Studies on purification of antibodies. II.

Abstract

This paper is the continuation of one delivered before this Society 1 describing the metal precipitation and purification of typhoid agglutinating extract. The extract is prepared by sensitizing typhoid bacilli with antityphoid horse serum, washing the complex with saline to remove horse serum and subsequently dissociating agglutinin from bacteria by means of weak alkali.

In that paper it was stated that a m/200 final concentration of CuCl₂ was used to precipitate agglutinin from the extract. We have found that at the proper hydrogen ion concentration, much smaller amounts of copper will precipitate all the agglutinin. The limiting concentration of copper seems to vary with different extracts, and with the same extract at varying hydrogen ion concentrations. For instance, an extract agglutinating in a dilution of 1:640 was precipitated with copper chloride. When 1.0 cc. N/40 HC1 was added to 10 cc. of extract before the addition of copper, the final concentration of M/4400 CuCl₂ was sufficient to bring down all of the agglutinin in the precipitate, while final concentrations of M/6600 and M/8800 brought down half the agglutinin and a final concentration of M/17600 did not bring down any.

When only 0.75 cc. N/40 HCl was added to 10 cc. of extract before addition of copper chloride, a final concentration of M/4400 brought down no agglutinin in the small amount of precipitate that formed and a final concentration of M/2500 of copper chloride was necessary to recover all the agglutinin.

In purifying the extracts by copper precipitation an orienting experiment is first performed. Portions of 10 cc. of extract are placed in centrifuge tubes and varying quantities of N/10 HCl added. To each tube is added 1 cc. of M/400 copper chloride solution and water to bring the total volume to 12 cc.