Abstract
Experiments with precipitates and supernatant fluids, obtained by bringing antibody-containing extracts of sensitized bacteria to a known range of hydrogen ion concentrations, revealed another method of purifying such antibody solutions of extraneous material, which method is different from that described in the preceding paper. It was found that at hydrogen ion concentrations between pH 7.0 and 7.6, that is, well to the alkaline side of the point of precipitation of antibody itself (about pH 4.0), pneumococcus protective antibody remained in solution, but a large amount of indifferent material, containing considerable nitrogen, was removed from solution. It was possible to remove a fraction of this indifferent material before adding copper chloride; and then, after addition of copper chloride, to remove a further fraction, as illustrated in the following protocol.
Two and a half liters pneumococcus antibody solution were brought to pH 7.0 by the addition of hydrochloric acid. A precipitate formed. This was allowed to stand in the ice box over night, and centrifuged. The supernatant was designated “M-20 C.” Trial precipitations of 10 cc. portions of this supernatant by addition of copper chloride indicated complete recovery of the antibody in the supernatant fluid, after removal of the precipitate formed at pH 7.2, with the copper chloride in final concentration of M/2100. Two liters of “M-20 C.” were then treated with 100 cc. of M/100 copper chloride and brought to pH 7.2. A pale green precipitate formed. It was allowed to stand over night in the ice box and then centrifuged. The original material, “isoelectric supernatant” (i. e., supernatant after removal of the first precipitate) and “copper supernatant” were tested on mice. Each mouse received 0.5 cc. of a 1/200 dilution of 18-24 hour pneumococcus Type 1 culture, (i. e., 0.0025 cc. of culture) and 0.5 cc. of the protective material diluted as indicated.
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