Application of cellular ELISA (CELISA) to the detection of human monoclonal autoantibodies Berta Sanchez, Josefa Melero, M. Mar Robledo, David Tarrago, Jose Yelamos, and M. Francisca Gonzalez Servicio de Inmunologia, Hospital Universitario Virgen del Rocio, Seville, Spain This work describes the application of cellular enzyme-linked immunosorbent assay (eEL/SA) for the detection of both polyreactive and monospecific human monoclonal antibodies against autoantigens. The eEL/SA is ideally suited for the screening of a large number of hybridoma culture supernatants, being, in this way, superior to other methods commonly used for the detection of autoantibody activity, such as indirect immunofluorescence on tissue sections and slide cell preparations, in terms of speed and sensitivity. This assay demonstrated higher sensitivity than ELISA using autoantigenic extracts from rabbit thymus, human spleen, nucleoprotamine, and salmon sperm nuclei, and enzyme immunoassays using ssDNA, dsDNA, and affinity purified autoantigens as substrate. The eELISA has been also successfully applied to the detection of autolymphocytotoxic antibody activity in heterohybridoma supernatants. Keywords: autoantibody; autocytotoxicity; ELISA; human monoclonal antibody; polyreactivity Introduction The generation of autoreactive human monoclonal au- toantibodies (MAbs) represents a better approach to defining the repertoire of autoreactive human B lym- phocytes in autoimmune patients, as compared with xenogeneic immunization of mice with purified or re- combinant human autoantigens, since the latter often recognize species-specific determinants rather than epi- topes relevant to the disease. 1 Autoreactive human MAbs have been also derived from B lymphocytes of individuals not suffering from autoimmune diseases. z - 4 The current most frequently used methods for screen- ing of autoantibodies are indirect immunofluorescence (IIF) on a tissue section screen of rat liver, stomach, and kidney, or using monolayers of human fibroblasts (HEp-2) as substrate, double immunodiffusion, and counterimmunoelectrophoresis. Autolymphocytotoxic antibodies, found in renal dialysis patients 5 as well as in individuals with certain autoimmune diseases,6 are detected by standard microlymphocytotoxicity (NIH). Address reprint requests to Dr. Berta Sanchez, Servicio de Inmuno- logia, Hospital Universitario Virgen del Roclo, Avda de Manuel Siurot sin, 41013-Sevilla, Spain. Received for publication 15 April 1993. © 1993 Butterworth-Heinemann 198 Hum. Antibod. Hybridomas, 1993, vol. 4, October All these assays are, in general terms, unsuitable for the screening of the large number of samples that are usually generated in routine hybridoma production. Al- though originally described for the detection of antibod- ies to cell-surface antigens,? the cellular enzyme-linked immunosorbent assay (CELIS A) can be successfully applied to the screening of heterohybridoma clones se- creting monospecific and polyreactive human mono- clonal autoantibodies, and even to the detection of autolymphocytotoxic human MAbs. Methods and materials Human autoreactive MAbs Human autoreactive IgM MAbs were generated by Ep- stein-Barr virus immortalization and hybridization with a human-mouse heteromyeloma of human peripheral B cells isolated from five different donors. Human MAbs BY and IRM were obtained from three hyperim- munized renal dialysis patients: BY-I, BY-2, BY-3, BY- 4, and BY-6 from patient APG;4 BY-7, BY-8, and BY- 12 from patient MOL;4 and those named IRM from' a hyperimmunized patient who exhibited autocytotoxic activity in her serum (manuscript in preparation). MAb CDC-l was generated from a scleroderma patient,8 whereas MAbs BOR-16, BOR-21, and ANA-45 were © 1993 Butterworth-Heinemann