Journal oflmmunological Methods, 86 (1986) 115-118 115 Elsevier JIM03766 A simplified ELISA for anti-receptor antibodies in myasthenia gravis Bansi L. Jailkhani 1,,, D. Asthana 1, Nargis F. Jaffery 2, Ramesh Kumar 3 and G.K. Ahuja 1 Neuroscwnces Center, "Biochemtstry Sectton, 1 Department of Neurology, " " " " " Centre for Ophthalmic Sciences, and 3 Department of Microbiology, A II India Institute of Medical Sciences, New Delhi 110029, India (Received11 June 1985,accepted30 September1985) Nicotinic acetylcholine receptor (nAchR) from triton extracts of muscle adsorbed specifically and optimally to microtitration plates at pH 7.4 rather than at pH 9.6. An ELISA for anti-receptor antibodies in myasthenia gravis based on direct adsorption of the receptor at pH 7.4 is described (direct assay). The direct assay compares very well in sensitivity and specificity with an indirect assay, in which the receptor was attached through a-bungarotoxin adsorbed on the solid phase (correlation coefficient 0.94). Key words: Acetylchofine receptor; ELISA; Myasthenia gravis Introduction Antibodies to nicotinic acetylcholine receptor in patients with myasthenia gravis are believed to play a pathogenetic role in the disease (Lindstrom and Lambert, 1978; Lindstrom, 1979). Antibodies in the sera of patients or animals immunized with purified receptor have been generally measured by a radioligand assay based on the use of 125 I-labelled a-bungarotoxin (Lindstrom et al., 1976; Vincent, 1981; Limberg et al., 1983). Some reports have appeared in which enzyme immunoassays have been used to detect the antibody in animals im- munized with nAchR (Norcross et al., 1980; Hin- man et al., 1983) and in patients with myasthenia gravis (Dwyer et al., 1983; Kawanami et al., 1984). The enzyme immunoassays described to date are based on direct coating of the purified receptor (Norcross et al., 1980; Kawanami et al., 1984) or on its attachment through et-bungarotoxin (Nor- * For correspondence and reprint requests: Dr. B.L. Jailkhani, Associate Professor of Biochemistry, Department of Neu- rology, A.I.I.M.S., New Delhi 110029, India. cross et al., 1980; Hinman et al., 1983; Jailkhani et al., unpublished) or a monoclonal antibody (Dwyers et al., 1983) adsorbed to the solid phase. We describe in this communication the unusual property of human muscle nicotinic acetylcholine receptor, from crude triton extracts, of adsorbing to microtitration plates at pH 7.4 and its applica- tion in an ELISA for the detection of antibodies in myasthenia gravis. Materials and methods Materials Microtitration Immulon 2 plates were from Dy- natech (U.S.A.), horseradish peroxidase, type VI RZ," 3.1., o-phenylenediamine, ghitaraldehyde, protease inhibitors and a-bungarotoxin were ob- tained from Sigma Chemical Co. (U.S.A.), heavy chain specific rabbit anti-human IgG was from Dakopatts (Denmark), other chemicals and re- agents were of analytical grade. Sera or plasma from patients with myasthenia gravis and from normal controls were stored at -20°C until analysis. 0022-1759/86/$03.50 © 1986 ElsevierSciencePublishers B.V. (BiomedicalDivision)