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Isolation and Characterization of Guinea Pig Properdin Using an EAC4^h3^g Intermediate

Harvard Medical School at the Robert B. Brigham Hospital, Boston, Mass. 02120

Abstract

The properdin assay consisted of incubating 0.1 ml of an EAC4^h3^g (1 x 10⁸) suspension in DGVB⁺⁺ containing limited factor B and excess , plus 0.1 ml of serum fractions for 5 min at 30C. Then 0.3 ml of guinea pig serum diluted 1:4 in 0.04 M EDTA buffer was added and the mixtures were incubated for 60 min at 37°C. One unit of properdin was defined as the reciprocal of the dilution of properdin which gave a Z of 1.0 above the background lysis caused by limited B and excess alone. The EAC4^h3^g intermediate was made by modifying the procedure described by Fearon et al. for making EAC4^h3^h (J. Exp. Med. 138:1305, 1975). Factors B and were purified from guinea pig serum by modifications of previously published methods (Brade et al., J. Immunol. 109:1174, 1972 and 112:1845, 1974). The factor B preparation contained 1495 units/O.D.₂₈₀; the factor preparation contained 10,450 units/O.D.₂₈₀.