Quantitative estimation of serum protein fractions by electroimmunodiffusion method

Abstract

An electroimmunodiffusion method has been developed for the quantitation of immunoglobulin in small samples of dilute biological fluids. It is, however, applicable to more concentrated solution and it requires much less time than the radial diffusion method.For the quantitation of serum IgA the optimal conditions were as follows: Antisera were diluted 30 times with 1% agar solution in veronal buffer at pH 7.6 and ionic strength 0.025. Slide glass, 5.2×7.5cm, was coated with 7.5ml of antibody containing agar gel. Electrophoresis was run at 100v, 30mA for 2 hours with veronal buffer at pH 7.6 and ionic strength 0.05. The measurable range for single determination of IgA was between 26 and 130mg/dl.The method is also applicable to the determination of IgM, IgG, α2-macroglobulin, ceruloplasmin, haptoglobin and β-lipoprotein. Optimal conditions were almost similar to those of IgA determination, but pH was 8.6 and electric current was 15mA. For fractions with high concentration in serum, such as IgG, the antigen should be diluted more than 40 times.The method is rapid, accurate and reproducible, and requires only 3μl of sample. The volume of sample, i. e. 3μl, filled in the well, should be rigorously observed for accurate determinations.