A new conformational epitope generated by the binding of recombinant 70‐kd protein and U1 RNA to anti–U1 RNP autoantibodies in sera from patients with mixed connective tissue disease

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Abstract
Objective: To establish an enzyme‐linked immunosorbent assay (ELISA) using a complex of in vitro–transcribed U1 RNA and recombinant 70‐kd, A, and C proteins (C‐ELISA) to detect anti–U1 RNP antibodies reactive in double immunodiffusion (DID), but not in ELISA using the proteins alone (P‐ELISA).Methods: Sera from 196 patients with mixed connective tissue disease were used to test reactivity in P‐ and C‐ELISAs, and the specificity of the sera was also tested by DID and immunoprecipitation (IP).Results: In P‐ELISA, 15 of 196 sera positive for anti–U1 RNP in DID did not react, while all sera reacted in C‐ELISA. The reactivity of 15 sera to the U1 RNA was tested by IP and ELISA, and only 3 sera reacted with the U1 RNA. These results indicated that the increased reactivity in C‐ELISA was not due to the U1 RNA itself. We confirmed that the 70‐kd and A proteins were bound directly to the U1 RNA by IP using antibodies to His‐tag, and we tested the reactivity of the sera to the U1 RNA–70‐kd protein complex and the U1 RNA–A protein complex by IP. All sera reacted with the U1 RNA–70‐kd protein complex, and 1 sample reacted with the U1 RNA–A protein complex.Conclusion: These results suggest that some anti–U1 RNP–positive sera specifically recognize the conformational structure altered by the binding of U1 RNA to the proteins, and the ELISA using U1 RNA and recombinant proteins is as useful as the DID method for detecting anti–U1 RNP antibodies.

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