Immunochromatography

Abstract

While the techniques of ion-exchange chromatography, on derivatives of cellulose(l) and dextran gel(2), and gel fitration chromatography (3-7) have proved valuable for the fractionation of serologically active proteins, they have not been used previously in direct combination with immunodiffusion. Recently proteins have been separated on a micro-scale by thin layer chromatography on dextran gels(8-10) and by paper chromatography on anion-exchange cellulose (11). This report describes the combination of chromatography and immunodiffusion on a microscope slide to separate and characterize serologically active compounds in a manner analogous to immunoelectrophoresis (12).

Materials and methods. The cross-linked dextran gels, diethylaminoethyl-Sephadex (DEAE) A-50 medium grade, carboxymethyl-Sephadex (CM) C-50 medium grade and Sephadex G-100 (G-100) 140-400 mesh, were prepared for use according to the manufacturer's instructions.§ In the experiments illustrated in Fig. 1, DEAE was equilibrated with sodium phosphate buffer (pH 5.8, 0.05 M), CM with sodium acetate buffer (pH 5.5, 0.1 M), and G-100 with sodium phosphate buffer (pH 8.0, 0.1 M in 0.5 M sodium chloride). For diffusion from CM and G-100 columns a 2% agar gel (Difco, Bacto-Agar) with 1% sodium azide in veronal-HCl buffer (pH 7.2, 0.2 M) was used, whereas, for diffusion from the DEAE columns phosphate buffer (pH 5.8, 0.1 M in 0.5 M sodium chloride) was necessary. Molten agar (2 ml) was pipetted onto a standard microscope slide which had been previously coated with 0.1% agar. After the agar had solidified a longitudinal halft was removed and a trough 1.4 mm wide × 7.2 cm long was cut 5 mm from the center of the slide in the remainder of the agar. Dextran gel columns of reproducible dimensions were prepard on the vacant half of the slide.