Antigen - Antibody Reactions: Dr.D.Bindu
Antigen - Antibody Reactions: Dr.D.Bindu
Antigen - Antibody Reactions: Dr.D.Bindu
REACTIONS
Dr.D.Bindu
Antigen-antibody reaction
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
One-dimensional single
electroimmunodiffusion test.
Was mainly used in the past for quantitative
estimation of antigens.
AGGLUTINATION
AGGLUTINATION
+
Applications of agglutination
Clumping
2 types : Direct
Indirect
Heterophile agglutination test
+
IMMUNOFLUORESCENCE
Immunofluorescence
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
Positive – NO colour
Negative – appearance of colour
Other type of ELISA
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
In vivo tests
In vitro tests
Neutralisation tests
INVIVO TESTS
Toxigenicity test
Schick test
INVITRO TESTS
ASO test
Virus neutralisation tests
Nagler reaction
Immunochromatography
CHEMILUMINESCENCE ASSAYS
IMMUNOBLOTTING