Abstract
Since the quantitative precipitin method of Boyden and Baier 1 has proved to be fairly simple, rapid, and reliable, it was thought advisable to investigate further some properties of this reaction itself. The volume of precipitate obtained in the reaction was studied as affected by (1) quantity of antigen, (2) temperature of the reaction mixture, (3) time and rate of centrifugation, and (4) length of incubation.
Only simple protein antigens have been used in this study (crystalline egg albumin). 2 All protein concentrations have been found by making modified Kjeldahls 3 on samples. The antisera were obtained from rabbits by intravenous injections of the antigen. They were bled from the heart 10 days after the last injection. Calibrated instruments only were used in making all dilutions of antigen and antiserum in buffered saline.
The technique in performing the reaction consisted in preliminary titer determinations (ring test) to ascertain the strength of the antiserum. Following this the quantitative nature of the reaction was studied, using van Allen thrombocytocrits as stated 1 with suitable modifications for particular experiments. The experimental results are shown graphically.
The quantitative technique employed has verified some of the conclusions of earlier workers with regard to antigen-antibody equilibrium. Fleishman and Michaelis 4 recognized that the formation of precipitate increases at first with addition of precipitinogen, then decreases and with a certain excess of the antigen approaches zero. Zinsser 5 also noticed the solvent action of excess of antigen. Opie 6 has drawn the same conclusions using crystalline egg albumin as antigen.
Figure 1 shows the definite effect of antigen dilution on precipitate volume. The antiserum was constant.(0.5 cc. of a 1:5 dilution) for all readings, while the antigen was diluted as shown using 0.5 cc. amounts.
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