Amino Acids, Peptides and Proteins - Devényi T., Gergely J. 1976

Methods of Immunochemical Analysis
Protein Analysis by Precipitation Reaction
Determination of the Equivalence Zone in the Antigen-Antibody Reaction

Principle of the method. The procedure determines the amounts of antigen and antiserum that react completely during the precipitation reaction after mixing, so that the supernatant contains neither antigen nor Antibodies after the precipitate is separated.

Scope of application. Preliminary Determination of the equivalence zone is required for performing quantitative precipitation assays (see p. 125) and Immunoelectrophoresis (see p. 137).

PROCEDURE

Prepare a series of twofold dilutions of an antigen solution of known concentration (see p. 121). Mix 0.1 ml of each antigen dilution with 0.5 ml of antiserum. Depending on its activity, the antiserum can be used either undiluted or at a 1:2 or 1:4 dilution. Incubate the mixture for 60 min at 37°C and leave overnight in the refrigerator. Sediment the resulting precipitate by centrifugation at 1500 rpm for 10 min, and determine the remaining antigen or antibodies in the supernatant as described on p. 120. The sample in which the supernatant contains neither antigen nor antibodies identifies the equivalence zone.

For a more precise determination of the equivalence zone, the supernatant from the tubes adjacent to the one indicating the equivalence zone should be further analyzed using intermediate dilutions of the antigen.