Casein is an essential cofactor in autoantibody reactivity directed against the C-terminal SmD1 peptide AA 83-119 in systemic lupus erythematosus

The C-terminal peptide SmD1_83-119 has been identified as an important autoantigen in systemic lupus erythematosus (SLE). ELISA studies have shown that roughly 70% of all sera from patients with SLE react with this peptide. Previous findings revealed that the addition of blocking agents and sample dilution buffers influences the discrimination between positive and negative anti-SmD1_83-119 sera in SLE. The aim of the present study was to identify possible cofactors in the anti-SmD1_83-119 reactivity. We therefore tested SLE sera (n=6) for anti-SmD1_83-119 reactivity by ELISA and analysed the effects of different blocking agents (1% skim milk, 1% gelatin, and 1% BSA). In our investigation, lipids were extracted from skim milk using dichlomethane, and the putative fraction was tested to assess the assay's ability to discriminate between positive and negative sera. The effects of enzymatic digestion by casein were analyzed, and different concentrations of casein were used to determine the role of this protein in the detection of anti-SmD1_83-119 antibodies by ELISA. Furthermore, rabbits were immunized with SmD1_83-119 adsorbed to casein and control proteins. One percent skim milk was the most effective blocking agent and sample dilution buffer for the discrimination between positive and negative sera. As demonstrated by SDS electrophoresis, the discriminative capacity was influenced by enzymatic digestion of skim milk proteins, but not by lipid extraction. Differences in anti-SmD1_83-119 reactivity upon variation of the casein concentration suggest that the protein plays a significant role in the detection of anti-SmD1_83-119 antibodies. However, our immunisation studies did not show any effect of casein on anti-SmD1_83-119 reactivity, suggesting that it has no immunogenic effect on the anti-SmD1_83-119 response. In conclusion, casein seems to be an important cofactor in autoantibody reactivity directed against the C-terminal SmD1_83-119 peptide and probably functions by changing the conformation of the peptide's critical epitope.