Immunoglobulin variable genes and epitope recognition of human monoclonal anti-Ro 52-kd in primary Sj gren's syndrome

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Abstract
Objective To clone and characterize human anti‐Ro/SSA autoantibodies from a patient with primary Sjögren's syndrome (pSS). Methods Monoclonal antibodies (mAb) were raised from the peripheral blood of a patient with pSS using Epstein‐Barr virus transformation and a hybridoma technique. Specificity was determined using cell extracts, recombinant Ro 52‐kd, Ro 60‐kd, and La proteins as well as Ro 52‐kd peptides in enzyme‐linked immunosorbent assay (ELISA) and Western blot. The immunofluorescence pattern was analyzed using cultured human and mouse cell lines. Complementary DNA was amplified by polymerase chain reaction, and Ig variable (V)–region genes were directly sequenced. Results Two human anti–Ro 52‐kd mAb of IgM isotype, denoted SG1 and SG3, were cloned from the peripheral blood of a patient with pSS. The 2 mAb reacted with the Ro 52‐kd antigen in cell extracts of human cell lines and mouse cell lines, and with purified human recombinant Ro 52‐kd protein in ELISA and Western blot. SG1 reacted specifically with 1 peptide, amino acids 136–156, of the Ro 52‐kd protein, and SG3 was mapped to react with a recombinant fragment representing amino acids 136–292. Immunofluorescence studies revealed cytoplasmic staining with both mAb. Both were encoded by VH3‐family genes. SG1 was highly homologous to the DP‐77 germ‐line gene, with 2 replacement mutations and 1 silent. It utilized the DPL‐11 germ‐line gene from the Vλ2‐family gene, with 1 silent mutation. SG3 was 100% homologous to the DP‐47 germ‐line gene, combined with a Vκ1‐family gene that was 100% homologous to the A30 germ‐line gene. Conclusion Two human mAb were demonstrated to be specific for the Ro 52‐kd protein and to be directed against 2 different epitopes, 1 linear and 1 conformation‐dependent, within a region previously described to be immunodominant. Somatic hypermutation appeared to be of minor importance in generating these 2 specificities.