A VERY SENSITIVE COUPLED LUMINESCENT ASSAY FOR CYTOTOXICITY AND COMPLEMENT-MEDIATED LYSIS

The demand for convenient and sensitive means of measuring cytotoxicit y and complement-mediated killing is likely to be increased by the rec ent identification of Complement Factor H, an important regulatory pro tein of both the classical and alternate pathways of complement, as a tumor-associated antigen. Here we describe a simple luminometric assay capable of detecting the death of similar to 0.03 nucleated human-cel l equivalent or similar to 1 rabbit-erythrocyte equivalent. The assay measures the release of glyceraldehyde-3-phosphate dehydrogenase (G3PD H) from dead or damaged cells by coupling its enzymatic activity to pr oduction of ATP, which in turn is measured by well-known methods invol ving firefly luciferase. This is accomplished by means of a reaction s eries in which the activity of G3PDH is coupled with that of phosphogl ycerate kinase, the next enzyme in the glycolytic pathway. As describe d, the assay uses inexpensive, commercially available reagents. This c oupled assay was used to demonstrate that an anti-factor-H antibody is capable of enhancing complement-mediated killing of the Raji cancer c ell line by > 1000%. (C) 1997 Elsevier Science B.V.