Determination of optimum amount of antigen in complement fixation tests

Abstract

A study of the quantitative relation between antigen and antibody in complement fixation, suggested a simple procedure for determining the optimum amount of antigen in these tests. Wassermann, protein and bacterial antigens were employed with their specific antisera. It was observed in the case of the Wassermann antigens (alcoholic, cholesterinized and Noguchi), that each one appears to possess an optimum concentration for binding complement with positive sera. This concentration could be determined only with weak positive sera, preferably those giving + and ++ reactions. The stronger positive sera do not seem to be markedly affected by the quantity of antigen employed.

The procedure consists in first determining the antigenic unit, or smallest quantity which gives complete fixation with some positive serum. A weekly positive serum is then pipetted into a series of 10 tubes, employing the same quantity used in the regular tests. The first tube then gets 1/4 unit antigen; the second, 1/2 unit; the third, 1 unit, etc.; the last tube getting about 10 units of this ingredient. After the fixation period, it will be observed, on adding sensitized cells, that certain tubes—not necessarily those containing the largest amount of antigen—will show the maximum amount of fixation. The number of antigenic units contained in these tubes being known, therefore, that number showing the maximum fixation, represents the optimum amount of antigen to be used in the daily tests. Necessarily this titration is to be repeated with 4 or 5 different sera.

In the case of the protein (edestin and phaseolin) and bacterial (B. abortus and B. mallei) antigen-antibody complexes, it was observed, after obtaining the antigenic unit, that increasing the number of units within the limitations of the complement fixation test, did not affect the strength of the reaction.