Enzyme immunoassay of calpain I and calpastatin and its application to the analysis of human erythrocyte hemolysate.
A highly sensitive sandwich enzyme immunoassay for a Ca2+-dependent cysteine proteinase (calpain I) and its specific endogenous inhibitor protein (calpastatin) was developed. The calpain I and calpastatin used as immunogens were purified from human erythrocytes. Anti-calpastatin antisera having sufficiently high titer were obtained only when the immunogen was purified by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The assay method was principally based on the report by M. Imagawa et al. (1982, J. Appl. Biochem. 4, 41-57), using a specific antibody-coated polystyrene ball and horseradish peroxidase-conjugated Fab' fragment of the antibody. The sensitivity was 0.1 ng of calpain I or calpastatin per assay tube. Starting with 50 microliter of the hemolysate from human erythrocytes, the method permitted direct and simultaneous determination of calpain I and calpastatin, without prior separation of these two enzymatically counteracting components by chromatography. The present method as applied to the erythrocytes from 14 healthy adults gave 120-170 micrograms for calpain I and 164-211 micrograms for calpastatin per gram of hemoglobin, respectively.
D002135:Calcium-Binding Proteins / Q000097:blood*; D002154:Calpain; D010450:Endopeptidases / Q000097:blood*; D004912:Erythrocytes / Q000201:enzymology*; D006461:Hemolysis; D006801:Humans; D007124:Immunoenzyme Techniques; D008722:Methods; D011480:Protease Inhibitors / Q000097:blood*
D002135:Calcium-Binding Proteins; D011480:Protease Inhibitors; C033668:calpastatin; D010450:Endopeptidases; D002154:Calpain