In the preceding report (1), it was shown that precipitating antibodies to streptolysin O concentrate were quite common in human sara, using the agar technic of Oudin (2). Although some sara showed several bands with this antigen, strong evidence was presented that the streptolysin O system was the most frequent in these tests.With rabbit anfisera to the streptolysin concentrate, up to four antigens could be found. The identity of the non-streptolysin bands could not be readily determined. Although the concentrates used were found to be contaminated with streptococcal desoxyribonuclease (3), and proteinase precursor (4), it was shown that the extra bands in the rabbit sara were apparently not due to these systems using the Oudin methods. The preparations were found to be essentially free of some of the other well characterized antigens or toxins produced by these organisms.Because of the inherent advantage and simplicity of the technic described by Ouchterlony (5-7), the above findings were further explored with this method. Special attention could thus be directed toward the attempted identification of the non-anfistreptolysin O systems found in individual human sara. Extensive study was made of sera from rheumatic fever patients, and these observations comprise a large portion of the following report. Because of the suprising multiplicity of antigen-antibody systems found, a few tests were made with Oakley's agar precipifin technic (8) for confirmation of this complexity.The application of agar precipitin technics for the study of human antibodies has thus far been only fragmentary. One test has been reported by Schiott (9), and four by Wodehouse (10). Both investigators were studying human allergies.

The antistreptolysin O activity of human sera has been under investigation for many yea~ (1-3). It has been generally assumed that this activity is due to antibody because of the changes in its titer that occur during disease and because antisera of this sort are so readily produced in animals by irnmlmi~tion procedures. High antistreptolysin O activity of human gamma globulin also has been reported (4, 5). In addition, the ready absorption of antistreptolysin activity of serum with streptolysin O containing preparations supports this view (6). Although cholesterol is capable of inhibiting streptolysin O in very low concentrations, the failure to affect antistreptolysin activity of some human sera by extraction with fat solvents at low temperatures (4, 6, 7) has also suggested the antibody nature of antistreptolysin O in human sera. However, several non-specific inhibitors are known to occur. In acute hepatitis sera, high levels of antistreptolysin O have been found which were dearly of non-antibody character (6, 7). The antistreptolysin O agent of human pleural exudate fluids has also been shown to be a non-antibody inhibitor (6). Rosendal and Bernheimer have shown that mice given sublethal doses of streptolysin O revealed a non-antibody inhibitor in the circulation (8). Stollerman (9) has recently shown that hypeflipemia in rabbits will result in nonspecific andstreptolysin O activity when the cholesterol-phosphofipid ratios are within a certain range. Robinson (5) has reported the development of non=specific antistreptolysin O activity in rabbits severely ill with Group A streptococcal infection. In addition, it may be recalled that acid or alkali treatment of human sera may result in striking increases in antistreptolysin O, presumably due to the effect on lipoprotein complexes (6). The above findings thus raise the question of the frequency and significance of these non= specific factors in the antistreptolysin O activity of sera from the population at large.

Five lens antigens of rabbits can induce antibodies homologously as evidenced by agar precipitin technics. Cross-reactions of lenses of other species (organ specificity) may be due to similarity of varying numbers of these antigens. Newborn and 26-day embryo rabbit lenses seem to have decreased concentrations of, or lack, one antigen present in adult rabbit lens. Streptolysin "O" concentrates appear to act as an adjuvant for homologous lens immunization in rabbits.

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