Kenneth Goodner

Ultrafiltration of Type I Antipneumococcal Sera

Elford, Grabar and Fischer 1 have reported that the “antibody activity” of antipneumococcal horse-serum is associated with the “larger complex protein in the serum.” Thus it was estimated by the method of optimal proportions that 75% of the antibody passed a 140mμ membrane, rather less than one percent passed an 80mμ membrane, while the filtrate from a 54mμ membrane showed no trace of antibody-activity. The membranes used in the present experiments were prepared by the method of Elford 2 with certain modifications described by Bauer and Hughes. 3 For filtration, Type I antipneumococcal horse-and rabbit-sera were diluted 1:5 in broth. Concentrated antipneumococcal horse-serum was diluted 1:10 in the same reagent. For evaluating the amount of antibody in the filtrate the quantitative precipitation method of Heidelberger, Sia, and Kendall 4 was used. Because of the difficulties in securing large amounts of filtrates complete analyses over a wide range of amounts of polysaccharide were impossible. Therefore an arbitrary amount (0.2 mg. per cc. of original serum) of the Type I acetyl polysaccharide∗ was employed for each filtrate. Determinations of nitrogen in the washed precipitates were carried out by the gasometric micro-Kjeldahl method of Van Slyke. 5 The end-points in filtration of the specific antibodies of Type I antipneumococcal horse- and rabbit-sera are shown in Fig. 1. The results are plotted in terms of the percentage of total specifically precipitable nitrogen recovered in the various filtrates against average pore-diameters. With antipneumococcal rabbit-serum no specifically precipitable protein passed through a membrane with average pore-diameter of 10.6mμ. A 13.8mμ filtrate contained 11.9% of the total specifically precipitable protein of the serum. Slightly greater amounts were then recovered as the pore-sizes were increased up to 73.0mμ. At this point the curve rose sharply until at 102.5mμ the filtrate contained 86.6% of the total amount of antibody.

The Purpuric Reaction Produced in Animals by Derivatives of the Pneumococcus

Julianelle and Reimann 1 have described the purpura that develops in white mice following the intraäbdominal injection of pneumococcal autolysates. This reaction is most evident in those portions of the skin on which hair is either scanty or absent. They concluded that some autolytic derivative of the bacterial cells damaged the endothelium of the capillaries in these areas. The experiments of Julianelle and Reimann have been largely repeated and extended. One of the most striking features of the reaction is that the purpura produced is seldom evenly distributed over the ears, feet, tail, snout, and genitalia. Even on the ears the distribution may be splotchy while along the tail the reaction is always most irregular, certain points showing a very intense reaction while others may be completely negative. These irregularities as well as the anatomical distribution of purpura suggested that some factor such as a mild trauma might be involved in the localization of the reaction. As a test of this hypothesis several experiments have been carried out of which 2 typical examples may be cited. Experiment 1. An area of one sq. cm. of the abdominal skin of a mouse was carefully shaved. Immediately thereafter 0.5 cc. of an autolysate of rough pneumococci prepared by the method of Julianelle and Reimann was injected intraäbdominally at a point removed from the shaved area. At 24 hours the shaved lesion was markedly purpuric but the removal of hair from neighboring areas showed these to be free of purpura. A second injection of the autolysate was then given. Twenty-four hours thereafter the freshly shaven areas were purpuric but further removal of hair showed that the adjacent skin was completely clear. It seems reasonable to conclude that the irritation associated with the shaving determined the localization of the purpura.

Relation of the Phospholipins to the Reactivity of Antipneumococcus Sera

Hardy and Gardiners method 1 for the removal of lipoids from sera is based upon the fact that extraction of protein solutions by alcohol or alcohol-ether at low temperatures does not cause denaturation. Using this method, Hartley 2 has shown that the removal of lipoids from certain antisera apparently abolishes their in vitro reactivity. Felton 3 has extracted antipneumococcus horse serum in a similar manner, and has demonstrated that the removal of lipoids does not diminish protective action. These findings have been confirmed for Type I antipneumococcus horse serum. Extracted sera fail to agglutinate homologous type pneumococci and to give a precipitate with the specific capsular polysaccharide. In vivo, however, they show the presence of protective antibody in unaltered concentration. Lipoid extraction was carried out in the following manner: The antiserum was introduced, with stirring, into 10 volumes of absolute alcohol at —10°C. After 6 hours′ extraction the precipitate was collected by centrifugation at a temperature below —2°C., and again extracted with an amount of chilled absolute alcohol equal to that first used. After 12 hours the precipitate was collected in the same manner, and was then extracted with anhydrous ether for 10 hours at —10°C. and for a second time with anhydrous ether for 10 hours at room temperature. The precipitate was then collected and freed of ether by vacuum distillation, and finally was dissolved in an amount of saline equal to the original serum volume. The resulting solution does not differ in appearance from untreated serum. Although failing entirely in vitro, the extracted antipneumococcus horse serum brought about agglutination in vivo as well as unextracted serum. As a consequence of this observation extracted immune horse serum was injected intraperitoneally into mice and the peritoneal fluid was withdrawn after 30 minutes.