Development of assays for measurement of blocking and modulating autoacetylcholine receptor antibodies using rhabdomyosarcoma cells

Autoimmune pathologies result from an immune response against autologous antigens. In myasthenia gravis autoantibodies are directed toward acetylcholine receptors (AChR) causing a disruption in neuromuscular transmission. Although testing is available for measurement of a heterogeneous binding autoantibody population, recent evidence suggests that specific modulating and blocking antibodies correlate more closely with disease status. General AChR binding antibodies are measured with 125I-bungarotoxin (125I-aButx) and solubilized human AChR in an immunoprecipitation RIA. Specific blocking and modulating antibodies are measured with a sequential RIA using rhabdomyosarcoma (RD) cells. The anchorage dependent cells are grown in vitro, enzymatically released and used as a "free" suspension. Expression of receptor was measured as was equivalence of RD-AChR to other sources. Performance of the specific systems was evaluated. Over 100 samples submitted for binding were also tested for blocking and modulating antibody. HPLC of endocytosed 125I1Vutx and AChR binding ability was measured. Binding antibody was also measured in autoimmune thyroid receptor antibody (TRAB) sample. Approximately 2.2 x 10(6) RD cells were used per test at a ratio of 10 mL patient sera/million cells. Maximum radiolabel binding to cells was observed in approximately 4 hours with a Kd of 5.3 x 10-(10)M. Within-assay imprecision was less than 14% and 18% for blocking and modulating assays respectively. Of the 107 samples tested, 57 were negative for all three antibodies and 31 testes positive. Using the binding assay only, 32% of the population were classified positive. When specific antibody tests were included 46% were positive. Endocytosed radiolabel lost 80% of receptor binding capacity and may be altered in size TRAB samples appeared to have higher AChR-binding than controls. The AChR binding assays is not capable of measuring all relevant antoantibodies. The RD assays proved added capability. However, there remain autoantibodies not measured by any of these assays. Future studies with clinically defined samples are necessary.