Download Serological reactions

Serological reactions
Department of Microbiology
Antigen-Antibody reactions
• The paratope of antobody molecule binds specifically to the
epitopes present on the antigen molecule.
• The interactions between Ag-Ab is highly specific but does not
cause any irreversible changes in either Ag or Antibody
molecule.
• The specificity between Ag-Ab binding has lead to the various
immunological assays.
• These immunological assays form the basis for various
diagnostic tests, measurement of immune response, detection
of various important biomolecules like hormone, drugs etc.
Serological reactions
Precipitation
Agglutination
Haemagglutination
Complement fixation
Radio-immuno assay (RIA)
Enzyme linked Immunosorbent
assay (ELISA)
Precipitation
• Interaction of antibodies with soluble antigen molecules leads
into formation of larger immune complexes which no more
remain in solution and precipitate out.
• Such antigen antibody interactions are called “precipitation”.
• Antibody molecule that participate in precipitation reaction are
also called as “precipitin”.
• Precipitation reactions are widely used in the diagnosis of
various infectious diseases.
Precipitation reaction is seen in Zone of
Equivalence
Precipitation in Gel
• Precipitation reactions can also be carried out in Agar or
Agarose Gel.
• Two important methods are:
- Single radial immuno-diffusion (Mancini’s method)
- Double immuno-diffusion (Ouchterlony’s method)
Agglutination
• Interaction of antibody molecules with antigen molecules of
particulate nature leads to formation of larger clumps.
• This immunological reaction is known as “Agglutination”.
• Agglutination results owing formation of larger immune complexes
by cross linking of polyvalent antigen molecules by antibodies.
• Antibodies taking part in agglutinations reactions are called
“agglutinins”.
• Ig M isotypes are considered as better agglutinins than IgG
molecules.
Agglutination
• Diagnosis of various bacterial disease
- Brucellosis
- Salmonellosis
• Serotyping of bacterial species
• ABO blood grouping
Agglutination
-
Antibody-Antigen interaction
antibodies in the specific antisera agglutinate with the bacteria
when the corresponding antigens are present
seen as particulate matter, or lumps forming
graded by strength as 1+, 2+, 3+, and 4+
4+ is a clear background with 100% agglutination
O agglutination is granular
H agglutination is loose and floccular
Haemagglutination (HA)
• Agglutination of RBCs is called “haemagglutination”.
• Agglutinations of RBCs caused by anti-RBC antibodies is an
example of immunological reaction.
• However, a number of agents can cause agglutination of RBCs
like plant lectins, viruses etc. Thus all haemagglutination
reactions are not immunological reactions.
• Interestingly, this agglutination of RBCs by viruses can be
inhibited by antibodies specific to the viral agent leading to
“haemagglutination inhibition (HI)”.
• Haemagglutination inhibition is an important diagnostic test.
Complement Fixation test (CFT)
• Binding of antibody with antigen molecule allows C 1 qrs
complement components to bind on antibody molecules.
• This lead to activation of complement cascade which
ultimately forms membrane attack complex.
• In a system which contain RBCs and hemolysin (Abs against
RBCs) with complement components, binding of antibody
molecules on RBC will lead to activation of complement
component and lysis of RBCs.
• This binding of complement with antibody molecules does not
show specificity.
Complement Fixation test (CFT)
• In complement fixation test, two antigen antibody systems are used:
- Test Ag and Ab system
- Sheep RBCs and Hemolysin
• Complement components from guinea pig is added to the system.
1. First test antigen and antibodies are allowed to react in presence of
complement.
- If Ags reacts with the Abs, then complement present will bind to Antigen-Antibody
complex.
- If Ags don’t react with Abs, then complemnent remain unbound and will take part in
hemolysis of SRBCs.
2. Thereafter, SRBCs and hemolysin are added.
• Result is interpreted on the basis of hemolysis of RBCs.
Interpretation of CFT
• In CFT:
a) Lysis of SRBCs indicate negative result:
Binding of complement with SRBCs-hemolysis complex indicates no reaction between
test Antigen and Antibody.
b) Absence of lysis of SRBCs indicate positive result:
This happens because of the binding of complement components to the test antigenantibody complex. Thus, no complement is available free for hemolysis of SRBCs.
Enzyme Linked Immuno Sorbant Assay (ELISA).
• The solid-phase ELISA is a very widely used
immunological assay which can be adapted to
quantitate either antibodies or antigens.
• It is probably the most widely used diagnostic assay.
Immunofluorescence
• It is possible to attach fluorescent molecules (fluorochrome)
chemically with antibody molecule without affecting its specificity
for the antigen.
• Fluorescent molecules absorb light of lower wavelength (higher
energy) and emit light of higher wavelength (lower energy).
• When these fluorochrome conjugated antibodies are allowed to react
with antigen, the antigen-antibody complexes formed could be
detected by a special optical instrument called “Fluorescent
microscope” which is equipped with UV light source.
• The most commonly used fluorochromes are Fluorescein
isothiocyanate
(FITC),
Rhodamine,
Phycoerythrin
etc.
Immunofluorescence technique is of great use in detecting antigens
in tissue sections.
Neutralization
•
When a virus is allowed to react with specific antibodies, loss
of infectivity of virus is observed. Such reactions are called
“Neutralization”.
• Loss of viral infectivity is assessed by inoculating virusantiserum mix into a suitable system like animal or egg
embryo or cell culture.
• Depending on the properties of virus, neutralization is detected
by
-Protection from disease,
- Absence of lethality,
- Absence of haemagglutination,
- Plaque formation etc
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