Abstract
A technique combining thin-layer gel filtration with single radial immuno-diffusion (SRID) is described, which was designed for characterization according to molecular size and for quantitation of proteins in biological fluids. Based on methodological experiments it is recommended to use boiled and autoclaved Sephadex G-200 superfine at about 3.2% in tris-HCl buffer, a plate-slope of 7° and 3 cm hydrostatic pressure between the buffer reservoirs. With these conditions proteins could be characterized in regard to migration rate as compared to a human serum reference. It was possible to separate fragments, monomers and polymers of albumin as well as 7S IgG and 7S and 11S IgA. Visualization of gel-filtered material was effected by overlaying agarose slabs containing antiserum monospecific for the protein to be analyzed. SRID reactions occurred and the halo precipitates were measured. Albumin, transferrin, immunoglobulins A and G in human serum as well as 7S and US IgA in human serum-colostrum mixtures could be quantitated. Quantitation of samples from 0.25 to 8.0 <i>μ</i>l in size, each containing 20 <i>μ</i>g of albumin, gave an error of the method less than ± 5 %.
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