illness and death
transmission (i.e., person-to person spread) who will upon infection unless antibiotics are administered.
become infected. often change interchangeably
Another term that is used with similar meaning is Immunogenicity other term
contagious.
- ability to produce a specific immunity or the
For example, the measles virus is extremely infections has the ability to produce a specific
contagious and has a high degree of infectivity. immunity
has high proportion to establish an infection For example,
Pathogenicity Measles virus is able to produce a lifelong immunity
● Inherent capacity of an organism to cause to the host so once you get infected you will produce
disease antibodies or specific immunity against organism and
if that organism is able to produce specific immunity,

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meaning it is IMMUNOGENIC or it has a high This results in inflammation, increased heart rate,
immunogenicity increased body temperature (fever), and a decrease
in blood pressure.

Bacterial Virulence Factors

Bacterial properties or features that determine Unlike endotoxin, which has multiple effects on the
whether an organism is pathogenic and able to cause body, exotoxins have a very specific and targeted
disease are referred to as “virulence factors.” activity.
Several factors that increase a bacterium’s virulence Endotoxin has multiple effects on body and have a
may be classified as either structural components very specific and targeted activity so they are protein
(e.g., endotoxin is a component of the cell walls of molecules that are released from a living bacteria and
certain bacteria that protects them from our immune are considered to be the most potent molecules
system) or as extracellular substances produced by known to harm the to living organisms because they
the bacteria, such as exotoxins. can act as “superantigens” are NOT processed by
Genetic determinants located on the bacterial antigen presenting cells (APCs).
chromosome are generally responsible for the
a “superantigen” induces activation of up to 20% of T
production of structural or surface molecules, which
help the organism to attach to and colonize the host cells, resulting in a massive release of cytokines.

MECHANISM OF EVASION The systemic events brought on by the release of

large amounts of cytokines leads to what is referred
Evasion - ability of a certain organism to colonize to as “toxic shock syndrome.”
inside a host
Examples include the TSST-1 toxin produced by
● Structural components
Staphylococcus aureus and the superantigens
● Genetic determinants
produced by S pyogenes, the cause of streptococcal
● Extracellular Virulence Factors
● Endotoxin and Exotoxins toxic shock syndrome (STSS).
● Extracellular Virulence Factors Immune Defense Mechanisms
Extracellular substances produced by bacteria also - How the body responds to certain microorganisms
contribute to an organism’s virulence by breaking Both innate and adaptive responses may occur after
down primary or secondary defenses of the body, an encounter with foreign antigens
damaging the host tissue and cells, or facilitating the
growth and spread of the organism. Against potential pathogens the first line of defense is
natural immunity or structural barriers which
Substances that perform the latter function are
includes the Intact skin and mucosal surfaces
called invasins. Several of the invasins include
hyaluronidase, collagenase, phospholipases, ● Intact skin and mucosal surfaces
lecithinases, coagulase, and various kinases.
Antimicrobial defense peptides
○ Lysozyme, alpha defensin
● Endotoxin and Exotoxins
- produce by certain bacteria Epithelial surface may have enzymes and nonspecific
antimicrobial defense peptides (ADPs) and proteins
Bacteria may also produce two types of toxins— that have antimicrobial activity.
endotoxin and exotoxins.
One example of an enzyme with specific antimicrobial
Endotoxin (lipid A) - is found in the LPS layer of the activity is lysozyme, which is found in many
cell walls of all gram-negative bacteria. secretions, including tears and saliva.
It helps to facilitate the invasion and induces a variety Lysozyme destroys the peptidoglycan found in the
of responses cell wall of bacteria, especially gram-positive bacteria.

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One group of soluble peptides is the defensin Cell-mediated immunity (CMI) - the other branch of
peptides. the adaptive immune response, is helpful in attacking
intracellular bacteria, such as Mycobacterium
Defensins - are produced constitutively by the cells in tuberculosis, Legionella pneumophila, Listeria
the body. monocytogenes, and Rickettsia species.
The three main classes of defensins are alpha, beta, ● Acute phase reactants
and theta. ● Production of antibodies directed against
Alpha defensins - are produced by neutrophils, bacterial antigens
certain macrophage populations, and Paneth cells of ● Cell-mediated immunity (CMI)
the small intestine. This class of defensins is believed Mechanisms of Evasion
to disrupt the microbial membrane. Ways of certain organism to inhibit our immune
Beta defensins - are produced by neutrophils as well system and to make it more difficult for our immune
as epithelial cells lining the various organs, including response to occur to survive
the bronchial tree and genitourinary system. They involves how the bacteria avoid the immune
are believed to increase resistance of epithelial cells response and how they successfully colonize and
to colonization. invade+
Theta defensins - are NOT found in humans. grow, colonize and produce in a host.
Antimicrobial proteins Evade antibodies - – antigenic variation → coat
Many antimicrobial proteins contribute to the innate themselves with host’s protein or fibronectin for a
immune response. treponema pallidum to hide their antigenic
determinants
For example,
S. pyogenes uses hyaluronic acid capsule to protect
Complement proteins - can promote chemotaxis. themselves from detection of our immune response
Interleukins - are involved in the regulation of bacteria can also invade the production of antibodies
immune responses and inflammatory reactions. because our body would not recognize them as
Prostaglandins are involved in the dilation and foreign and does no specific immune response that
constriction of blood vessels and modulation of could be elicited such as proteases to degrade some
inflammation. antibodies specifically IGA antibodies

Leukotrienes are involved in inflammation and fever. Block phagocytosis can mount a defense at several
stages of a phagocytic process, can block the steps of
Acute phase reactants also play important roles. phagocyte
against certain microorganisms
PROCESS PHAGOCYTIC
For example,
before the attachment of antigen to the phagocytic
C-reactive protein (CRP) - activates the complement cell
system and promotes phagocytosis by macrophages.
diapedesis
increasing during bacterial infection
chemotaxis
acts as opsonin in macrophages and haptoglobins
which deprives the bacteria of iron and ceruloplasmin adhesion or attachment
which are also bactericidal which has bactericidal engulfment
activity
digestion
Adaptive immune responses - include the production
of antibodies directed against bacterial antigens or excretion
extracellular products produced by bacteria such as Example:
exotoxin.

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N. gonorrhea - inhibits the release of chemotactic 3. Treponema pallidum subspecies endemicum

factors and preventing chemotaxis, it would now
prevent the phagocytic cells toward the area of ● causes: Non venereal endemic syphilis - found
infection in desert regions

Presence of polysaccharide capsule which are found in 4. Treponema carateum

N.meningitidis, estromonie, uracinopestis and H. ● Agent of pinta - found in central and south
influenzae with the binding of neutrophils and other america
macrophages needed to initiate phagocytosis
5. Treponema cuniculi
digestion can be inhibited by bacteria by blocking the
diffusion of lysosomal granules or these digestive ● Rabbit syphilis - which is zoonotic infection
enzyme that helps the digestion of antigen
Salmonella, M.Tuberculosis and M.labrae Treponema spirochetes
important mediators - phagocytic cell
➔ long, slender and they are helically coiled
Inactivate the complement cascade - c3b acts as bacteria containing Hacial filaments or
opsonin is inactivated periplasmic flagella and they have corkscrew
motility
enhancing the phagocytosis ➔ gram negative, microaerophilic and they
blocking the action of the complement that produce exhibit characteristics corkscrew
capsule and do not bind to the complement ➔ have similarities including localized skin
component infection
➔ disease that progresses to the heart and a
Protein H produced by S. pyogenes binds to disease cause a latent stage of cardiac and
complement c1 complement protein and will not
neurological involvement and many others
allow complement cascade to proceed further
complement is a cascade
strep. agalactiae and other group B strep. that are rich
in sialic acid which cause the degradation of c3b in
making the organisms resistant as well to complement
phagocytosis
Immunologic Response to Several Important Bacteria
SYPHILIS
● Genus Treponema contains four principal
species of pathogenic organisms:
○ causative agent of syphilis
○ also known as French disease,
spanish disease, great box or evil box
1. Treponema pallidum subspecies pallidum
● causative agent: human syphilis
● member or spirochetes or spirochetal
infections
● three in this group are more pathological and SYPHILIS
antigenically similar to our T. pallidum species Mode of transmission:

2. Treponema pallidum subspecies pertenue ● Sexual transmission - primary mode of

transmission
● yaws found in tropic ● Parenteral exposure through contaminated
● needles and blood
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● Congenital infections during pregnancy didn’t know she has chancre or ealy sign of
● Blood tranfusion syphilis
● in this stage, serological test are non reactive
The first diagnostic blood test for syphilis was the or negative that’s why dark field microscopy
Wassermann test (1906) - based on a complement showing positive coiled organisms with
fixation test corkscrew motility are the method of choice
● classic procedure and have been of detection of syphilis
subsequently replaced by variety of method ● hanapin ang coiled organisms
to detect triponema ● serolo= negative
● treatment: penicillin continues to be or
remains to be the drug of choice for the
treatment of syphilis
● and the first treatment are heavy metals with
such as arsenic and because of harmful effects
and toxic effects of arsenic, it was then
replaced by discovery of penicillin in the 1940s
In the treatment of syphilis, heavy metals, such as
arsenic, were replaced by penicillin in the 1940s.
STAGES OF SYPHILIS
Untreated syphilis can undergo for primary,
secondary and tertiary
B. SECONDARY SYPHILIS
The incubation period of syphilis can last about 3
It is usually observed 1 to 2 months after the
weeks but can range from 10 - 90 days that depends
disappearance of primary chancre
on immunity and immune response of the host and
the person is highly contagious during primary stage ● usually observed 1 to 2 months after the
of syphilis disappearance of chancre
● Systemic dissemination of the organism
at the end of incubation, it usually develops primary
occurs.
syphilis between 10 - 90 days after inoculation with
○ usually you have fever and rashes and
the average of 21 days of incubation, patient develops
that is because of immune response to
a chance that the primary sign of early syphilis
our body
○ 15% of reported cases - primary
lesions may be present
A. PRIMARY SYPHILIS (Early syphilis) ○ highly contagious patient
● Patient develops a characteristic, primary ○ SYMPTOMS: lymphadenopathy which
inflammatory lesion called a chancre at the is enlargement of lymph nodes, malay
point of initial inoculation and multiplication fever sore throat, rashes in the skin
of the spirochetes. because of systemic dissemination and
● Serological tests usually give non reactive rashes may all over the body and may
result (negative) at the time be the open source on the mucus
● chancre - usually painless solitary relation membrane that contains pus which
(one only) characterized by raised well called condylomata lata
defined borders and these lesions during early ○ palms and soles of the feet
syphilis heal spontaneously with or without ○ involvement of central nervous system
treatment and can persist for one to six weeks and now earlier that previously
and will heal completely after suspected during the secondary or
● in men, chancre usually occur at the outside primary syphilis and thereby CSF is
of the penis needed to confirm the presence of
● in women, it may appear in the vagina or on triponims on our CNS and persons that
the cervix and thus may go undetected so she has CNS invasion may exhibit

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neurological signs such as visual bones, skin, on mucus or subcutaneous

disturbances, hearing loss and facial tissues and spontaneously heal without
weaknesses scarring or they mar remain destructive areas
○ lesions usually persist from few days to of chronic inflammation
up to eight weeks spontaneously heal ○ likely or usually represents the hosts
like chancre in primary stages a response to infection of fever and
○ DIAGNOSTICS: rashes while also in the tertiary or
■ serological test now may chronic stage or syphilis there would
become positive because it also be a complications in the
has systemic dissemination ➔ cardiovascular system causing aortic
and positive through dark field aneurysm angina pectoris
microscopy from the fluid ➔ Neurosyphilis most often associated with
from rashes tertiary stage
● Patients may develop Condylomata lata (flat ◆ invasions in CNS but it can also occur
wart-like lesions) anytime after primary stage
Diagnosis: ◆ Immunodeficient individuals or
patients with HIV are usually
● Darkfield microscopy of the fluid of the rash susceptible to early neurosyphilis
● Serological tests because of their lack of immunity or
immune response
◆ Specimen of choice is CSF and the test
is usually preferred
◆ → VDRL using CSF (specimen of
choice)
CONGENITAL SYPHILIS AND NEUROSYPHILIS
A. CONGENITAL SYPHILIS
● T.pallidum crosses the placenta from the 18th
of gestation
● Causes abortion or stillbirths; (live born)
C. LATENT SYPHILIS
disfigurement, blindness, deafness and other
● Characterized by lack of clinical symptoms complications
● Patients are non-infectious at this time, ● fetus is may affected during second and third
except pregnant women who can pass the trimester
infection to the fetus ● those infants have live born have no clinical
● Diagnosis can be made only by serologic signs of the disease during the first few weeks
methods of life and they remain asymptomatic but on
○ dark field is not recommended the later if the symptoms develop and not
● follows the disappearance of syphilis treated at birth will exhibit now and some of
● those symptoms are hemorrhagic rhinitis,
skin eruptions, popular rash and other
D. TERTIARY OR LATE SYPHILIS symptoms such as lymphadenopathy,
● Characterized by the appearance of lesions hepatosplenomegaly, jaundice, anemia, and
called (three stages) Gummas, CVD and other abnormalities
Neurosyphilis ● early stage of congenital syphilis is seen
● Congenital syphilis occurs younger than two years who are untreated
● Diagnosis is done by reactive serological test and late stage is seen in children older than
and a reactive spinal fluid test (Neurosyphilis) two years old who are untreated
● Often occurs between 10 - 30 years following ● symptoms of these untreated late stage
the secondary stage ●
● Gummas - localized areas of granulomatous
inflammation that are most often found on
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Hutchinsonian Triad: triad of notched teeth, keratitis a. Wasserman antigen

and deafness
● Made up of cardiolipin which is isolated from
B. NEUROSYPHILIS cardiac muscle
● A hapten
● Syphilis of the central nervous system ● cardiolipin used to detect the reagin antibody
● may occur in up to 60% of infants with because reagin reacts with cardiolipin
congenital diseases
b. Treponemal antigen
● Reiter treponeme
○ non-virulent, cultivable variant of
T.pallidum
○ Commonly used in CF tests and TPI
○ used to remove a cross-reacting
antigens against treponema
● Nichol’s treponeme
○ Virulent strain of T.pallidum
LABORATORY DIAGNOSIS
○ used in fluorescent treponemal
Diagnostic markers for Syphilis: ANTIBODIES
antibody absorption (FTA-ABS)
a. Reagin antibodies or non-treponemal antibodies
○ used for confirmatory test for syphilis
● Antibody-like substances which in theory are which is based based on the principle
the results of interaction of the Treponema of FTA-ABS
with host tissues.
LABORATORY DIAGNOSIS
● Non-specific antibodies of Treponema because
Direct Detection
they are directed against the protein antigen
DIRECT DETECTION:
group common to pathogenic spirochetes
● IgG and IgA types Direct observation of Organism
● not specific
● they are not directed or not produced in ● primary syphilis and secondary can be
response to treponema but they are directed diagnosed by demonstrating the bacteria in
against a protein antigen that is formed due to exudates from skin lesions
the interaction of treponema with host issue I. Darkfield Microscopy
● reagin is produced with SLE, RNA in pregnant
women ● Darkfield condenser is used to keep all
○ false positive production are prone in incidental light out of the field except for that
the production of region antibody captured by the organisms themselves
that’s why test detection region ○ mas makta at mas mademonstrates
antibody are just screening for ang organisms
diagnosis of syphilis ● Motility is a key to identification
○ since you need to confirm it the cause ● A negative test does not exclude a diagnosis of
of the formation of region antibodies is syphilis.
because of interaction of treponema ● should have a good specimen in the form of
serous fluid from the lesions and usually the
b. Anti-treponemal antibodies (ATA) (confirmatory) serous fluid obtained by cleaning the lesion of
● Specific antibodies developed in response to chancre with sterile saline and then rubbing it
T.pallidum infection. with clean gauze and then using dark field
● IgM in primary syphilis - which can be seen microscopy pathogenic treponemes are
during primary syphilis identified on the basis of the morphology and
● IgG type during secondary syphilis the corkscrew of motility
● motility - key identification for treponema, the
LABORATORY DIAGNOSIS specimen must be examined quickly before
Syphilis Antigen they dry out so can identify or we can use
Syphilis: ANTIGEN
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motility as basis in characterizing or identifying A. Non-Treponemal Test: VDRL

treponemas Venereal Disease Research Laboratories
● false negative - when there is delay in
evaluating the slide and also an insufficient Principle: flocculation
specimen or specimens treated with antibiotic Reagents:
because corkscrew or the morphology of
bacteria could be affected Cardiolipin (0.03%)

II. Fluorescent Antibody Testing ○ Main reacting component

○ Alcoholic extract of normal beef heart
● A sensitive and highly specific alternative to extract
darkfield microscopy.
● Live specimens are not required Lecithin (0.21%)
● Monoclonal antibodies can still cross your out ○ Helps neutralize the anti-
without the subspecies of the pallidum and complementary properties of
must be taken into account cardiolipin
LABORATORY DIAGNOSIS Cholesterol (0.9%)
Serological tests
A. Non Treponemal tests: VDRL & RPR (consist of clc ○ Provides adsorption centers
○ Increases the reacting surface of
● Detect REAGIN Antibodies cardiolipin
○ produced because of interaction of ○ Increases the complement-fixing
treponema capacity of cardiolipin with reagin
● Not specific for syphilis (not confirmatory) ● 1% benzoic acid to stabilize the antigen at 6-
● Inexpensive to carry out 10C
● Reagent: CLC (cardiolipin lecithin and
cholesterol)
○ used in the reaction to detect the non
treponemal
● Disadvantage: reagin disappears soon after Procedure of VDRL
syphilis is cured a. Qualitative Serum VDRL
● VDRL - venereal disease research laboratory
test ● 50uL or 0.05mL serum
● RPR - rapid plasma reagin tests ● 56C for 30 mins to inactivate complement
○ both test are based on flocculation ● One drop (1/60 mL) of VDRL antigen is added
○ principle: flocculation reactions which to the ring
based on the patients complexes with ● Rotated for 4 minutes at 180rpm
the cardiolipin antigen and ● Determine presence of flocculation
flocculation is the specific type of ○ Non-Reactive (NR)
precipitation that occurs over a narrow ■ No flocculation or no
range of concentrations clumping
■ flocculation - formation of fine ○ Weakly Reactive (WR)
particles that clump together ■ Slight flocculation (small
in positive reactions clumps)
○ Reactive
False-positive reactions: ■ Definite flocculation (medium
and large clumps)
Transient: Hepatitis, IM, Varicella, herpes, measles,
malaria, TB and pregnancy b. Quantitative Serum VDRL
Sustained: SLE, Leprosy, IV drug use, Autoimmune ● Needle used shall deliver 75 drops of antigen
arthritis suspension per 1 mL
● 100 drops of saline per 1 mL
Serological Tests

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● The highest serum dilution that gives reactive

result is reported as the endpoint titer
● Often used as a therapeutic index in the
treatment of syphilis
● Diagnostic for Syphilis if reactive in dilution
1:16 or higher
c. CSF VDRL
● Boerner agglutination slide which has concave
wells 16mm in diameter and 1.75mm deep is
used
B. Treponemal tests
● The serum is not heated before testing
● Specific treponemal antigens
● The antigen must be diluted before use with
● Used to detect Treponemal antibodies
10% saline solution
developed in response to Treponemal
● Antigen delivery: 21 or 22g (1/100 mL)
infections
ROTATION: ● Highly specific and sensitive
● Used as verification procedures
VDRL serum: 180 rpm for 4 mins
a. Agglutination tests:
VDRL CSF: 180 rpm for 8 mins
● Treponema pallidum Agglutination tests (TPA)
Serological Tests ● T.pallidum Hemagglutination (TPHA)
A. Non-Treponemal Test: RPR ● Microhemagglutination- Treponema pallidum
Rapid Plasma Reagin (RPR) (MHA-TP)
● Hemagglutination Treponemal Test for
● Modified VDRL test Syphilis (HATTS)
● More sensitive but less specific than VDRL
● Any positive result must be followed by a
VDRL or other more specific procedures
Antigen is similar to VDRL with the addition of the
following:
b. Immunofluorescence
a. Charcoal
● Fluorescent Treponemal Antibody Absorption
● Makes test easier to read (FTA-ABS)
b. EDTA c. Complement fixation tests
● Prevents oxidation of lipids d. Immobilization tests (TPI)
c. Thimerosal B. Treponemal tests
● Preservative Fluorescent Treponemal Antibody A
● Indirect fluorescent antibody test
d. Choline Chloride ● Test of choice to run with with darkfield
microscopy
● Inactivate the complement
● Earliest serological test to be positive
Serological Tests ● Patient serum is heat inactivated and made
with a sorbent consisting of non-pathogenic
Treponemes (Reiter strain)
A. Non-Treponemal Test: ● Nichol’s strain of T.pallidum have been fixed
RPR to slides used for the test

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Fluorescent Treponemal Antibody Absorption Test

(FTA-ABS) FIGURE 21–5 TP-PA test results. Row A: positive control.
Row B: negative control. Row C: serum from a positive
patient. T pallidumsensitized gel particles were placed in
column 3 of each row and unsensitized gel particles were
pipetted into column 4 of each row. Positive wells (A3 and
C3) are indicated by a diffuse mat of particles that spread
over the surface of the well, whereas negative results are
indicated by a compact button. (Linda Miller.)

C. True Treponemal tests

Immobilization tests (TPI)
● Considered as standard or reference to which
all other treponemal tests are evaluated
● Involve mixing of patient serum with live,
actively motile T.pallidum extracted from
testicular chancre of a rabbit and complement
● Test is considered positive if >50%

B. True Treponemal tests

Particle Agglutination (PA) tests ● Treponemes are immobilized
● Originally used sheep RBCs coated with T ○ Positive: If 50% or more Treponemes
pallidum antigen are immobilized
● Serodia T pallidum particle agglutination (TP- ○ Negative: If fewer that 20% are
PA) test immobilized
○ “Doubtful result”: range of 20-50%
Testing Algorithms
● The traditional testing algorithm for syphilis
involves screening the sample with a
nontreponemal test and confirming any
positive results with a more specific
treponemal test

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Diagnostic Tests for Streptococcal Infection

CULTURE
● Appear as small translucent colonies
surrounded by a clear zone of β hemolysis
● Identification is made on the basis of
susceptibility to bacitracin, testing for L-
pyrrolidonyl-β-naphthylamide (PYR) activity,
or through Lancefield typing

GROUP A STREPTOCOCCAL INFECTION

● Caused by Streptococcus pyognes
● major sites of infection are the upper
respiratory tract and skin with pharyngitis and
empetigo being the most common
● May lead to rheumatic fever and post-
streptococcal glomerulonephritis

FIGURE 20–10 Throat culture plate showing a positive result

for beta hemolytic Group A streptococci (S pyogenes).
Bacterial colonies not producing beta hemolysis represent
indigenous microbiota of the oropharynx. (James Vossler.)

Detection of Group A Streptococcal Antigens

VIRULENCE FACTORS A. Streptolysin O antigen
● The M protein is the major virulence factor
● Immunity to Group A streptococci (GAS) ● Oxygen-labile hemolysin active against human
appears to be associated with antibodies to and rabbit RBC
the M protein ● Its reduced form, capable of lyzing human or
● Additional virulence factors include various rabbit rbc
exotoxins that may be produced during the ● It unites with an antibody (ASO) and thereby
course of a infection loses its lytic activity
● Additional extracellular substances include • DNAse B
the enzymes streptolysin O,
deoxyribonuclease B (DNase B), • Hyaluronidase
hyaluronidase, nicotinamide adenine
• Streptokinase
dinucleotide (NAD), and streptokinase

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● Lateral flow immunochromatographic assays ○ 0 = total loss of color [negative

(LFA) are increasingly being used for the result]
detection of bacterial, viral, fungal, and
parasitic antigens in clinical samples 3. Streptozyme testing

The most diagnostically important antibodies are the ● Slide agglutination screening test for
following: Anti-streptolysin O (ASO), anti-DNase B, detection of antibodies to several
streptococcal antigens
anti-NADase, and anti-hyaluronidase (AHase).
● Sheep RBC’s are coated with streptolysin,
1. Anti-Streptolysin O titer streptokinase, hyaluronidase, DNAse and
NADase so that antibodies to any of the
● Based on neutralization of the hemolytic streptococcal antigens can be detected
activity of Streptolysin O
● Normal: Titer of 166 Todd units or below
● Moderately elevated: if the titer is atleast 240
Todd units in an adult and 320 in child
● ASO increases within 1-2 weeks after infection
and peak between 3 to 6 weeks following
initial symptoms
Anti-Streptolysin titer
Traditional Method
● Involves dilution of the unknown serum to
which a measured amount of Streptolysin O
antigen is added
● 5% suspension rabbit or human RBC are
added as indicator
● The tube containing the least amount of
serum (one with highest dilution) which
demonstrate no hemolysis is the ASO TITER
[use of 14 tubes → tube 13 does not contain
ASO control]
● Test tube 13: RBC Control no hemolysis
→ no hemolysis
● Test tube 14: ASO Control complete
hemolysis → complete hemolysis
Detection of Streptococcal Antibodies
2. Anti-Dnase B Testing
● Sometimes appear earlier than ASO in
streptococcal pharyngitis
● Measurement is based on neutralization
● If anti-DNAse B antibodies are present they
will neutralize the reagent DNAse B
● Presence of DNAse is measured by its effect
on DNA-methyl green conjugate.
● The conjugate is in its intact form, but when
hydrolyzed by DNAse, the methyl green is
reduced and becomes colorless
● Tubes are graded for color:
○ 4+ = intensity of color is unchanged
[positive result]
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Serology and Molecular Detection of Viral Infections

Immune Defenses Against Viral Infections

● Viruses are obligate intracellular pathogens
that rely on the host cell for their replication
and survival
● Innate immunity provides the first line of
protection against viral pathogens
● Type I interferons and natural killer (NK) cells
● Virus-specific antibodies are produced by B
cells and plasma cells
● Cell mediated Immunity

Viral Escape Mechanisms

● Frequent genetic mutations and Antigenic
variation
● Escape the action of components of the
innate immune system
● Suppresses the adaptive immune system

Hepatitis Type/ Family Transmission

Viruses

Hepatitis A Picornavirus Fecal-Oral

(HAV)

Hepatits B Hepadnavirus Parenteral

(HBV) (needle),
sexual,
perinatal
(infected
mother to
infant)

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● High vertical transmission

Hepatitis C Flavivirus Parentera
(HCV) 3. Hepatitis B Core Antigen (HBcAg)

Hepatitis D Parentera ● Not detectable in serum because of the viral

(Delta Virus) (or similar to envelope that masks it
Hepatitis B) ● Detected only through biopsy of the infected
liver
Hepatitis E Calicivirus Fecal-oral ● Not considered as a serologic marker
(HEV)
HBV antibodies:
1. IgM Anti-HBc
Hepatitis A
● Known as infectious hepatitis ● 1st antibody to be produced
● Short incubation hepatitis (ave of 28 days) ● Indicator of current or recent infection
● Useful in detecting infection during the “core
HAV Antigens: window” period
1. HAV antigens 2. IgG Anti-HBc
● Shed in feces of infected individual ● Life-long marker of HBV infection
HAV Antibodies: 3. Anti-Hbe
1. IgM Anti- HAV ● Indicates that the patient is recovering from
● Marker of acute hepatitis A HBV infection
● Peak during 1st month of illness and decline ● Marker of convalescence and favorable
within 6-12 months prognosis
● Routinely detected by solid-phase antibody 4. Anti-HBs
capture ELISA
● Appears during the recovery period of acute
2. IgG Anti-HAV hepatitis B, weeks to months after HBsAg
● Produced as a result of natural infection or produced
immunization ● Persists for years and provide protective
● Indicate immunity to HAV immunity
● Detected by competitive inhibition ELISA Tests ● Also produced after immunization with
hepatitis B vaccine
Hepatitis B
● Serum Hepatitis Serological markers in acute hepatitis
● Dane particle: complete HBV that causes
infection
HBV antigens:
1. Hepatitis B surface antigen (HBsAg)
● First marker to appear
● Indicator of active infection or chronic
infection
● Important marker in screening blood donors
2. Hepatitis B Envelope Antigen (HBeAg) FIGURE 23–4 Typical serological markers in acute hepatitis
B. Solid lines represent viral antigen concentrations,
● Present during periods of active replication of whereas dashed lines indicate antibody concentrations.
the virus Each antigen shares the same color with its associated
● Indicates a high degree of infectivity when antibody.
present
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● Super-infection: When an HBV carrier is

exposed to infected blood from co-infected
patients then the exposure results in super-
infection of the existing HBV infection with
delta virus; this may result in development of
acute hepatitis (due to delta virus) in an HBV
chronic carrier.
○ Positive for IgG anti-HBc

FIGURE 23–5 Typical serological markers in chronic hepatitis

B.

Hepatitis E
● Usually presents as an acute, self-limiting
hepatitis without progression to a chronic
Hepatitis C carrier state
● Previously classified as “non A –non B” ● Associated with a high rate of mortality in
hepatitis pregnant women
● Major cause of post-transfusion hepatitis
● Hepatitis C is transmitted mainly by exposure “water-borne hepatitis”
to contaminated blood, with intravenous drug HEV Antibodies
use being the main source of infection
● HCV has an average incubation period of 7 ● IgM anti-HEV is typically present during the
weeks acute infection but rapidly declines in the
● Majority of infections are asymptomatic early recovery period
● ELISA, Western blot and fluorescent antibody
Hepatitis D blocking assay
● Unclassified, single-stranded RNA virus
● Incomplete virus HEV RNA
● Parenterally transmitted infection that can
● Detected in feces of most patients for about 2
only occur in the presence of hepatitis B
weeks after the onset of illness, but may
● Infection with two virus occur either:
persist longer in some cases
○ Simultaneously as → Coinfection
● Identified by means of PCR
○ Sequentially → Superinfection in
chronic HBV carriers HUMAN IMMUNODEFICIENCY VIRUS
● Co-infection: Where a person who is ● Etiologic agent of the Acquired
susceptible to HBV is exposed to someone Immunodeficiency Syndrome (AIDS)
who is co-infected with HBV and delta virus,
this results in acute co-infection with both the HIV-1
viruses at the same time. ● Formerly called human T cell lmphotropic
○ positive for HDV antibodies and IgM virus-type III (HTLVIII), Lymphadenopathy-
anti-HBc
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 IMMUNOLOGY AND SEROLOGY
LECTURE/FINALS
1st SEM, 2022

associated Virus (LAV), and AIDSassociated ● It is based on the recognition of the major HIV
retrovirus (ARV) proteins (p24, gp41, gp120/160) by
● Causes AIDS in US, Europe fractionating them according to their weight
by electrophoresis and then visualizing their
HIV-2 binding with specific antibodies over
● Endemic in West Africa nitrocellulose sheets.
● Less pathogenic and has a lower rate of
transmission
● A positive result: presence of any two of the
Main Structural Genes: following bands—p24, gp41, and gp120/160
1. Env (envelope)
● gp120, gp41 (markers; proteins involve in ● If the test is positive for bands gp41 and/or
attachment of cd4 to HIV) p24 in conjunction with a positive EIA test
● Attachment and fusion to CD4 cells result, it is regarded as a confirmatory test.
2. Gag (group antigen) gene
● p55, p15, p17, p24 ● A negative result demonstrates the absence
of bands

3. Pol (polymerase)
a. Reverse transcriptase
b. Integrase
c. RNAse
d. Protease
Laboratory testing for HIV Infection
A. SCREENING
1. ELISA – sandwich ELISA principle (positive result →
bound for confirmatory tests)
• Agglutination Tests
• Dot-Blot Testing
2. Agglutination Tests
- Western Blot Testing (Standard confirmatory
test for number years but replaced by humor Heterophile Antibodies associated with Infectious
methods) Mononucleosis (IM)
3. Dot-Blot Testing ● Heterophile antibodies are antibodies capable
of reacting with similar antigens from two or
B. CONFIRMATORY more unrelated species
1. Western Blot Testing Infectious Mononucleosis
WESTERN BLOT ● Caused by Epstein Barr virus
● Target: B cells (CD21)
● Atypical lymphocyte: T cells reacting to B cells
infected with EBV
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 IMMUNOLOGY AND SEROLOGY
LECTURE/FINALS
1st SEM, 2022

Disease Monitoring ERYTHROCYTES

Two laboratory markers:
Antibody to (+) adsorption (-) not adsorbed
● CD4 T-cell count - → prevent infection in Forsmann
progression to AIDS; <200/uL → stage 3 HIV
Antibody to Serum (+) adsorption (+) adsorption
infection, <200-499/uL → stage 2, <500/uL Sickness
● →1
● HIV-1 RNA level, or “viral load,” - (based on Antibody to IM (-) not adsorbed (+) adsorption

amplification methods that increase number AGGLUTINATION

of RNAs),” → response to ART (anti-retroviral WITH SHEEP RBC
treatment); successful therapy can result to
Antibody to NO AGGLUTINATION AGGLUTINATION
62undetectable viral load with the help of ART Forsmann

Antibody to Serum NO AGGLUTINATION NO AGGLUTINATION

Sickness

Antibody to IM AGGLUTINATION NO AGGLUTINATION

Agglutination with GPK and no agglutination with

Heterophile Antibodies associated with Infectious beef = infectious mononucleosis
Mononucleosis (IM)
● Heterophile antibodies are antibodies capable 3. Monospot test
of reacting with similar antigens from two or ● based on the agglutination of horse
more unrelated species erythrocytes by heterophile antibody present
Infectious Mononucleosis in infectious mononucleosis.
●
● Caused by Epstein Barr virus ● Horse red blood cells (RBCs) exhibit antigens
● Target: B cells (CD21) directed against both Forssman and infectious
● Atypical lymphocyte: T cells reacting to B cells mononucleosis antibodies, a differential
infected with EBV absorption of the patient’s serum is necessary
to distinguish the specific heterophile
Laboratory Diagnosis
antibody from those of the Forssman type.
1. Paul- Bunnell test
● Detect heterophile antibodies in patient
serum when mixed with antigen-bearing
sheep erythrocytes.
● Dilutions of inactivated patient serum are
mixed with sheep erythrocytes, incubated,
centrifuged, and macroscopically examined
for agglutination.
● Positive reactions are preliminary
1. Davidson Differentialtest
● Distinguishes between the heterophile
antibodies that agglutinate the antigen-
bearing erythrocytes of sheep
● Performed only if the preliminary Paul-
Bunnell test is positive in a titer of 1:56 or
greater
Laboratory Diagnosis

ADS WITH GPK CELLS ADS. WITH BEEF/OX

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