Antigen-Antibody Reactions in Agar

Abstract

Summary A micromodification of the agar gel precipitin reaction of Oudin is presented; the method uses the optical densities of antigen-antibody precipitate bands formed in reaction tubes for determining the antibody concentration of rabbit antiprotein antisera in absolute weight units, mg N/ml. When microreaction tubes were used, it was possible to obtain standard O.D. vs. antiserum concentration curves (1.56% to 12.5% antiserum) which required only 0.4 ml of antiserum and less than 0.2 ml of 1% antigen in agar. Optical density values could be obtained at a single antiserum concentration (6.25%) through the use of 0.05 ml of antisera and less than 0.02 ml of 1% protein antigen in agar. These values were used to obtain total precipitate nitrogen values from a standard curve relating O.D. to total precipitate nitrogen at equivalence in liquid media. Total nitrogen values were converted to antibody nitrogen values by substituting them in an equation which expresses antibody nitrogen as a function of total nitrogen and the ratio of antibody to antigen at equivalence. This method was used to monitor weekly the production of antisera to human serum albumin in 12 rabbits. Optical density measurements made at single antiserum concentrations were used to predict the final antibody content of two pools of antisera. The mieromodification of the agar gel precipitin reaction offers considerable savings in time and materials, since a complete analysis can be obtained from O.D. measurements at a single antiserum concentration, using a minimum of antigen, antiserum and other reagents after only 2 days of incubation at 25°C.