Journal oflmmunological Methods, 50 (1982) 227--231 227 Elsevier Biomedical Press A ONE-STEP PROCEDURE FOR PREPARATION OF CLASSICAL PATHWAY (Clq) AND ALTERNATIVE PATHWAY (FACTOR D) DEPLETED HUMAN SERUM F. PRAZ, M.C. ROQUE ANTUNES BARREIRA 1 and P. LESAVRE Clinique Nephrologique and INSERM U-25, Hbpital Necker, 161, rue de Sgvres, 75730 Paris Cedex 15, France (Received 2 October 1981, accepted 23 October 1981) A simple method for preparation of a serum depleted in both Clq and Factor D is described. The hemolytic activities of both pathways are completely abolished and can be fully restored using the respective purified complement components. Furthermore, this serum is useful for studying cell systems since blocking either complement pathway does not require chelating agents. Key words: complement -- alternative pathway -- classical pathway -- hemolytic assay -- complement technology INTRODUCTION Several techniques have been used to inhibit either the classical or the alternative pathway in studying human complement activation. Classical pathway (CP) inhibition due to Ca 2~ chelation by Mg-EGTA was described earlier by Platts-Mills and Ishizaka (1974). Alternative pathway (AP) inhi- bition was accomplished by Factor B depletion subsequent to heating serum for 30 min at 50°C according to G5tze and Miiller-Eberhard (1971). Both methods are widely used by many authors for their simplicity. We developed a simple method to deplete selectively Clq and Factor D from human serum. This method is based on the particular behavior of Fac- tor D (Lesavre et al., 1979) and Clq (Tenner et al., 1981) with regard to ion exchange chromatography. It is the purpose of this paper to report that such 1 Recipient of CNPq: Conselho Nacional de Desenvolvimento Cientffieo e Tecnol6gico (Brasil). Present address: Faculdade de Medicina de Ribeir~o Preto; Universidade de S~o Paulo, S~o Paulo, Brasil. 0022-1759/82/0000--0000/$02.75 © 1982 Elsevier Biomedical Press