Journal oflmmunologicalMethods, 133 (1990) 199-206 199 Elsevier JIM05713 Functional assay of C5-activating and nonactivating cobra venom factor preparations in the mouse system Carmen W. Van den Berg, Piet C. Aerts and Hans Van Dijk Eijkman- Winkler Laboratory of Medical Microbiology, Department of Experimental Microbiology, Faculty of Medicine, University of Utrecht, Utrecht, The Netherlands (Received 30 January 1990, revised received 19 April 1990, accepted 22 June 1990) This paper deals with a new, functional assay of cobra venom factor (CVF) preparations with or without C5-activating property. Existing methods lack sensitivity and use diluted human complement as target of inactivation. An adapted assay using diluted mouse serum as complement source was hampered by underestimation of C3 depletion by bystander lysis and an overvaluation of C5 consumption resulting from C3 inactivation in the reagent used. These disadvantages prompted us to develop the new assay which is based on the incubation of CVF preparations with undiluted mouse serum. After incubation, residual total C activity, as well as functional C3 and C5 are estimated by titration. The procedure permits the assessment of CVF activities with minimal interference from undesired processes. The conditions in the new assay approach the in vivo situation in mice by the use of undiluted serum from the same animal species. Key words: Cobra venom factor; Complement, mouse; C3; C5 Introduction Since the turn of the century it has been known that cobra venoms possess strong anti-comple- mentary activity (Ewing, 1894; Flexner and Noguchi, 1903). At least three factors are involved in the anti-complementary effect: (i) the C3- activating cobra venom factor (CVF), (ii) a high M~ factor (H-CVF) interfering selectively with classical pathway activation, and (iii) a low M r complement inhibitor binding to native as well as activated C3 and C4 and preventing alternative Correspondence to: C.W. Van den Berg, Eijkman-Winkler Laboratory of Medical Microbiology, University Hospital HP G04.614, P.O. Box 85500, NL-3508 GA Utrecht, The Nether- lands. pathway (AP) activation (Ballow and Cochrane, 1969; Von Zabern et al., 1981). CVF is the most common anti-complementary principle in cobra venom. It is a glycoprotein of 144 kDa composed of three different chains held together by disulfide bridges (Miiller-Eberhard and Fjellstr6m, 1971; Miyama et al., 1975; Eggertsen et al., 1980; Von Zabern et al., 1980, 1982). CVF has C3b-like activity in forming a Mg2+-depen - dent complex with factor B which is subsequently activated by factor D to CVF, Bb, an enzyme with C3 convertase activity (Mtiller-Eberhard, 1967; G/Stze and Miiller-Eberhard, 1971; Cooper, 1973; Hunsiker et al., 1973; Hensley et al., 1986). Dif- ference between CVF,Bb and the AP-dependent C3b,Bb C3-convertase is the resistance of the former to inactivation by factors H and I resulting in a much longer half life (t~[37°C]-~ 7 h versus 0022-1759/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)