Methods of preparing supplementing fractions from fresh unheated bovine sera for use in modified direct complement-fixation tests.

TWO METHODS OF PREPARING PARTIALLY PURIFIED, RELATIVELY STABLE SUPPLEMENTING FACTOR FROM FRESH BOVINE SERUM ARE DESCRIBED: gel filtration through Sephadex G-25, and anion exchange chromatography on a diethylaminoethyl (DEAE) cellulose column. In both procedures, an active, reconstituted precipitate prepared by dialysis of fresh unheated normal serum in the cold for 18 hours against phosphate buffer pH 6.2, 0.02 M, serves as the starting material. The Sephadex G-25 column is equilibrated with acetate buffer pH 5.4, 0.2 M. The most actively-supplementing material appears in the eluates in which the pH has risen to 7.5 or higher. For the DEAE cellulose chromatography a gradient system is used: initial phosphate buffer 0.03 M, pH 8.0, limiting buffer Na H(2)PO(4), 0.3 M. The greater part of the supplementing activity is eluated between pH 5.6 and 6.0, although some of the earlier fractions are also reactive. Pooled active eluates stored in the frozen state for nine months or longer maintained their supplementing titre in modified complement-fixation tests of two bacterial antigen-bovine antibody systems.