ANTIGEN – ANTIBODY

INTERACTION
ANTIGEN – ANTIBODY INTERACTION

The antigen–antibody reaction is a

bimolecular association where the
antigen and antibody combine with each
other specifically and in an observable
manner
Reversible alteration in either antibody
or in antigen.
 CHARACTERISTICS OF
ANTIGEN – ANTIBODY INTERACTION

Specicity  Reaction is specific; an antigen combines only

with its homologous antibody and vice-versa.
However, cross reactions may occur due to
antigenic similarity.

Strength 01 02 03 04

Diagnostic Use  Hydrogen bonds

 Electrostatic
Antigen
Affinity interactions
Immunoassay
Non Covalent  Hydrophobic
Antibody
Avidity interactions
Immunoassay
Interaction  Van der Waals forces.
IMMUNOASSAYS & DIAGNOSTIC USE

by specific Ab
Ag
Detection
assays

Y Ab
Detection
assays
by specific Ag

Serological test is done in serum sample. Can also be done in CSF & Urine sample
 CHARACTERISTICS OF
ANTIGEN – ANTIBODY INTERACTION

Specicity

Qualitative vs
Quantitative

Strength 01 02 03 04 05

Diagnostic Use

 Qualitative - Positive or Negative

Non Covalent  Quantitative - Titration
Interaction
Marrack’s Lattice
Hypothesis
Evaluation of Immunoassays
SENSITIVITY

Sensitivity is defined as ability of a test to identify

correctly all those who have the disease,
i.e. true-positives.

SPECIFICITY

Specificity is defined as ability of a test to identify

correctly all those who do not have disease,
i.e. true negatives.
 Ag-Ab Reaction Types

CONVENTIONAL METHODS
PRECIPITATION

AGGLUTINATION

COMPLEMENT FIXATION
TEST

NEUTRALISATION
 Ag-Ab Reaction Types
NEWER METHODS
ELISA ELFA

IMMUNO FLUORESCENCE
RADIO IMMUNO ASSAY
(IFA)
(RIA)

CHEMI LUMINESCENCE
ASSAY (CLIA) IMMUNOHISTO
CHEMISTRY

RAPID TEST
IMMUNO BLOTTING

IMMUNO ELECTRON
MICROSCOPY
Ag-Ab Reaction Types

OTHER METHODS
CONGLUTINATION

OPSONISATION
 SOLUBLE Ag INSOLUBLE
Optimal t ◦ PRECIPITATE
PRE
CIPI
Electrolytes(NaCl)
Y
TAT
ION

Optimal pH
Y SPECIFIC Ab
PRECIPITATION
 OBSERVATION
 Insoluble precipitation band/ring when
gel or agar containing medium is used
(called immunodiffusion)
PRE
CIPI
 Eg. Ascoli’s thermoprecipitin test
TAT
ION (for Anthrax)
RING/BAND PRECIPITATION TEST
 OBSERVATION
 Insoluble precipitation band when gel
or agar containing medium is used (called
immunodiffusion)
PRE
CIPI
 Eg. Ascoli’s thermoprecipitin test
TAT
ION (for Anthrax)

 Insoluble floccules when liquid medium

is used (called flocculation test).
 Eg. VDRL / Syphilis
(for Syphilis)
Flocculation slide test
 AGGLUTINATION
INSOLUBLE Ag Particle are
Suitable t◦ clumped
AGG
LUTI
Electrolytes(NaCl)
Y
NAT
ION

Suitable pH
Y SPECIFIC Ab
AGGLUTINATION
 Advantages:
 Visible
 Easy to Read
AGG
LUTI
Types:
NAT
ION  Direct
 Indirect
Performed in:
 Card / Slide / Microtiter plate
 Test tube
  Direct Agglutination Reaction
It is usually performed to confirm the identification
and serotyping of bacterial colonies grown in
culture. It is also the method used for blood
grouping and cross matching.

II
AGG
LUTI
NAT
ION
 MASTER SERUM DILUTION

1:12.5
Serum Dilution 0.2ml of Serum
2.3ml of Saline
 CONTROL
0.2m 0.2m 0.2m 0.2m TUBE
l l l l

S.Typhi
0.
2
ml
DISCARD

O Ag
AddO.2ml
O.2ml of
S. Typhi
Mix &OTransfer
NORMAL
Ag
MASTER
O.2ml
From 1 st of Serum
to 7th
tube
SALINE
SERUM DILUTION 1st 2nd 3rd 4th 5th 6th 7th
From 2nd
to 6 th 0.2m 0.2m 0.2m 0.2m
From
Add st
& 2 S. to
1O.2ml nd
7th
tube
Paratyphi l l l l
tube
H Ag
0.
2

S.Paratyphi A
DISCARD
ml

H Ag
1st 2nd 3rd 4th 5th 6th 7th
 INCUBATE THE RACKS
IN WATER BATH AT 37`C FOR 18hrs

OBSERVE THE TUBE AFTER 18 HOURS

Heterophile Agglutination test
Detection of Antibody using cross reacting Antigen
Typhus fever (Weil Felix reaction)
Infectious mononucleosis (Paul Bunnell test)
Mycoplasma pneumonia (Cold agglutination test).

Microscopic Agglutination test

Performed in Microtiter plate & results read under
microscope
Microscopic Agglutination test (MAT) - Leptospirosis
  Indirect Agglutination Reaction
As agglutination test is more sensitive and better interpreted
than precipitation test, attempt has been made to convert a
precipitation reaction into an agglutination reaction. This is
possible by coating the soluble antigen on the surface of a
carrier molecule (e.g. RBC, latex or bentonite), so that the
AGG
antibody binds to the coated antigen and agglutination takes
LUTI place on the surface of the carrier molecule.
NAT
ION
Passive Agglutination Test
For Antibody Detection

Reverse Passive Agglutination Test

For Antigen Detection
  Indirect Agglutination Reaction

Passive Agglutination Test

For Antibody Detection
AGG
LUTI
NAT  Indirect Hemagglutination Test
ION

 Latex Agglutination Test

ASO Test
  Indirect Agglutination Reaction
Reverse Passive Agglutination Test
For Antigen Detection

AGG
 Reverse Passive Hemagglutination Test
LUTI
NAT
ION HBsAg
 Latex Hemagglutination Test
RA Factor
Capsular Ag (CSF)
 Coagglutination Test
Protein A Staph aureus
 Hemagglutination test
Antibody
Direct against
Hemagglutination RBCs Used Infectious
Test
agent
EBV
Paul Bunnel Test Sheep RBCs Infectious
Mononucleosis
Cold
Agglutination Human RBCs Mycoplasma
Blood grouping Rh Typing
Coombs Test Anti Rh Rh
Antibodies Incompatibility
AGG
LUTI
NAT
ION
 Ag-Ab Reaction Types

CONVENTIONAL METHODS
PRECIPITATION

AGGLUTINATION

COMPLEMENT FIXATION
TEST

NEUTRALISATION
COMPLEMENT FIXATION
TEST
 Detects the antibodies in patient’s serum
that are capable of fixing with
complements.
 Application:
 Wasserman Test – Syphilis
 Detection of Complement fixing Antibodies
 Rickettsia
 Chlamydia
 Brucella
 Mycoplasma
 Some Viral Infection
NEUTRALIZATION TEST
 Viral neutralization test: It detects the
presence of neutralizing antibody in patient’s
serum
 Plaque inhibition test: This is done for
bacteriophages
Toxin–antitoxin neutralization test
 Schick test: It is a diphtheria toxin–antitoxin neutralization
test
 Nagler’s reaction: It is used for detection of α-toxin of
Clostridium perfringens
 ASO test: Antistreptolysin O antibody was detected before
by neutralization method; however, it is now replaced by
latex agglutination.
 APPLICATION OF
ANTIGEN – ANTIBODY INTERACTION
In the Body or in Vivo
(i) It forms the basis of immunity against infectious
diseases.
(ii) It may lead to tissue injury in some hypersensitivity
reactions and autoimmune diseases.
In the Laboratory or in Vitro
(i) For diagnosis of infections
(ii) Helpful in epidemiological studies
(iii) For identification of non-infectious agents such as
enzymes
(iv) Detection and quantitation of either antigens or
antibodies.
 APPLICATION OF
ANTIGEN – ANTIBODY INTERACTION
 The most common use is the determination of blood groups i.e.
blood typing.
 Rapid diagnosis test kits used for pregnancy detection as well
as detection of several diseases such as malaria, dengue, etc. are
based on this principle. They require very little time for the tests.
 Serological ascertainment of exposure to infectious agents.
 Quantification of drugs, hormones, viral antigens, etc.
 Detection of presence or absence of proteins in serum.
 To study the characteristics of different immunodeficiency
diseases.
 To perform confirmatory tests for infections such as Western
Blotting for HIV infection.
 APPLICATION OF
ANTIGEN – ANTIBODY INTERACTION
In Vivo Reactions In Vitro Reactions
•Agglutination •Agglutination
•Precipitation •Precipitation
•Complement fixation •Complement fixation
•Neutralization •Neutralization
•Ab Dependent Cell-Mediated •ELISA
Toxicity
•Immobilization •Radioimmunoassay (RIA)
•Opsonization •Western Blotting
•Immunochromatograhy (ICT)
•Immunofluorescence