Absence of high-affinity calreticulin autoantibodies in patients with systemic rheumatic diseases and coeliac disease

Calreticulin has been reported to be an autoantigen in various autoimmune connective tissue diseases and in coeliac disease. Previous studies have used incubation buffers with low salt and low detergent concentrations (low stringency conditions) with serum albumin or other proteins as a blocking agent. Using these conditions we found a relatively high level of non-specific binding in many sera. Antibodies to proteins that are used as blocking reagents in ELISA (bovine serum albumin (BSA), ovalbumin, skimmed milk powder) are frequently present in sera, and these may cause false-positive results. Moreover, the low isoelectric point of calreticulin and its chaperone properties may give rise to false-positive results under low stringency conditions. We report that the use of a simple buffer without protein (50 mM Tris, pH 7.5, 1% Tween 20, 0.3 M NaCl) removes most of the problems with unwanted binding (high stringency conditions). Using the high stringency conditions, we screened sera from 107 patients with systemic lupus erythematosus, sera from patients with other systemic autoimmune diseases and from children with coeliac disease for the presence of high-affinity calreticulin autoantibodies by immunoblotting and ELISA. None of the sera contained high-affinity calreticulin antibodies. It is concluded that calreticulin is not a common autoantigen in patients with autoimmune connective tissue diseases or coeliac disease.