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Immunochemical Analysis of Human Serum and Its Fractions

II. Qualitative and Quantitative Analysis of the Fraction Soluble in Two–Thirds Saturated Ammonium Sulphate

From the Laboratorie d'Immunochimie Analytique, Institut Pasteur, Paris, France

Abstract

1. The antigenic composition of the fraction of human serum soluble in saturated ammonium sulfate and insoluble in saturated ammonium sulfate has been qualitatively and quantitatively studied. This fraction has been subfractionated in order to lower the competition of the main component toward the others in the immunization.

2. Rabbits have been immunized against a number of fractions and subfractions. The immune sera have been allowed to react with fractions and subfractions, using precipitation in gels (simple diffusion in one dimension). The observed precipitation zones have been identified mainly by a method previously described. This method, based on the quantitative laws of penetration, requires no previous manipulation, e.g., preabsorption or reaction of several antigen solutions in the same medium.

3. Eight distinct antigens have been demonstrated in the fraction studied. The concentration of six of them is larger in this fraction or in its subfractions than in any other fraction of human serum studied. These six antigens, considered to be the major components of the analyzed fraction, which corresponds to the fraction formerly called albumin, have been named A1, A2, A3, A4, A5 and A6. This order is probably not greatly different from the decreasing order of their concentrations in the serum. By suitable immunizations and absorptions, immune sera giving a single reaction could be prepared against seven of the eight listed antigens.

4. The concentrations of five antigens in human serum and in the fractions have been quantitatively studied, using measurements of the penetration of the precipitation zones and the laws of penetration, previously described. Antigen A1 is the main component; it can be prepared in an almost pure state by techniques described long ago by other authors. Antigen A2 is very labile on aging even in the cold and on fractionation. It was not studied quantitatively. Antigens A3, A4, A5 and A6 have been quantitatively studied. Because of the lack of pure preparations of these antigens, their concentrations in the fractions have been expressed only in arbitrary units. Antigen A4 gives precipitation zones which vary somewhat from fraction to fraction. This can be explained by the heterogeneity of this antigen.