Practical Immunology And Serology Antigen and Antibody reactions

1. Practical Immunology
And Serology
Antigen and Antibody
reactions
By
Ahmed Riyadh Abdul Rahman
Al-Noor University College

2. Antigen and Antibody reactions
Precipitation Reactions
 Precipitations involve combination of soluble antigen with soluble antibody to produce
insoluble complexes that are visible (Principle).
 A visible antigen-antibody complex is called a precipitin.
 Precipitin occur only in the zone of equivalence.

3. Prozone: Ab excess, precipitate
dose not form (soluble immune
complexes).
Zone of
equivalence
Optimal ratio of
Ag/Ab insoluble
precipitate
Post zone: excess of Ag,
precipitate dose not form
(soluble immune complexes).

4. Example of Precipitation Reactions
1.Immunodiffution: Ab and Ag react in agar to make precipitin and
or ring, eg:
a.Ouchterlony (double diffusion)
b.Radialimmunodiffusion (single diffusion).
2-Immunoelectrophoresis (IEP): complex mixture of Ags
separated, then reacted with the Antibodies, eg:
a.Counterimmunoelectrophoresis (CIEP).
b.Rocket Electroimmunodiffusion(RID).

5. Ouchterlony (double diffusion)
• A precipitation technique in agar, usually accomplished in a Petri dish or glass
slides in which antigen and antibody are allowed to diffuse against each other and
permit the formation of a precipitin line between the sample wells.
Principle:
• Antibody dilutions and specific soluble antigens are placed in adjacent wells. (If
the well size and shape, distance between wells, temperature, and incubation time are
optimal), these solutions diffuse out, bind to each other, cross-link, and form a visible
precipitate line at the point of equivalence between the wells. The precipitation bands
will be compared with a standard antigen.

6. The effect of relative
molecular size of an
antigen on the shape of
the precipitin lines
formed in double
immunodiffusion
(Ouchterlony technique)

7. If two or more sample (antigen) wells are diffused against a single antiserum
(antibody) well, the following distinctions can be made:
•
• Lines of identity: a single precipitin line indicating uniformity and
identity of the antigens in the sample wells.
• Lines of nonidentity: two distinct and crossing precipitin lines indicating
no cross-reactivity or identity between the antigens in the two sample wells.
• Lines of partial identity: a spur formation, indicating that some of the
antigenic determinants (epitopes) are shared between the antigens in the two
sample wells.

9. • Objective:
1. Detect, identify, and quantify Ab and Ags.
2. Determine the relationship between antigen and antibodies.
3. To compare antigens.
4. For disease diagnosis for example, Antibodies associated with autoimmune
disorders such as rheumatoid arthritis and systemic lupus erythematosus
can be identified by double diffusion.
5. In general, double diffusion assays are useful for determining the presence
or absence of a given antigen or antibody in any kind of biological fluid.

10. PROCEDURE
 Prepare 25 ml of 1.2% agarose (0.3 g /25 ml) in 1X assay buffer by boiling to dissolve
the agarose completely.
 Wipe the glass plate with alcohol to make it grease free for even spreading of agarose.
 Cool the solution to 55-60°C and pour 4 ml/slide on to grease free glass slide placed
on a horizontal surface. Allow the gel to set for 30 minutes.
 Punch wells by keeping the glass plate on the template.
 Fill the lower well with 10µl of antiserum and the upper two wells with 10 µl each of
Antigen 1 and 2
 Keep the glass plate in a moist chamber overnight at 37°C.
 After incubation, observe for opaque precipitin lines between the antigen and antisera
wells.

11. INTERPRETATION OF RESULTS
Observe for the presence of precipitin lines between the antigen and antisera wells.
 If pattern A or ‘pattern of identity’ is observed between the antigens and the
antiserum, it indicates that the antigens are immunologically identical.
 If pattern B or ‘pattern of partial identity’ is observed, it indicates that the antigens
are partially similar or cross-reactive.
 If pattern C or ‘pattern of non-identity’ is observed, it indicates that there is no
cross-reaction between the antigens. i.e., the two antigens are immunologically
unrelated.

12. Fig 2: Glass plate showing pattern of lines obtained following
Ouchterlony double diffusion.

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