AUTOIMMUNE SERA REACT WITH MULTIPLE EPITOPES ON RECOMBINANT 52 AND 60KDA RO(SSA) PROTEINS

Objective. To determine the reactivity of recombinant 52 and 60 kDa Ro (SSA) (Ro) proteins with sera from 3 subsets of patients with Ro autoa ntibody associated disease. Methods. Complementary DNA (cDNA) clones t hat encode the human 52 and 60 kDa Ro autoantigens were isolated by th e polymerase chain reaction and utilized to express recombinant glutat hione S-transferase (GST) fusion proteins. Double immunodiffusion (ID) defined Ro positive autoimmune sera from 12 patients with neonatal lu pus erythematosus (NLE), 16 with subacute cutaneous lupus erythematosu s (SCLE) and 12 with primary Sjogren's syndrome (SS) were tested by en zyme linked immunosorbent assay (ELISA) against the recombinant 52 and 60 kDa Ro fusion proteins. Results. Seventy-five percent of NLE, 56% of SCLE and 83% of SS sera reacted with the 52 kDa fusion protein. Sev enty-five percent of NLE, 63% of SCLE and 83% of SS sera reacted with the 60 kDa fusion protein. Seventeen percent of NLE sera, 25% of SCLE sera and 8% of SS sera were nonreactive to both full length fusion pro teins. Eight (57%) of 14 ID defined Ro negative NLE, SCLE and SS sera were reactive with both Ro fusion proteins by ELISA. ELISA studies wit h recombinant 52 and 60 kDa Ro protein fragments revealed at least 2 m ajor epitopes on each Ro protein. A fragment of the 52 kDa Ro protein that contains a putative leucine zipper motif reacted with 100% of ID defined Ro positive SS sera. Conclusion. Our data demonstrate that the ID assay and the recombinant Ro ELISA together are more sensitive in detecting Ro antibodies than either assay alone, and that multiple epi topes are present on both Ro proteins.