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

Methods of Immunochemical Analysis
Analysis of Proteins by Gel Diffusion Methods
Comparative Protein Analysis by the Osserman Method

Principle of the method. If, after Immunoelectrophoresis of the protein mixture under study, a solution of a known protein is introduced into one of the parallel troughs of the Agar plate, and a specific antiserum to this protein is added to the other, a longitudinal precipitation line forms where the diffusing Antigens and Antibodies meet. If the protein mixture under study contains a component identical to the known protein, the precipitation line corresponding to this component will merge at its ends with the longitudinal line, demonstrating the Ouchterlony phenomenon of identity (see p. 131).

Applications. If the researcher has access to known Proteins (reference proteins) and their corresponding antisera, this method can be used to identify individual components of a protein mixture of unknown composition.

Comparative protein analysis by the Osserman method uses the same equipment and accessories as immunoelectrophoresis (see p. 137).

PROCEDURE

1. Preparation of the agar plate: see p. 142.

2. A well for the sample under study is cut in the center of the agar plate, and two parallel troughs are made on either side of the well, at a distance of 3 mm from it.

3. After immunoelectrophoresis of the protein mixture under study (see p. 141), a solution of the reference protein is introduced into one trough, and the immune serum against it is poured into the other (in Fig. 30, this is the upper trough). Precipitation lines form where the components of this system, diffusing in the agar gel, meet. If the protein mixture under study shares common antigens with the reference protein solution, the longitudinal precipitation line merges with the lines previously formed during immunoelectrophoresis, appearing as an extension of them (Fig. 30).

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Fig. 30. Comparative protein analysis by the Osserman method [16] (for explanation, see text).

NOTES

For the successful Analysis of Protein solutions of unknown composition, the following Methods can be used: 1) immunoelectrophoresis, in cases where the precipitation lines obtained with the immune serum can be identified (Fig. 31, A); 2) the Osserman method, in cases where the researcher has reference protein solutions and the corresponding antiserum (Fig. 31, C); 3) the so-called depletion method (Fig. 31, B), which briefly consists of the following.

The protein solution of unknown composition under study is incubated with a specific immune serum; the resulting precipitate is removed by centrifugation, and the supernatant is used as the "depleted" immune serum in immunoelectrophoresis of a known protein mixture. In the resulting immunoelectrophoregram, precisely those components that are shared by the protein solution under study and the known protein mixture will be absent, as they have removed the corresponding antibodies from the immune serum.