A Quantitative Study of a Technique of Double Diffusion in Agar

Summary A technique of double diffusion is described. Warm liquid agar is layered onto serum in tubes and after the agar has cooled and solidified antigen is added. Tubes approximately 1.7 mm inside diameter are used, making it possible to use quantities as small as 0.01 ml. Egg albumin in concentrations as low as approximately 0.0001% could be detected in this way. A theoretical treatment of double diffusion is given which predicts the relation between band position, antigen and antibody concentration, antigen and antibody diffusion coefficients, time, and agar length. Experimental results are compared with these predictions and found to agree in most respects. Zones of precipitation, however, failed to move as rapidly as predicted. In particular, it was shown that band position is linearly related to the logarithm of the ratio of the antigen to antibody concentrations. Bovine gamma globulin and azo-substituted bovine gamma globulin were used to show the interaction between immunologically related proteins in double diffusion. Non-specific effects and the identification of bands were considered.