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

Methods of Immunochemical Analysis
Protein Analysis Using Precipitation Reactions
Qualitative Precipitation Reaction

Principle of the method. Mixing a soluble protein antigen with a specific immune serum results in The formation of a precipitate.

Applications. Detection of specific Antibodies and detection of protein Antigens used as immunizing material using a specific immune serum.

PROCEDURE

Depending on the available volume of immune serum, aliquot 0.1–0.5 ml into test tubes. Add an equal volume of antigen solution to each tube: undiluted to the first tube, and serial dilutions (1:2, 1:4, 1:8, 1:16, etc.) to the subsequent ones. After mixing the solutions, incubate the tubes in a Water bath at 37°C for 60 min. If the serum is sufficiently active, clearly visible precipitates will appear at this stage. After incubation, leave the tubes in a refrigerator overnight. When using weak immune sera, precipitates can be detected after centrifugation at 1500 rpm for 10 min.

Control I: to 0.1–0.5 ml of 0.9% NaCl solution, add an equal volume of antigen solution in the same manner as in the experimental sample.

Control II: to 0.1–0.5 ml of immune serum, add 0.9% NaCl solution in the same volume as the antigen added in the experimental sample.

NOTES

1. The reaction is considered positive if a precipitate forms in the tube. In the case of a known immune serum, this indicates the presence of the corresponding protein antigen; in the case of a known protein antigen, it indicates the presence of specific antibodies in the analyzed antiserum.

2. The reaction is considered negative if no precipitate is formed. This may mean that: 1) the antigen specific to the antiserum used is absent in the test system; 2) antibodies to this antigen are absent in the antiserum; 3) there is an excess of antigen in the system, which dissolves the precipitate. To avoid a false-negative evaluation due to precipitate dissolution in an excess of specific antigen, serial dilutions of the antigen solution should be added to the same volume of antiserum, or its amount should be gradually decreased.

3. In principle, for a visible precipitate to form, it is sufficient for 0.1–0.5 mg of antibody nitrogen to participate in the reaction. With a smaller amount of antibodies, the precipitate can still be detected by centrifuging the reaction mixture. However, if The amount of antibody nitrogen is less than 0.01 mg, the precipitation reaction does not occur.

4. If a limited amount of immune serum is available, the precipitation reaction can be performed as follows. Aliquot 0.1–0.5 ml of this serum into a centrifuge tube, then add 0.01 ml of antigen solution, mix, and leave for 30 min at 37°C. If no precipitate forms, add another 0.03 ml of antigen solution to the reaction mixture and continue incubation for another 30 min. While waiting for the precipitate to appear, this procedure can be repeated further by adding increasing amounts of antigen (0.1, 0.3, 1.0 ml, etc.).

5. By centrifuging the reaction mixture in a positive precipitation reaction, the formed precipitate can be removed to determine which of the two system components, antigen or antibody, remained in the supernatant. This allows identifying which component was in excess during the reaction. To do this, half of the supernatant is mixed with an equal volume of immune serum, and increasing amounts of antigen solution are added to the other half (see the previous section). Precipitate formation in the first case indicates an excess of antigen, while In the second case, it indicates an excess of antibodies. In this manner, the optimal antigen-antibody ratio for a positive precipitation reaction can be determined.