Calpastatin autoantibodies: detection, epitope mapping, and development of a specific peptide ELISA

Autoantibodies against calpastatin (CAST) were detected in a 53-year-old female patient with a history of arthritis and thrombosis. The specificity of the autoantibodies was determined by screening expression cDNA libraries, sequence analysis of positive clones, and subsequent Western blotting against recombinant antigen. Because the Western blot lacked satisfactory reproducibility, an ELISA for anti-CAST antibodies was established. The major epitope recognized by 24 Western blot-positive sera was located within the C-terminal 27 amino acids. The ELISA was therefore based on a synthetic peptide representing these amino acids. The assay was calibrated with serial dilutions of a positive reference serum. Intraassay precision is high with a CV of approximately 4% for low- and high-titer samples. Interassay precision (CV) was 5.6-8.2% for sera with low and intermediate titers [10-60 arbitrary units (AU), where 1:100 dilution of the positive reference serum = 100 AU, 1:1000 dilution = 10 AU, etc.], which increases with higher titers ( > 60 AU). Among 205 healthy blood donors, the mean + 3 SD (after logarithmic transformation) was 30 AU; higher values were seen in 2.9% of 138 hospitalized patients. The newly developed ELISA will be a useful tool for further clinical studies on the association of anti-CAST antibodies to disease, because it permits rapid and reproducible analysis of patient sera.