Enzyme-linked immunosorbent assay for the detection of cathepsin-kininogen complexes in human plasma

A simple and sensitive enzyme-linked immunosorbent assay (ELISA) for the determination of cathepsins B-, H- and L-kininogen (B-KG, H-KG, and L-KG) complexes was constructed using a microtiter plate based sandwich technique. The assay range was 7.5-480 ng/ml with three cross-linked cathepsin-kinin-free low molecular weight KG (LMWKGf) complexes. The average within-run coefficients of variation (CV) using three concentrations were 7.5, 7.2 and 9.9% for B-LMWKGf, H-LMWKGf, and L-LMWKGf complexes, respectively. The between-run CV values indicated satisfactory reproducibility for the method. Recoveries of cross-linked B-LMWKGf, H-LMWKGf, and L-LMWKGf complexes from negative control plasma were 127 +/- 7.1, 112 +/- 5.8 and 102 +/- 8.8% (n = 10, mean +/- SD), respectively, whereas the average recovery from phosphate buffer (pH 7.2) containing 0.15 M NaCl, 0.05% Tween-20 and 0.1% bovine serum albumin was 93.1 +/- 8.2% (n = 10, mean +/- SD). It was possible to detect B-KG and H-KG complexes in the plasma of 5/280 and 14/280 patients manifesting acute phase responses. Levels in plasma from healthy individuals were negligible. This ELISA should permit the study of cathepsin metabolism in acute phase disorders.