4. Purpose
In-vivo : Basis of
• Antibody mediated immunity
• Tissue injury in hypersensitivity
• Autoimmune diseases
In-vitro : For
• Diagnosis of infections
• Epidemiological surveys
• Identification of infectious agents & non-infectious agents such
as enzymes
• Detection /quantitation of antigens/antibodies
5. General Features
• Reaction is specific
• Entire molecules react ,not fragments
• No denaturation
• Combination occurs at surface
• Combination is firm but reversible
• Both antigens and antibodies participate
• Combination is in varying proportions
6. Precipitation Reaction
When a soluble antigen combines with its
antibody in the presence of electrolytes
(NaCl) at a suitable temperature & pH, the
antigen – antibody complex forms an
insoluble precipitate
• Flocculation – when precipitate remains
suspended as floccules.
7. Mechanism of precipitation
• 1954 : Marrack proposed lattice hypothesis
• Multivalent antigens combine with bivalent
antibodies in varying proportions ,depending
on the antigen –antibody ratio in the reacting
mixture
• Precipitation results when a large lattice is
formed consisting of alternating antigen and
antibody molecules
9. Zone phenomenon
– Prozone – Zone of antibody excess
– Zone of equivalence
– Post zone – Zone of antigen excess.
Prozone
Sera rich in antibody may sometimes give a false
negative precipitation or agglutination result,
unless several dilutions are used.
11. Applications of precipitation
• Ring test
Ascoli’s thermoprecipitation test
Grouping of Streptococci by Lancefield
technique.
• Slide test – VDRL test for syphilis
• Tube test – Kahn test for syphilis
12. Immunodiffusion- Precipitation in
gel
•1% soft Agarose gel used in Immunodiffusion
•Results in formation of visible bands instead of floccules and
can be preserved for longer time
•It can differentiate individual antigens from a mixture , each
antigen forms a separate bands after reacting with specific
antibody
13. Principle
Based on 2 principles
1. Whether only antigen diffuses (single diffusion) or
both Ag and Ab diffuse (Double diffusion)
2. When Ag or Ab diffuses in one dimension (vertical
diffusion when test is done on a tube layered with
gel) or 2 dimension (diffusion in both X and Y axis
which occurs when test is done on a slide or petri
dish layered with gel)
14. Applications of precipitation (cont) –
Immunodiffusion
1.Single diffusion in one dimension
2.Double diffusion in one dimension
3.Single diffusion in two dimensions
4.Double diffusion in two dimensions
5.Immunoelectrophoresis
15. 1. Single diffusion in one dimension
when Ag solution is poured over a layer of
gel containing Ab, only Ag diffuses in one
direction towards Ab to form a band
2. Double Diffusion in one Dimension
(Oakley-Fulthorpe Procedure), Column of
Plain agar is placed between the antigen
layer and layer of gel incorported with Ab, then
Ag-Ab move towards each pother in opposite
directions and precipitate band is formed at the
line they meet in the plain agar
16.
17. 3. Single diffusion in two dimension (Radial Immunodiffusion):-
Gel is incorporated with Ab is placed on a slide and several
wells are cut
when drop of Ag is placed in the wells
they diffuse radially in all directions to meet with Ab and ring
shaped bands are formed around the well
The diameter of the ring is directly proportional to the
concentration of the antigen in the well
4. Double dimension in two dimension (Ouchterlony procedure):-
on a slide poured with agar gel, Ab is placed in the central well
and surrounding wells are filled with Ags
both Ag and AB moves towards each other in all the directions
and forms a band
18. The adjacent precipitate bands interact in 3 ways
1. They may fuse with each other (indicates both antigens are
identical)
2. They may cross one another (indicates antigens are
unrelated)
3. They may cause spur formation (indicates cross reaction or
Partial Identity)
19. Precipitation reaction in Gel in presence of
Electric Field
1. Electro-immunodiffusion –
The development of precipitin lines can be
speeded up electrically driving the antigen &
antibody.
2. Counter-immunoelectrophoresis ( one
dimensional double electroimmunodiffusion)
3. Rocket electrophoresis (one dimensional
single electroimmunodiffusion)
20. Electroimmunodiffusion
When electric current is applied to a slide layered with gel
the serum proteins placed in a well are separated into
individual antigens
antiserum is present in trough moves towards the
antigen components resulting in formation of separate
precipitin lines in 18-24 hours
21. Counter current Immunoelectrophoresis
Takes 30 minutes, Faster
More sensitive because it involves simultaneous electrophoresis of
the Ag and Ab in gel in opposite direction
Uses:- alpha feto protein in serum and Capsular Ags of
Cryptococcus neoformans and Meningococci in CSF.
