Higher incidences of anti-SS-A/Ro and anti-SS-B/La autoantibodies in Japanese patients with autoimmune disorders ?studies of antigens and antibodies using the immunoblotting method

Abstract

A specific and sensitive assay was performed to detect both anti-SS-A/Ro and anti-SS-B/La antibodies in sera of patients with autoimmune diseases, including systemic lupus erythematosus (SLE), progressive systemic sclerosis (PSS), Sjögren's syndrome (SS), discoid lupus erythematosus (DLE), mixed connective tissue disease (MCTD), generalized morphea (GM), and dermatomyositis (DM). The SS-A/Ro and SS-B/La antigens were prepared from human spleen (HSE) and cultured human cell line (KB cells, KBE), white rabbit thymus extract (RTE) was used as the SS-B/La antigen marker. The antigens were partially purified by DEAE cellulose column chromatography. Immunoblotting showed that the SS-A/Ro antibody reacts mainly with the 58-kDa peptide of the partially purified antigen. Sera containing both the SS-A/Ro and SS-B/La antibodies reacted with the 40-kDa peptide of RTE, and the 58-kDa, 42-kDa, and 40-kDa peptides of HSE and KBE. We found that some of the SS-A/Ro antisera could further react with the 64-kDa peptide of HSE and KBE. The 58-kDa peptide is rich in its cytoplasmic fraction of KB cells, and the 4-kDa peptide in nucleoplasmic fraction. The KB cell line is a better source of the antigens than human spleen extract. The immunoblotting method clearly showed that the positivity rates of SS-A/Ro and/or SS-B/La autoantibodies were higher in sera from Japanese patients with SLE compared with titers reported for Caucasians but not in sera from healthy volunteers.

Antibodies targeting extractable nuclear antigens: historical development and current knowledge

“…The presence of anti‐Ro/SSA is associated with antinuclear antibody‐negative LE, 43 complement‐deficient LE, 44 SS/LE overlap, 40 vasculitis, 45–47 and a decreased frequency of lupus nephritis 48,49 . The frequency of anti‐Ro/SSA is increased in Japanese lupus patients 50 …”

A Novel Anti‐ENA Antibody in Sera of Patients with Childhood Idiopathic Thrombocytopenic Purpura

“…Ouchterlony double immunodiffusion using rabbit thymus and human spleen extracts was performed as previously described (6). Further investigation by immunoblot was carried out by established methods (7,8). Briefly, extracts of rabbit thymus or KB cells were subjected to electrophoresis on 10% SDS polyacrylamide gel slabs (9) in duplicate.…”

“…Samples were reduced in SDS-sample buffer containing 2% SDS and 5mM p-mercaptoethanol. One gel slab was stained with Coomassie brilliant blue R-250 and the other slab was used for e1ectroblotting according to Towbin's procedure (7,8). Diluted (1:100) sera were allowed to react with antigens on the membrane at 25°C for one hour.…”