ANTIGEN–

ANTIBODY
REACTION
 GENERAL PROPERTIES OF ANTIGEN -
ANTIBODY REACTIONS

 Specificity: Specific interaction b/w epitope of an Ag with paratope of its homologous antibody.

 Noncovalent Interactions: Hydrogen bonds, Electrostatic interactions ,Hydrophobic interactions ,Van

der Waals force

 Strength: The combination is firm but reversible.

 Affinity

 Avidity

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DIAGNOSTIC USE
 Diagnostic tests based on Ag-Ab reactions are K/a immunoassays

 2 types-

 Ag detection assays

 Ab detection assays

 Qualitative and quantitative methods.

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MARRACK’S LATTICE
HYPOTHESIS
 Ag-Ab reaction is weak or fails to occur when the number of Ag and Ab
are not proportionate to each other.

In prozone/post zone -
Lattice does not enlarge

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 TYPES OF ANTIGEN–ANTIBODY
REACTIONS
Conventional techniques - Newer techniques -

 Precipitation reaction  Enzyme linked immunosorbent assay (ELISA)

 Enzyme linked fluorescent assay
 Agglutination reaction
 Immunofluorescence Assay (IFA)
 Complement fixation test  Chemiluminescence-linked immunoassay (CLIA)
 Neutralization test  Immunohistochemistry
 Rapid tests-
 Lateral flow assay (Immunochromatographic test)
 Flow through assay

 Western blot

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CONVENTIONAL
IMMUNOASSAYS

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PRECIPITATION REACTION

When a soluble Ag reacts with its Ab in the presence of optimal temperature,

pH and electrolytes, it leads to formation of the Ag-Ab complex.

 Seen as floccules or bands

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CLINICAL APPLICATIONS
 Slide Flocculation Test (for Syphilis)

 VDRL (Venereal Disease Research Laboratory)

 RPR (Rapid Plasma Reagin) test

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AGGLUTINATION REACTION

When a particulate or insoluble Ag is mixed with its Ab in the

presence of electrolytes at a suitable temperature and pH, the
particles are clumped or agglutinated.

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AGGLUTINATION REACTION

 Classified as:

 Direct

 Indirect (passive)

 Reverse passive agglutination

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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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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.

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TUBE AGGLUTINATION-
APPLICATIONS
 Typhoid fever (Widal test)

 Acute brucellosis (Standard agglutination test)

 Coombs Antiglobulin test

 Heterophile agglutination tests:

 Typhus fever (Weil Felix reaction)

 Infectious mononucleosis (Paul Bunnell test)

 Mycoplasma pneumonia (Cold agglutination test)

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 Reverse Passive Agglutination Test (for Ag
Detection)
Ab is coated on a carrier molecule which detects Ag in the patient’s serum.

Test Carrier molecule Clinical applications

Latex agglutination test Latex particles  CRP (C reactive protein),

 RA (rheumatoid arthritis factor),
 Capsular antigen detection in CSF (for pneumococcus,
meningococcus and Cryptococcus)
 Streptococcal grouping

Coagglutination test Staphylococcus aureus  Used in past for antigen detection test in the past (e.g.
Salmonella Ag detection from blood and urine).

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HEMAGGLUTINATION TEST

 Refers to the agglutination tests that uses red blood cells (RBCs) as source
of antigen.

 Types of hemagglutination tests:

 Direct hemagglutination test

 Indirect hemagglutination test

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DIRECT HEMAGGLUTINATION TEST

Serum antibodies directly agglutinate with surface antigens of RBCs to

produce a matt.

 Paul Bunnell test

 Cold agglutination test

 Blood grouping

 Coombs test or Antiglobulin test

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 NEWER
TECHNIQUES
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 IMMUNOASSAYS AND THE TYPES OF MOLECULE
USED FOR LABELING

Immunoassay method Molecules used for Type of visible effect

labeling
ELISA Enzyme linked immunosorbent assay Enzyme- substrate/ Color change is detected by
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 immunoassay Chemiluminescent Emits light, detected by luminometer
compounds
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IMMUNOASSAYS AND THE TYPES OF MOLECULE
USED FOR LABELING

Abbreviation Immunoassay method Molecules used for labeling Type of visible effect

IHC Immunohistochemistry Enzyme or Fluorescent dye Color change (naked eye) or

Fluorescence microscope
WB Western blot Enzyme Color band (naked eye)

Rapid test Immunochromatographic test Colloidal gold or silver Color band, (naked eye)

Flow through assay Protein A conjugate Color band, (naked eye)

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TYPES OF ELISA

 Direct ELISA

 Indirect ELISA

 Sandwich ELISA

 IgM Antibody Capture (MAC) ELISA

 Competitive ELISA

 ELISPOT Test
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DIRECT ELISA
• Used for detection of antigen in serum.
• Primary antibody is labeled with the enzyme

Well + Antigen (Test serum) + Primary antibody-Enzyme +

Substrate-chromogen>>>>>> Color change
INDIRECT ELISA

• Used for detection of Ab/Ag in serum.

• Secondary antibody is labelled with enzyme

Wells coated with Ag + primary Ab (Test serum) + secondary Ab enzyme + Substrate

chromogen>>>>Color Change
SANDWICH ELISA

 Wells coated with capture Ab + Ag (test serum) + primary Ab-enzyme + substrate–

chromogen → color

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IGM ANTIBODY CAPTURE (MAC) ELISA

 Use of avidin-biotin system helps in amplifying

the signal generated between enzyme-antibody
complex, thus increases the sensitivity of the
assay.

 Wells coated with capture anti-IgM Ab + IgM Ab

(test serum) + recombinant antigen + secondary
Ab-biotin +avidin-enzyme + substrate–chromogen
→ color 27
IGM ANTIBODY CAPTURE (MAC) ELISA
 Enzymatically amplified sandwich-type immunoassay.

 Uses:

 Dengue

 JE

 West Nile virus

 Scrub typhus

 Leptospirosis

 Toxoplasmosis. 28
COMPETITIVE ELISA

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ADVANTAGES OF ELISA
 Method of choice for detection of antigens/ antibodies in serum big
laboratories - large number of samples can be tested together

 Economical

 Takes 2–3 hours for performing the assay

 High sensitivity

 More specific.

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DISADVANTAGES OF ELISA

 ELISA is less preferred than rapid tests in small labs

 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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APPLICATIONS OF ELISA

 ELISA used for antigen detection – HBsAg for hepatitis B, NS1

antigen for dengue etc.

 ELISA can also be used for antibody detection against hepatitis B,

hepatitis C, HIV, dengue, EBV, HSV, toxoplasmosis, leishmaniasis, etc.

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 RAPID TESTS

 Point of care (POC) tests,

 POC can be performed independent of laboratory equipment and deliver

instant results.

 2 formats:

 Lateral flow assay

 Flow through assay.

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RAPID TESTS
For the diagnosis of :

 Malaria

 hepatitis B

 hepatitis C

 HIV

 leptospirosis,

 Helicobacter pylori

 syphilis
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IMMUNOCHROMATOGRAPHIC TEST (LATERAL FLOW
ASSAY)
 Based on lateral flow technique.

 Simple, low-cost and rapid.

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

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PRINCIPLE OF ICT (ANTIGEN DETECTION)

 The test system consists of a

nitrocellulose membrane (NCM) and
an absorbent pad.

 Two formats are available: cassette or

strip.

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 PRINCIPLE OF ICT (ANTIGEN
DETECTION)
 Test band: At the test line, the Ag-labeled Ab complex is immobilized by binding
to the monoclonal Ab in the test line to form a colored band

 Control band: The free colloidal gold labeled Ab can move further and binds to
the anti-human Ig to form a color control band.

 If the control band is not formed, then the test is considered invalid irrespective of
whether the test band is formed or not

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