Quantitative radioligand assays using de novo-synthesized recombinant autoantigens in connective tissue diseases - New tools to approach the pathogenic significance of anti-RNP antibodies in rheumatic diseases

Objective. To describe new assays for the detection and quantification of a ntibodies to RNPs in rheumatic diseases, using soluble nuclear antigens syn thesized de novo in reticulocyte lysates, Methods. Sera from 381 patients with various rheumatic diseases, including 212 patients with systemic lupus erythematosus (SLE), were analyzed in orde r to evaluate the sensitivity and specificity of serum autoantibody reactiv ities to several recombinant soluble autoantigens: U1-A RNP, Sm-B, SSA/Ro 5 2 and SSA/Ro 60, SSB/La, and Ku, Radioligand assays (RLAs) were performed f ollowing the in vitro transcription and translation of each autoantigen fro m the corresponding complementary DNA, labeled with S-35-methionine. The ra diolabeled protein was then bound by the specific serum autoantibody, formi ng immune complexes that were captured by protein A-Sepharose beads and qua ntified by counting the radioactivity. Results. Among the SLE patients, 44% were positive for anti-U1-A RNP activi ty, 34% for anti-Sm-B, 44% for anti-SSA (32% for Ro 52 and 46% for Ro 60), 32% for anti-SSB/La, and 11% for anti-Ku reactivities. SSA antibodies had a high frequency in patients with mixed connective tissue disease (MCTD) (80 %); 65% these patient sera reacted,vith Ro 52, 45% with Ro 60, and 45% with U1-A RNP, Twenty percent of the MCTD patients also exhibited antibodies to Sm-B and Ku, In patients with Sjogren's syndrome, anti-SSA was the main an ti-RNP antibody (63%), together with SSB/La antibodies (44%), Among patient s with inflammatory myopathy, only antibodies against Ro 52 (36%) and Ro 60 (36%) were present. These new RLA allowed observation of a strong correlat ion (P < 0.0001) between Sm-B antibody levels and the severity of SLE las m easured by the SLE Disease Activity Index), and establishment of a correlat ion between anti-U1-A RNP antibodies and the occurrence of SLE nephritis (P < 0.02), All RLAs were highly specific for the antigen tested and displaye d, in the disease groups studied, a higher sensitivity than conventional im munodiffusion assays. Conclusion. These highly sensitive, specific, and quantitative RLAs represe nt new tools for the detection of autoantibodies to RNP antigens in rheumat ic diseases, and may be useful for (differential) diagnosis in clinical pra ctice.