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

Methods of Immunochemical Analysis
Protein Analysis by Precipitation Reaction
Determination of Precipitating Antibody Titer by Antigen Dilution

Principle of the method. When a soluble protein antigen is added to a specific immune serum, a precipitation reaction occurs. The amount of precipitating Antibodies in undiluted or 10-fold diluted antiserum can be characterized by the highest antigen dilution that is still capable of eliciting a precipitation reaction with the given serum; this dilution is referred to as the precipitin titer.

PROCEDURE

1. Preparation of serial antigen dilutions. Depending on the amount of serum available to the researcher, a series of two-fold, four-fold, or ten-fold antigen dilutions is prepared. To prepare a series of two-fold antigen dilutions, 16 Wassermann tubes are placed in a rack; 0.5 ml of undiluted antigen is added to the first tube, and 0.5 ml of a 0.9% NaCl solution is added to all the others. In the second tube, 0.5 ml of undiluted antigen is added, and after mixing, 0.5 ml of the mixture is pipetted into the third tube. After mixing the Contents of the third tube, 0.5 ml is transferred to the fourth, and so on, to the end of the row. From the last tube, 0.5 ml is discarded. This yields a series of antigen dilutions ranging from the initial concentration to a 1 : 32 768 dilution. (By varying the aliquots of the antigen and sodium chloride solutions, dilution series of other factors can be prepared in a similar manner.)

2. Addition of immune serum. Equal volumes of undiluted or 10-fold diluted immune serum are added to each tube containing the diluted antigen solution.

3. Control samples. Control I: 0.5 ml of immune serum is mixed with an equal volume of 0.9% NaCl solution. Control II: 0.5 ml of 0.9% NaCl solution is mixed with an equal volume of antigen solution.

4. Incubation and reading of reaction results. After mixing the solutions, the tubes are incubated at 37°C for 60 min, then transferred to a refrigerator, and the results are read the following day. The precipitin titer is defined as the highest antigen dilution at which a precipitate is still formed with the test immune serum.

NOTES

1. Immune sera prepared for immunochemical Analysis of Proteins must precipitate the corresponding protein Antigens diluted to at least 1 : 10 000 — 50 000.

2. The disadvantage of this method is that relatively large amounts of the test material are required. If the researcher has only small quantities of antigen or antiserum available, the volumes of the system components can be reduced to 0.2 ml, or another method requiring smaller amounts of components can be used.

3. For the successful conduct of certain immunochemical studies, the precipitation reaction must occur in the so-called equivalence zone, i.e., at optimal quantitative ratios of the reacting antigens and antibodies. Using the dilution method described above, this zone can be determined as follows. A series of two-fold dilutions of the immune serum is prepared in a volume of 0.2—0.5 ml, and an equal volume of the antigen solution at the dilution where precipitation still occurs is added to each tube. The tubes are incubated at 37°C for 60 min, left overnight in a refrigerator, and the reaction is evaluated the next day. The highest dilution of the immune serum that still forms a precipitate with the antigen used at its titer is determined. Under these conditions, the precipitation reaction is considered to occur in the equivalence zone.