Antigen - Antibody Reactions: Dr.D.Bindu

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ANTIGEN – ANTIBODY

REACTIONS
Dr.D.Bindu
Antigen-antibody reaction

 Bimolecular association where the antigen


and antibody combine with each other
specifically and in an observable manner .
 Ag-Ab reaction involves specific interaction
of epitope of an antigen with the
corresponding paratope of its homologous
antibody
Antigen-Antibody Reactions

 PRECIPITATION

 AGGLUTINATION

 COMPLEMENT FIXATION

 NEUTRALISATION TESTS

 IMMUNO FLUORESCENCE

 ELISA

 OTHER TESTS
Precipitation
 When a soluble antigen reacts with its antibody in the
presence of optimal temperature, pH and electrolytes , it
leads to formation of the antigen-antibody complex in the
form of:
o Insoluble precipitate band when gel containing medium
is used or
o Insoluble floccules when liquid medium is used
(precipitate remains suspended as floccules)
 Ring test: In a narrow tube (e.g. capillary tube), antigen
solution is layered over an antiserum;
o Precipitate ring appears at the junction of two liquids.
o Example: Streptococcal grouping by Lancefield
technique, and Ascoli’s thermoprecipitin test done for
anthrax.
Precipitation in liquid medium
 Flocculation test- When a drop of antigen is mixed with
a drop of patient's serum, then floccules appear.
o Examples of slide flocculation test- VDRL and RPR
tests used for diagnosis of syphilis
o Examples of tube flocculation test- Kahn test used
previously for syphilis
Precipitation in gel
 Using 1% soft agarose gel for precipitation reaction has
many advantages over liquid medium-
o Results in formation of clearly visible bands instead of
floccules that can be preserved for longer time.
o Can differentiate individual antigens from a mixture
as each antigen forms a separate band after reacting
with specific antibody.
Precipitation in gel

 Whether only Ag diffuses (single diffusion) or


both Ag and Ab diffuse (double diffusion)
 Whether Ag or Ab diffuses in one dimension
(i.e. vertical diffusion when test is done on a
tube layered with gel) or two dimensions (i.e.
diffusion in both X and Y axis when test is
done on a slide or a petri dish layered with
gel)
OUDIN procedure
Double diffusion (oakley and Fulthorpe
procedure)
RADIAL IMMUNODIFFUSION
Double diffusion in double dimensions
(Ouchterlony procedure)
1. Electroimmunodiffusion (EID)
2. CIEP (Countercurrent
immunoelectrophoresis)
3. Rocket electrophoresis
Electro immunodiffusion

 Electric current is applied to a slide layered


with gel.
 Helps in identification and approximate
quantitation of various proteins present in
the serum.
Counter electrophoresis

 Test is even faster (takes 30 minutes) and


more sensitive than EID.
 Popular in the past - detecting alpha
fetoprotein in serum and capsular antigens of
Cryptococcus and meningococcus in the
cerebrospinal fluid.
Rocket electrophoresis

 One-dimensional single
electroimmunodiffusion test.
 Was mainly used in the past for quantitative
estimation of antigens.
AGGLUTINATION
AGGLUTINATION

 Particulate antigen combines with its


antibody producing clumping
 Incomplete antibodies donot cause
agglutination
 Blocking antibodies inhibit agglutination

+ 
Applications of agglutination

 Slide agglutination test


 Tube agglutination test
 Coombs test
 Passive agglutination test
Slide agglutination

 Clumping

 Also used for blood grouping, cross


matching
Tube agglutination test

 Serological diagnosis of typhoid, brucellosis


and typhus fever
Coombs test
 Detection of incomplete anti-Rh antibodies

 They only coat the erythrocytes and donot agglutinate


Rh positive erythrocytes in saline

 Principle : Such coated RBCS are treated with Coombs


serum ( rabbit antiserum against human
gammaglobulin), cells are agglutinated

 2 types : Direct
Indirect
Heterophile agglutination test

 Weil Felix reaction


 Antigenic sharing between Proteus and Rickettsial
strains
 Paul Bunnel test
 Infectious mononucleosis
 Streptococcus MG agglutination test
 Primary atypical pneumonia
Passive agglutination test

 Carrier particles : RBCs, Latex, Bentonite

 Latex agglutination test : ASO. RA. CRP. HCG


 Haemagglutination test : Rose waaler test
 Co agglutination tests

+ 
IMMUNOFLUORESCENCE
Immunofluorescence

 Property of absorbing light rays of one


particular wavelength and emiting rays with a
different wavelength

 Direct
 Indirect
DIRECT INDIRECT

Fluorochrome Fluorochrome
Labeled Ab Labeled Anti-Ig
Unlabeled
Ab

Ag
Tissue Section
Ag
Tissue Section
 Fluorescent dyes –
 fluosrecein isothiocyanate
 Lissamine rhodamine
ELISA – Enzyme Linked Immuno Sorbent
Assay
ELISA

 Principle
 Used to detect antigen or antibody
 MACRO ELISA – done in polystyrene tubes
 MICRO ELISA – in polyvinyl microtitre plates
 Types of ELISA –
 Sandwich ELISA
 Indirect ELISA
 Competitive ELISA
 Casette / Cylinder ELISA
Detecting Ab’s against HIV- HIV
coat protein is the Ag
SANDWICH ELISA
Competitive

 Used for detecting HIV antibodies


 2 specific antibodies – one conjugated with
enzyme and another present in the serum
 Competition occurs between the two
antibodies for same antigen

 Positive – NO colour
 Negative – appearance of colour
Other type of ELISA

 CASSETTE ELISA / CYLINDER ELISA


 Rapid
 Detection of HIV 1 and 2 antibodies

Enzymes and substrates :


▪ Alkaline Phosphatase – para nitro phenyl phosphate
▪ Horse radish peroxidase – ortho phenylene diamine
dihydrochloride
USES OF ELISA

 Detection of HIV antibodies in serum


 Detection of mycobacterial antibodies in
Tuberculosis
 Detection of rotavirus in stool samples
 Detection of hepatitis B markers in serum
 Detection of enterotoxin of Escherichia coli in
stool samples
Radio immuno assay

 RIA is based on
competition for a
fixed amount of
specific Ab between
a known
radiolabelled Ag
and unknown
unlabelled (test) Ag
NEUTRALISATION
Neutralisation test

 Bacterial exotoxins produce neutralising


antibodies (antitoxins)
 Viruses may also be neutralised by antibodies
– Virus neutralisation tests

 In vivo tests
 In vitro tests
Neutralisation tests

 INVIVO TESTS

 Toxigenicity test
 Schick test

 INVITRO TESTS
 ASO test
 Virus neutralisation tests
 Nagler reaction
Immunochromatography

 Popular form of rapid immunoassays


 Strip based test – one step procedure
 Within 30 minutes
Immunochromatography
IMMUNOELECTRON MICROSCOPIC TESTS

 Immuno electron microscopy

 Immuno ferritin test

 Immuno enzyme test


OTHERS

 CHEMILUMINESCENCE ASSAYS

 IMMUNOBLOTTING

1. Western blot - Proteins


2. Southern Blot - DNA
3. Northern Blot - RNA
THANK YOU

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