2- Precipitation Reaction
 A precipitation reaction is a secondary serological reaction, in which
soluble Ag or Ab are in solutions ( not particulate) are mixed
together to form an insoluble compound that settles out of solution as
a solid. That insoluble compound is called a precipitate.
 When Ag and Ab meet specifically at an optimal ratio they will form
the maximum amount of precipitate (equivalence zone).
 When Ag is in excess, the Ab binding site will be blocked and cannot
cross-link, thus preventing precipitation and causing small molecules.
 It can be done using:
1- Tube or Capillary tube precipitin test
2- Precipitation in gels : single radial immunodiffusion RID (Mancini
test)
3- Precipitation in gels: Double immunodiffusion (Ouchterlony test)
4- Immunoelectrophoresis
1-Tube or Capillary tube precipitin test
In this test a clear solution containing the test Ag is carefully layered into a clear
antisera in capillary tube. After incubation, if +ve this will give a visible line of
precipitate between the two layers.
2-Precipitation in gels : single radial immunodiffusion RID
(Mancini test)
In radial immunodiffusion antibody is incorporated into the agar gel ; holes are cut
and test solutions of Ag are placed in them.
After few hours, as the antigen diffuses into the gel, it reacts with the antibody and
when the equivalence point is reached a ring of precipitation is formed around the
wells and gradually grow outwards.
The size of the ring at any time is proportional to the Ag concentration in the well.
Thus by adding standard solutions Ag in some of the wells, this method can be
caliprated and used to determine the conc. Of the same Ag in test solutions.
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Applications:
1- determine of IgG in human serum.
2- determine of C3
3-Precipitation in gels: Double immunodiffusion (Ouchterlony
test)
This method was described by Ouchterlony. In this test, antibody and possible
antigens are placed in wells in agar plates and allowed to diffuse toward one another.
The antibody is placed in a center well and antigens (specific or nonspecific) are
placed in surrounding wells. When an antibody and its specific antigen meet one
another and are at the proper concentrations, the precipitate will form a visible white
line between the two wells. This line is called a precipitin line.
Result:
 There are three characteristic precipitin reactions which may occur in the
Ouchterlony analysis: identity, non-identity, and partial identity.
 The demonstration of these reactions requires that the test wells be positioned
closely enough to allow precipitin lines to fuse or cross.
 When the two antigens are different, yet both react with the antibody, a pattern
of non-identity results. Precipitin lines are seen to cross at their ends.
 A partial identity reaction occurs when the two antigens involved have common
antigenic determinants, but one antigen also has a unique determinant. Antigens
which are closely related chemically often produce a pattern of partial identity
when diffused against antibody homologous to one.
 Indications of partial identity have proven useful for systematists interested in
determining evolutionary relationships between organisms
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