Antigen-Antibody Reactions in Agar V Precipitate Density Studies Using the Oudin Technique

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Abstract
Summary and Conclusions: The leading edge of the precipitate band obtained in the simple diffusion tube technique of Oudin has been measured over a wide range of antigen concentrations, antiserum concentrations and diffusion times. Five different antigen-antibody systems, egg albumin, bovine serum albumin, bovine γ-globulin, hog thyroglobulin, and lobster hemocyanin, and their respective rabbit antibodies were employed. All systems showed that at the higher antigen and lower antiserum concentrations the O.D. is independent of the time of diffusion and of the antigen concentration. At the lower antigen and higher antibody concentrations, however, the O.D. increases with increasing time of diffusion and decreasing concentration of antigen. For the egg albumin, bovine serum albumin, bovine γ-globulin and hemocyanin systems, at sufficiently high antigen and low antibody concentrations the relationship between O.D. and antiserum concentration is a straight line extrapolating to the origin. Thyroglobulin under the same circumstances gives a straight line that does not extrapolate to the origin. Under conditions in which the O.D. is independent of the antigen concentration and time of diffusion and directly proportional to the antiserum concentration, there is a linear relation between O.D. of the leading edge of the precipitate band in the Oudin test and the total nitrogen concentration of the precipitate as found by the quantitative precipitin reaction carried out in liquid media. The same linear relationship was found for egg albumin, bovine serum albumin, bovine γ-globulin and hemocyanin, but not for thyroglobulin. No effect on the O.D. of the addition of nonspecific protein to the antiserum layer was observed. Reasons were given for expecting that under the proper conditions there should be no effect of adding nonspecific protein to the upper layer.