Steep Gradients of Inert Substances as Supports for Precipitin Reactions

Abstract

Precipitin reactions were conducted in the wells of micro titer plates and the light “absorbance” at 405nm plotted as a function of the neg log₂ dilutions of the antigens using the Titertek Multiscan apparatus. The procedure followed was to incorporate antibody in 20% sucrose and to diffuse the mixture into serial two-fold dilutions of antigen overlaid on the sugarantibody columns in the wells. Where the antigen met its antibody in equivalence precipitin bands formed. The bands of precipitates remained suspended. The precipitates formed by mixing the antibody sugar mixture with the serial two-fold dilutions of the antigen were also recorded in the Titertek Multiscan apparatus. Tobacco mosaic virus and its IgY type antibody produced simoidal precipitin curves with the layering as well as the mixing techniques. The haemocyanin of the whelk and its IgY antibody yielded a precipitin diagram with the layering technique which gave evidence to three antigens. The three antigens in the haemocyanin was confirmed by two dimentional Laurell-electrophoresis. Semi-purified human colostral dimeric IgA and its IgY antibody produced a precipitin diagram which showed evidence of four of five antigens. With the mixing technique a single precipitin maximum was obtained preceded by a prozone. Rabbit Ig and its IgY antibody gave a precipitin curve with two maxima. The precipitin curve obtained when the reagents were mixed had one peak preceded by a prozone.