Abstract
Several differences in the immunological activities of Type I anti-pneumococcal rabbit and horse sera have been reported. 1 However, little is known about the difference, if any, in the manner of union between the polysaccharide and its precipitin in the sera of these two animals. In this report, we shall show that such a difference exists. When the immune precipitates were washed with normal saline solution, and the amounts of polysaccharide present in the combined washings were estimated by the method described in the preceding paper, it was found that practically all the polysaccharide in the immune-rabbit precipitate could be accounted for in the washings. On the other hand, no appreciable amount of polysaccharide could be removed from the immune-horse precipitate.
Twenty cc. of Type I pneumococcal polysaccharide (0.1 mg. per cc.) were added to 20 cc. of homologous horse or rabbit antiserum. The mixture was incubated at 37°C. for half an hour. The super-nate was poured off and was found to contain an excess of antibody. The precipitate was washed with four 50-cc. portions of 0.85% NaCl solution, dissolved in 5 cc. of water and 5 cc. of N/140 NaOH, and the alkaline solution was poured into 400 cc. of 0.85% NaCl solution (designated as P). The polysaccharide-content of the P-fraction and the combined washings (W) was determined by the complement-fixation method. The results are shown in Table I.
In another experiment, the washed immune-rabbit precipitate containing 50 mg. protein was found to contain 0.025 mg. polysaccharide. The precipitate was suspended in 20 cc. of water and dissolved with 5 cc. of N/70 NaOH. The clear alkaline solution was brought to slight turbidity with N/70 HC1 and allowed to stand in ice-water overnight. On the following morning the solution was neutralized, solid NaCl was added to 0.85%, and it was centrifuged. The supernatant fluid which contained 25% of the original protein was found to have a higher titer in agglutination, precipitation with homologous polysaccharide, and mouse-protection, than the combined washings. We have found that the addition of 0.025 mg. polysaccharide to 50 mg. of immunologically pure precipitin 2 caused no visible precipitation.
We have stated elsewhere 2 that the low immunological activity of the antibody-polysaccharide complex is due to some alteration in the antibody-protein itself and not merely to the combination of the antibody with the polysaccharide. If the antibody had not been altered during precipitation, the protein in the washings of immune-horse precipitate which contained no polysaccharide should have the same activity as pure precipitin. But this is not the case. Furthermore, it is difficult to conceive that the immune-rabbit precipitate after losing nearly all its polysaccharide should still retain the original low solubility and low activity of the precipitate. The increase in solubility and immunological activities of the washed precipitate can be brought about not by the removal of the polysaccharide but by treating with alkali under conditions suitable for the reversion of denatured protein.
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