3 th Immunity
practical
class
Lab 4
Hani Hassan
Precipitation
Precipitation: assays are performed in semi-solid media such as agar or
agarose where antibodies and antigens can diffuse toward one another
and form a visible line of precipitation.
Precipitation reactions: are based on the interaction of antibodies and
antigens. They are based on two soluble reactants that come together to
make one insoluble product. These reactions depend on the formation of
lattices (cross-links) when antigen and antibody exist in optimal
proportions. Excess of either component reduces lattice formation and
subsequent precipitation. Precipitation reactions differ from agglutination
reactions in the size and solubility of the antigen and sensitivity. Antigens
are soluble molecules and larger in size in precipitation reactions. There
is several precipitation methods applied in clinical laboratory for the
diagnosis of disease. These can be performed in semisolid media such as
agar or agarose.
Precipitation Reaction
Precipitation Curve
● Prozone – antibody excess, many antibodies coat all antigen sitesresults in false negative.
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3 th Immunity
practical
class
Lab 4
Hani Hassan
● Prezone – antigen excess, antibody coats antigen but cannot get lattice
formation, results in false negative.
● Zone of Equivalence – antigen and antibody present in optimal
proportions to bind and give visible reaction.
Precipitation methods:
1- Radial Immunodiffusion (Mancini)
In radial immunodiffusion antibody is incorporated into the agar gel as
it is poured and different dilutions of the antigen are placed in holes
punched into the agar. 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.
● Application:
1-
Measure
the
concentrations
immunoglobulin's in a person’s serum.
2
of
specific
antibodies
or
3 th Immunity
practical
class
Lab 4
Hani Hassan
2- Produce anti-antibodies (antibody) - by inject human antibodies
(antigen) into an individual of another species where they will be
antigenic and because production of antibodies directed against the
human antibodies.
•
Method
–
Ab in gel
– Ag in a well
• Interpretation
– Diameter of ring is
proportional to the
concentration
• Quantitative
– Ig levels
2-
Immunoelectrophoresis
In immunoelectrophoresis, a complex mixture of antigens is placed in
a well punched out of an agar gel and the antigens are electrophoresis
so that the antigen is separated according to their charge. After
electrophoresis, a trough is cut in the gel and antibodies are added. As
the antibodies diffuse into the agar, precipitin lines are produced in the
equivalence zone when an antigen/antibody reaction occurs.
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3 th Immunity
practical
class
Lab 4
Hani Hassan
● Application: Used to evaluate purity of isolated serum proteins.
Countercurrent electrophoresis
In this test the antigen and antibody are placed in wells punched out of
an agar gel and the antigen and antibody are electrophoresis into each
other where they form a precipitation. This test only works if
conditions can be found where the antigen and antibody have opposite
charges. This test is primarily qualitative, although from the thickness
of the band you can get some measure of quantity. Its major advantage
is its speed.
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