Some observations on the reaction between horse serum and a pool of rabbit antihorse serum. II. An analysis of the antigens concerned in the production of multiple zones

Abstract

1. The reaction horse serum and a pool of rabbit antihorse serum exhibiting four α procedure optima has been investigated with a view to analysing the antigens involved in the production of the multiple zones.2. The following conclusions have been reached regarding the antigenic constitution of horse serum, a horse serum-albumin preparation and a horse serum-globulin preparation: Horse serum consists of at least five antigens. The horse serum-albumin preparation contains three antigens, two of which are probably impurities, one of these being a globulin. The horse serum-globulin preparation consists of three antigens and evidence was obtained of a fourth. Electrophoretic analysis of these protein preparations gave concordant result.3. Multiple zones of rapid particulation in the precipitation reaction are due to the independent activity of multiple antigens and their homologous antibodies.4. The absorption of precipitin from a mixture of antibodies by a mixture of antigens at a single α procedure optimum is both a useful and a practicable procedures.5. Attention is drawn to the fact that the method of antigenic analysis used in the present series of experiments, involving the correlation of α; procedure optima occurring in the parallel titrations of a single antiserum against a mixed antigen and equivalent concentrations of physically or chemically prepared fractions of it supplemented by absorption of precipitins at individual and multiple α precedure optima and retitration of the absorbed sera, could be applied to the identification and nomenclature of proteins occurring in natural mixtures, and also to the quantitative and qualitative appraisal of the purity of proteins prepared by physical or chemical means. The method does not rely on the preparation of highly purified protein samples containing a single antigen.

The formation of precipitates on mixing anti-horse sera

“…When anti-horse sera are titrated against preparations of horse-serum albumin and horse-serum globulin, multiple zones are also frequently observed (Taylor et al 1932;Naylor, 1950), indicating that preparations of horse-serum derivatives contain a number of antigens. * Crystalbumin, a highly purified derivative of horse serum, shows two zones of reaction with the majority of anti-horse sera when titrated by the method of optimal proportions.…”

“…Therefore the main zone of the reaction, occurring with far lower concentrations of the mixed antigen than the secondary zone, probably represents a reaction involving the major antigenic component of the crystalbumin preparation. Naylor (1950), working with preparations of horse-serum albumin and horse-serum crystalbumin of the same protein content and a pool of rabbit anti-horse sera, showed that * Since the nomenclature of the proteins present in animal sera is capable of misinterpretation, definition of the terms used is necessary . In this paper the term albumin refers to a protein constituent of horse serum which, though not necessarily homogeneous, probably consists of very similar molecules and is considered to behave as a single antigen.…”

“…Naylor (1950) notes that horse-serum crystalbumin prepared by the method of Hewitt contains little impurity as shown by the wide separation of the two zones of reaction obtained with a pool of rabbit antihorse sera. All the sera and fractions were stored frozen at -20°C.…”

“…If an antiserum, which contains antibodies to many antigens, is absorbed by a mixture of antigens, which exhibits multiple zones, in the proportions of the optimum occurring with the lowest concentration of the mixed antigen, the antibody responsible for this zone is completely removed without the complete removal of other antibodies (Naylor, 1950). The optimal ratio of serum B0988 to 2 9 % horse-serum crystalbumin, in the main zone, was 1:50, so serum B 0988 was absorbed in these proportions with crystalbumin.…”