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

Objective, To clone and characterize human antiRo/SSA autoantibodies from a patient with primary Sjogren'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 hybrido ma technique, Specificity was determined using cell extracts, recombinant R o 52-kd, Ro 60-kd, and La proteins as well as Ro 52-kd peptides in enzyme-l inked immunosorbent assay (ELISA) and Western blot. The immunofluorescence pattern was analyzed using cultured human and mouse cell lines. Complementa ry DNA was amplified by polymerase chain reaction, and Ig variable (V)-regi on genes were directly sequenced. Results, Two human anti-Iio 52-kd mAb of IgM isotype, denoted SG1 and SG3, were cloned from the peripheral blood of a patient with pSS, The 2 mAb reac ted with the Ro 52-kd antigen in cell extracts of human cell lines and mous e 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 recombina nt fragment representing amino acids 136-292. Immunofluorescence studies re vealed cytoplasmic staining with both mAb, Both were encoded by V(H)3-famil y genes. SG1 was highly homologous to the DP-77 germ-line gene, with 2 repl acement mutations and 1 silent. It utilized the DPL-11 germ-line gene from the V(lambda)2-family gene, with 1 silent mutation. SG3 was 100% homologous to the DP-47 germ-line gene, combined with a V(kappa)1-family gene that wa s 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 co nformation-dependent, within a region previously described to be immunodomi nant. Somatic hypermutation appeared to be of minor importance in generatin g these 2 specificities.