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.