I052 ISOLATION OF SUBSTANCES WITH IMMUNE PROPERTIES 



peatedly with 7V/i,ooo sulphuric acid, the extracts are pooled, flocculated by neutralization, 

 and the suspension centrifugated. The flocculation is quantitative as hemolysin is almost 

 insoluble in pure water. The precipitated material is extracted with physiological salt solu- 

 tion and solutions of any desired hemolysin content may be obtained by varying the 

 amount of salt solution used. 



The protein content, per unit of hemolysin, of the most avid anti-sheep erythro- 

 cyte whole serum purified by this method was approximately 0.07 mg. The protein 

 content, per unit of hemolysin, in the final, purified extract, was approximately 

 0.000044 rng-/ representing a purification of 150,000 per cent. The average de- 

 gree of purification obtained with less avid serums was approximately 10,000 per 

 cent. The minimum degree of purification, obtained with a very poorly avid serum 

 prepared by a procedure of prolonged intraperitoneal immunization, was about 5,ood 

 per cent. It was apparent that the character of the purified preparations yielded by 

 the method was largely dependent upon the character of the immune sera from which 

 they were derived. 



The character of an immune serum is largely dependent upon the procedure of its 

 preparation.^ Individual, homologous, immune sera differ widely in their titre or 

 immune-substance content, their avidity or combining afhnity for specific antigen, 

 their specificity, stability, and their content of associated and of partly denatured 

 immune substances. 



A formed antigen such as the stroma of the sheep erythrocyte rarely elicits the 

 production of a single type of immune substance. A variable concentration of he- 

 magglutinin usually accompanies the hemolysins formed, reaching its maximum late 

 in the immunity period when the hemolysin titre is retrogressing from its maximum 

 value.3 Rabbits which have been used repeatedly for hemolysin production not only 

 produce sera of inferior hemolysin titre and abnormally high hemagglutinin titre, 

 but these sera may contain a large concentration of "degenerate" or partially de- 

 natured immune substances. The hemolysins of a serum prepared as the result of a 

 series of gradually increased injections of sheep cells are frequently less than 15 per 

 cent adsorbed by a i per cent suspension of that antigen, while the hemolysins of a 

 serum prepared as the result of a single injection occasionally may be more than 90 

 per cent adsorbed. The latter serum is said to have a greater avidity, per unit of 

 hemolysin content, than the former. If the two sera are aged or heated at identical 

 temperatures (below 60° C.) for identical periods, the latter undergoes a greater 

 loss in titre and combining capacity than the former, and when the respective ca- 

 pacities of the two sera to combine with heterologous antigens are compared, the 

 latter is found to be the less specific. 



The different immune fractions in a hemolysin serum may be separated by suc- 

 cessive extractions with small amounts of specific antigen. The most avid, the least 

 stable, and the least specific fractions are adsorbed first, and the least avid, the most 

 stable, and the most specific fractions resist adsorption to the very last. It is apparent 



'Locke, A., Main, E. R., and Hirsch, E. F.: /. Infect. Dis., 39. 126. 1926. 



» Locke, A., Main, E. R., and Miller, F. A.: ibid., 41, 32. 1927. 



i Okell, C. C, and Parish, H. J.: Brit. J. Exper. Path., s, 355- 1924- 



