Precipitation tests and types

Gaurab Karki — Fri, 01 May 2020 14:05:13 +0000

Precipitation test

When bivalent antibody combines with multivalent soluble antigen, visible precipitation is formed which is indicator of antigen-antibody reaction.
If precipitate remains suspending instead of sedimentation, it is called flocculation test.
In order to occur precipitation Ag-Ab must be in appropriate concentration.
When antibody concentration is too high and antigen concentration is too low, visible precipitate is not found. This inhibition of precipitation by excess antibody is called prozone effect.
On the other hand when antibody concentration is too low and antigen concentration is too high, visible precipitate is not formed.
This inhibition of precipitates by excess antigen is called post-zone effect.
Precipitation occurs only when antigen and antibody are in appropriate concentration (4:1)
Region is the graph where precipitate occurs maximally is called equivalence zone.
Formation of precipitate can be described by lattice hypothesis.
When antigen and antibody are in appropriate concentration, maximum cross linking of antigen by antibody occurs so that visible precipitate is formed.
Either excess antigen or excess antibody prevents extensive cross linking of antigen by antibody so that visible precipitate is not formed.
This is the reasons why the precipitate occur only in equivalence zone but not in prozone and post zone.

This test is carried out on glass slide.
One drop of reagent (either antigen or antibody) is placed on a slide. Then one drop of serum sample is added to it.
Then the slide is rotated to mix the serum and reagent.
If precipitate is formed it indicates a positive test.

A clear solution of sample containing antigen is layered slowly as to a clear antibody solution in a narrow test tube
After certain period a white ring of precipitate appears at the junction of two liquids.

In this method precipitate is carried out in gel.
It is single diffusion test in one direction.
Antibody is incorporated into gel in a test tube.
Sample containing antigen is placed into the test tube.
Antigen diffuses into the gel and form line of precipitate where antigen and antibody met in appropriate concentration.

ii. Okley-fulthorpe variation of Oudin

It is double diffusion test in one direction.
Antibody is incorporated into gel in a test tube.
Above this gel, a plane gel is solidified.
Sample containing antigen is placed above the plane gel.
Both the antigen and antibody diffuse into the plane gel and form visible precipitate where they met in appropriate concentration.

It is single diffusion test in two dimension.
It is quantitative test and is used to determine concentration of antigen or antibody in sample.
Wells are made on agar gel by using template and sample containing antigen is placed into the well.
Specific antibody is incorporated into gel and form visible ring of precipitate around the well where antibody and antigen met in appropriate concentration.
Diameter of ring of precipitate is directly proportional to concentration of antigen in sample.

It is double diffusion test in two dimension.
Wells are cut an agar gel using a template.
Serum containing antibody is placed into central well and different antigens are placed into surrounding well.
Both antigen and antibody diffuse from the well and form visible precipitate.
This technique is used to identify relationship between two antigens.
Three different pattern of precipitation occur in different cases
- If two antigen are similar, line of precipitate fused
- If two antigen are non-identical, line of precipitate crossed
- If two antigen are partially identical, spur formation of precipitate occurs.

This technique is used to detect and isolate particular protein in serum of patient.
For example, to detect cancerous mixture of proteins. At first serum containing mixture of protein is placed in well on gel. Then proteins present in serum are separated by electrophoresis.
After electrophoresis protein present in serum form distinct band. Then a parallel trough is cut in gel and solution of specific antibody is placed in it.
A precipitate band is formed where antigen and antibody met in appropriate concentration.

It is a quantitative test and is used to measure concentration of antigen or antibody in sample.
Antibody is incorporated in gel and sample containing antigen is placed in well. Then electrophoresis is carried out.
Antigen and antibody combine to from rocket shaped precipitate band where length is directly proportional to concentration of antigen.

Agar gel is taken and two wells are made at opposite end of gel.
Sample containing antigen is placed in one well and specific antibody is placed in another well.
During electrophoresis negatively charged antigen migrate toward anode and positively charged antibody migrate towards cathode.
Antigen and antibody combine to form line of precipitate where they met in appropriate concentration.
This technique is rapid and more sensitive than simple immune-diffusion method.
pH of gel should be 8.6 at which antibodies have positively charged and antigen have negative charge

to identify microbes (bacterial cells)
to detect antigen in serum or other sample
to detect antibody in serum or other sample
to detect toxin or anti-toxin
to detect food adulteration
It can be used to measure concentration of antigen or antibody. Eg by radial immuno-diffusion