Precipitation test
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If a solution of soluble antigen is mixed with a strong antiserum, the mixture becomes cloudy within a few minutes and then flocculent. Finally a precipitate which consists of antigen – antibody complexes settles to the bottom of the tube.
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Antibodies that aggregate soluble antigens are called precipitins.
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For formation of an antigen – antibody lattice or complex (i) antibody must be bivalent (ii) antigen must be either bivalent or polyvalent i.e. it must have at least two copies of the same epitope or have different epitopes that react with different antibodies present in polyclonal antisera.
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If increasing amounts of soluble antigen are mixed with a constant amount of antibody, no obvious precipitate is formed at low antigen concentration. As the amount of antigen increases, larger quantities of precipitate form until the amount are maximal. With further addition of antigen, the amount of precipitate gradually diminishes until none is observed in tubes with excess antigen (Horse antibodies produce a distinct flocculation over a very narrow range of antigen concentration due to IgG (T) subclass).
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Where there is excess antibody, each antigen molecule is covered with antibody, preventing cross linkage and precipitation. When the reactants are in optimal proportion (i.e. in the equivalence zone), the ratio of antigen to antibody is such that cross linking and lattice formation is extensive. As this lattice grows it becomes insoluble and eventually precipitates. In antigen excess, each antibody binds two antigen molecules and further cross linking is impossible, and since these complexes are small and soluble, no precipitation occurs.
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Last modified: Thursday, 26 August 2010, 7:26 AM
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