ANTIGEN-ANTIBODY

REACTION
Prepared by Reviewed by
Mr. SHUHAIB P PPT Review
Assistant Professor Committee,
YSAHS
Department of MLT
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SPECIFIC LEARNING OBJECTIEVES
At the end of the session, the students will be able to understand:

• General properties of antigen antibody reactions

• Types of antigen antibody reactions

• Conventional immunoassays

• Newer techniques

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 ANTIGEN–ANTIBODY REACTION
• Bimolecular association where the antigen and antibody combine
with each other specifically and in an observable manner .

• Specific interaction between epitope of an antigen with the

corresponding paratope of its homologous antibody.

• Exception- cross reactions which may occur due to sharing of

epitopes among different antigens.

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In such cases, antibody against one antigen can cross react with a
similar epitope of a different antigen.
• Reaction is specific
• Entire molecule is involved
• No denaturation of the antigen or the antibody
• Combination occurs at the surface
• Combination is firm but reversible

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DIAGNOSTIC USE
• Specific and observable

• Diagnostic tests based on antigen-antibody reactions are called as

immunoassays

• Immunoassays can be broadly categorized into two types-

Antigen detection assays
Antibody detection assays
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QUALITATIVE VS QUANTITATIVE IMMUNOASSAYS
Immunoassays can be performed by both

• Qualitative method

• Quantitative methods.

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QUALITATIVE ASSAYS
• Undiluted specimen containing the antibody is directly mixed with
the suspension of antigen or vice versa.

• Result is read as ‘positive’ or ‘negative’ based on presence or

absence of antigen or antibody in the clinical specimen.

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QUANTITATIVE ASSAYS
• When the qualitative test turns positive, the exact amount of
antibody in serum can be estimated by serial dilution of the
patient’s serum and mixing each dilution of the serum with a
known quantity of antigen.

• Measurement of antibody is expressed in terms of titer.

• Quantitative tests are more reliable as they can differentiate

between true negative and false negative results.
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TYPES OF ANTIGEN–ANTIBODY REACTIONS

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Newer techniques -
Enzyme linked immunosorbent assay (ELISA)
Enzyme linked fluorescent assay (ELISA)
Immunofluorescence Assay (IFA)
Chemiluminescence-linked immunoassay (CLIA)
Immunohistochemistry
Rapid tests-
 Lateral flow assay (Immunochromatographic test)
 Flow through assay
 Western blot
Immunoassays using electron microscope
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PRECIPITATION REACTION
• Definition-When a soluble antigen reacts with its antibody in the
presence of optimal temperature, pH and electrolytes (NaCl), it leads
to formation of the antigen-antibody complex in the form of:
Insoluble precipitate band when gel containing medium is
used or
Insoluble floccules when liquid medium is used (precipitate
remains suspended as floccules)
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Clinical Applications:

• Slide Flocculation Test (for Syphilis)

Examples - VDRL (Venereal Disease Research Laboratory) and
RPR (Rapid Plasma Reagin) tests

• Elek’s Gel Precipitation Test ( Detecting Diphtheria Toxin)

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AGGLUTINATION REACTION
When a particulate or insoluble antigen is mixed with its antibody in
the presence of electrolytes at a suitable temperature and pH, the
particles are clumped or agglutinated.
 Advantage:
• More sensitive than precipitation test and the
• Clumps are better visualized and interpreted as compared to bands
or floccules.

Universities Press Pvt Ltd.

Applications:

• Classified as direct, indirect (passive) and reverse passive

agglutination reactions.

• All these agglutination tests are performed - on a slide, or in tube

or in card or sometimes in microtiter plates.

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Direct Agglutination Test :

• The antigen directly agglutinates with the

antibody.
1. Slide Agglutination :
• Performed to confirm the identification and
serotyping of bacterial colonies grown in culture.
• Method used for blood grouping and cross
matching.

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2. Tube Agglutination
• Standard quantitative test for estimating antibody
in serum.

• Antibody titer can be estimated as the highest

dilution of the serum which produces a visible
agglutination

• Eg: Typhoid fever (Widal test)

Acute brucellosis (Standard agglutination test)

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Indirect (Passive) Agglutination Tests

• If the antigens are adsorbed onto particles (e.g. RBCs, latex beads,
bentomile clay), soluble antigens can respond to agglutination test.
• Antibody reacts with the soluble antigen adhering to the particles.
• Therefore, the particles agglutinate with each other as these do in
the direct agglutination tests

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Examples:
Hemagglutination
Latex agglutination
Reversed passive agglutination
Co-agglutination test

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Latex agglutination

• Positive result is indicated by formation

of visible clumps.

• LAT is one of the most widely used tests

at present as it is very simple and rapid.

• Used for detection of ASO

(antistreptolysin O antibody).

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Haemagglutination
• It is process of clumping of RBCs.
• When the RBCs are agglutinated by certain
viruses such as those causing mumps,
measles, influenza, etc. it is called viral
haemagglutination.
• This test is used for the diagnosis of a
number of viruses affecting human body.

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COMPLEMENT FIXATION TEST

• Detects the antibodies in patient’s serum that are capable of fixing with
complements. It was once very popular, now is almost obsolete.

Applications

• Detection of complement fixing antibodies in Rickettsia , Chlamydia ,

Mycoplasma infections and some viral infections, such as arboviruses.

• Used for various other serological tests such as: Treponema pallidum
immobilization test and Sabin-Feldman dye test for Toxoplasma
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NEUTRALIZATION TEST

• These are based on the action of antibodies to neutralise an end

product or the outcome of the action of an antigen
• Neutralization tests are also less commonly used in modern days.
Various examples are as follows:
Viral neutralization test
Plaque inhibition test
Toxin–antitoxin neutralization test

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NEWER TECHNIQUES

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• Uses a detector molecule to label antibody or antigen which in
turn detects the corresponding antigen or the antibody in the
sample by producing a visible effect.

• Most of the newer techniques use the same principle, but they
differ from each other by the type of labelled molecule used and
the type of visible effect produced.

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IMMUNOASSAYS AND THE TYPES OF MOLECULE USED FOR LABELING
Abbreviation Immunoassay method Molecules used for Type of visible effect
labeling
ELISA Enzyme linked immunosorbent Enzyme- substrate- Color change is detected by
assay chromogen complex spectrophotometer
ELFA Enzyme linked fluorescent assay Enzyme- substrate Fluorometric detection

IFA Immunofluorescence Assay Fluorescent dye Emits light, detected by

fluorescence microscope
RIA Radioimmunoassay Radioactive isotope Emits β and γ radiations,
detected by β and γ counters
CLIA Chemiluminescence-linked Chemiluminescent Emits light, detected by
immunoassay compounds luminometer
IHC Immunohistochemistry Enzyme or Fluorescent dye Color change (naked eye) or
Fluorescence microscope
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Enzyme-linked immunosorbent assay (ELISA)

• Immunoassay that detects either antigen or antibodies in the specimen,

by using enzyme–substrate– chromogen system for detection
Immunosorbent- Absorbing material used (e.g. polystyrene,
polyvinyl) that specifically absorbs the antigen or antibody present
in serum.
Enzyme is used to label one of the components of immunoassay (i.e.
antigen or antibody).
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• Substrate- chromogen system is added at the final step of ELISA.

• Enzyme-substrate reaction in turn activates the chromogen to produce a

color.

• Color change is detected by spectrophotometry in an ELISA reader.

• Intensity of the color is directly proportional to the amount of the

detection molecule (Ag or Ab) present in test serum.

• (Ag-Ab complex)-enzyme + substrate → activates the chromogen → color

change → detected by spectrophotometry
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PROCEDURE OF ELISA :

• ELISA kits are commercially available; contain all necessary

reagents (such as enzyme conjugate, dilution buffer,
substrate/chromogen, etc).

• Procedure involves a series of steps done sequentially; at each

step, a reagent is added - then incubated, followed by washing of
the wells [manually or by an automated ELISA washer.

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Most Common ELISA Types

Direct ELISA
Indirect ELISA
Sandwich ELISA
Competitive ELISA

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Advantages of ELISA

• Method of choice for detection of antigens/ antibodies in serum in

modern days, especially in big laboratories as large number of
samples can be tested together using the 96 well microtiter plate.

• Economical, takes 2–3 hours for performing the assay.

• ELISA has a high sensitivity - that is why, it is commonly used for

performing screening test at blood banks and tertiary care sites.

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Disadvantages of ELISA

• small laboratories having less sample load - ELISA is less preferred

than rapid tests as the latter can be done on individual samples.

• Takes more time (2–3 hours) compared to rapid tests , which take
10–20 minutes

• Needs expensive equipment such as ELISA washer and reader.

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A. ELISA reader (Biorad); B. ELISA washer

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ENZYME-LINKED FLUORESCENT ASSAY (ELFA)
• Modification of ELISA, differs from ELISA in two ways:
Automated system, all steps are performed by the instrument.

Ag-Ab-enzyme complex is detected by fluorometric method.

• VIDAS and mini VIDAS (bioMérieux) are commercially available systems based on
ELFA technology.

• The solid phase receptacle present in reagent strip (equivalent to wells in

microtiter plate in ELISA) serves as the solid phase; which is either coated with
capture antigen (for antibody detection) or antibody (for antigen detection).
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CHEMILUMINESCENCE-LINKED IMMUNOASSAY (CLIA)

• Emission of light (luminescence), as a result of a chemical reaction.

• Principle: similar to that of ELISA; however the chromogenic

substance is replaced by chemiluminescent compounds (e.g. luminol
and acridinium ester) that generate light during a chemical reaction
(luxogenic).

• Light (photons) can be detected by a photomultiplier, also called as

luminometer.
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• (Ag-Ab complex)-enzyme (e.g. HRP) + chemiluminescent substrate (e.g.
luminol and acridinium ester) → product+ light (photons) → detected
by luminometer or photomultiplier.

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Advantages of CLIA
• 10 times more sensitive than ELISA.
• Further modified by using an enhancer that potentiates the
chemical reaction.
• Most samples have no 'background' signal, i.e. luminol compounds
do not themselves emit light.
• Measurement of chemiluminescence is not a ratio unlike the
measurement of fluorescence (IFA) and or color (ELISA).
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RAPID TESTS

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• Point of care (POC) tests, because unlike ELISA and other
immunoassays, the Point of Care tests can be performed independent
of laboratory equipment and deliver instant results.

• Two principles of rapid tests are available:

Lateral flow assay
Flow through assay.

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• Both the formats are available for the diagnosis of various diseases
such as malaria, hepatitis B, hepatitis C, HIV, leptospirosis,
Helicobacter pylori, syphilis, etc.

• Based on lateral flow technique.

• Widely used in diagnostic laboratories because of its simplicity, low-

cost and rapidity.

• It can be used for both antigen and antibody detection in sample.

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SUMMARRY

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IMPORTANT QUESTIONS
• Name various antigen-antibody reactions and describe the principle
and application of precipitation reactions.

• Define agglutination reaction? Discuss the principle and application of

agglutination reactions.

• Principles and uses of immunofluorescence tests and enzyme linked

immunosorbent assay (ELISA).

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• Difference between precipitation and agglutination.

• Principles and uses of latex agglutination test and coagglutination.

• Principle of complement fixation test.

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REFERENCES
• Sastry AS, Bhat S. Essentials of medical microbiology. JP Medical
Ltd; 2018 Oct 31.
• Baveja CP. Textbook of microbiology. Arya Publications; 2019

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