A Comparison of Seven Procedures to Detect the Primary Binding of Antigen by Antibody

Abstract

Summary Seven procedures to detect and measure humoral antibody that depend entirely on the primary interaction between antigen and antibody were applied to the same human and rabbit antisera. Two tests, the precipitation of soluble antigen-antibody complexes by 50% saturated ammonium sulfate and by heterologous anti-γ globulin provided quantitative data with respect to the amount of bovine serum albumin bound by an antiserum. The percentage of antigen bound by antibody as determined by either method was remarkably similar at various serum dilutions and at two antigen concentrations. Early primary rabbit antisera bound slightly less antigen when anti-γ globulin data were compared to the ammonium sulfate data. Sera were also studied for the effect of antigen dilution on a serum's binding capacity and the results obtained by precipitating antigen-antibody complexes with either ammonium sulfate or anti-γ globulin were again comparable. These data confirmed that the precipitation of antigen-antibody complexes with heterologous anti-γ globulin is a highly sensitive and reliable procedure especially if sera are from hyperimmunized subjects. Two other quantitative tests that depend on the separation of antigen-antibody complexes from free antigen by ultracentrifugation or by electrophoresis in acrylamide gel were employed. Although data were based on objective determinations of radioactive antigen, results could be expressed only in terms of a serum dilution endpoint. The remaining three tests, radio-immunoelectrophoresis, radio-immunodiffusion and radio-gel-electrophoresis were qualitative radio-autographic procedures. Radio-gel-electrophoresis depends on the natural migratory characteristics of different serum proteins on slides covered with agar. The results of these three tests were also expressed in terms of a serum dilution endpoint but depend on subjective visualization on sensitive x-ray film of radioactive antigen associated with antibody. The procedures in which electrophoresis was involved appeared to be less sensitive than the other primary methods. Within the limits of comparison, the five other primary tests were remarkably similar to each other in sensitivity. Data derived from the P-80 test which measures spontaneous precipitation of antigen-antibody complexes, a secondary manifestation of a primary reaction, were compared with data from the primary tests. The antigen-precipitating efficiencies of the antisera were estimated and expressed as the ratio of the precipitating capacity of a given antiserum determined by the P-80 test, to the serum's binding capacity as determined by a quantitative antigen-binding test. As in previous studies, antibodies in human sera were sometimes not detected except by a primary binding test. Accordingly, it is suggested that if the interpretation of an experiment is dependent upon the presence or absence of detectable antibody, the protocol should include at least one primary test.