CRYSTALLINE PNEUMOCOCCUS ANTIBODY

Abstract

The immune precipitate formed by antipneumococcus horse serum and the specific polysaccharide is not hydrolyzed by trypsin as is the diphtheria toxin-antitoxin complex, and purified pneumococcus antibody cannot be isolated by the method used for the isolation and crystallization of diphtheria antitoxin. Type I pneumococcus antibody, completely precipitable by Type I polysaccharide, may be obtained from immune horse serum globulin by precipitation of the inert proteins with acid K phthalate. The antibody obtained in this way may be fractionated by precipitation with (NH4)2SO4 into 3 main parts. Fraction 1 is insoluble in neutral salts but soluble from pH 4.5 to 3 and from pH 9.5 to 10.5. This is the largest fraction. A 2d fraction is soluble in 0.05 to 0.2 saturated (NH4)2SO and the 3d fraction is soluble in 0.2 saturated (NH4)2SO4 and precipitated by 0.35 saturated (NH4)2SO4. The 2d fraction can be further separated by precipitation with 0.7 saturated (NH4)2SO4 to yield a small amt. of protein which is soluble in 0.17 saturated (NH4)2SO4 but insoluble in 0.25 saturated (NH4)2SO4. This fraction crystallizes in poorly formed, rounded rosettes. The crystallization does not improve the purity of the antibody and is accompanied by the formation of an insoluble protein as in the case of diphtheria antitoxin. None of the fractions obtained is even approx. homogeneous as detd. by solubility measurements. Purified antibody was also obtained by dissociating the antigen-antibody complex. The protective value of the fractions is quite different, that of the dissociated antibody being the highest and that of the insoluble fraction, the lowest. All the fractions are immunologically specific since they do not precipitate with Type II polysaccharide nor protect against Type II pneumococci. All the fractions give a positive precipitin reaction with antihorse rabbit serum. The dissociated antibody gives the least reaction. Comparison of the various fractions, either by their solubility in salt solns. or through immunological reactions, indicates that there are a large number of proteins present in immune horse serum, all of which precipitate with the specific polysaccharide but which have very different protective values, different reactions with antihorse rabbit serum and different solubility in salt solns.