An improved assay for antibody dependent cellular cytotoxicity based on time resolved fluorometry.
A new and faster assay for antibody dependent cellular cytotoxicity based on release of europium from target cells is described. This has a number of important advantages over the traditional assays based on release of chromium-51 (51Cr). The new method involves labelling of Wein 133 target cells (B cell non-Hodgkin's lymphoma cells) which express the antigen, CDw52, with the chelate europium diethylenetriaminopentaacetic acid (EuDTPA) according to the method of Blomberg et al. (1986). Labelled cells are sensitised (coated) with the anti-lymphocytic monoclonal antibody, Campath-1H. Human peripheral blood mononuclear cells are added to mediate lysis of EuDTPA labelled Wein 133 cells by ADCC. Release of EuDTPA from lysed cells is determined by mixing supernatants with enhancement solution containing 2-naphthoyl trifluoroacetone, 2-NTA, to form a highly fluorescent chelate which is measured using time resolved fluorometry. Results obtained with the new EuDPTA release assays were comparable to traditional assays based on the release of the radioisotope 51Cr. It is anticipated that this assay will have a widespread application among laboratories performing ADCC assays. The method is non-hazardous and has been used routinely for over 2 years to monitor production and purification of Campath-1H.
Patel
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AK
Biotechnology Analytical Labs., Wellcome Foundation, Beckenham, Kent, UK.
Boyd
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PN
D000818:Animals; D000920:Antibody-Dependent Cell Cytotoxicity; D015703:Antigens, CD / Q000276:immunology; D000951:Antigens, Neoplasm; D000074301:CD52 Antigen; D016466:CHO Cells; D002455:Cell Division; D002860:Chromium Radioisotopes / Q000378:metabolism; D006224:Cricetinae; D003601:Cytotoxicity Tests, Immunologic / Q000379:methods*; D005063:Europium / Q000378:metabolism*; D005470:Fluorometry / Q000379:methods; D006023:Glycoproteins; D006801:Humans; D008228:Lymphoma, Non-Hodgkin / Q000276:immunology; D004369:Pentetic Acid / Q000378:metabolism*; D015203:Reproducibility of Results; D014407:Tumor Cells, Cultured
D015703:Antigens, CD; D000951:Antigens, Neoplasm; D000074301:CD52 Antigen; C000613862:CD52 protein, human; D002860:Chromium Radioisotopes; D006023:Glycoproteins; D005063:Europium; D004369:Pentetic Acid