Direct measurement of calpastatin subtypes by sandwich enzyme immunoassay using monoclonal antibodies.
Six stable hybridoma cell lines secreting monoclonal antibodies to human calpastatin were established. All monoclonal antibodies belong to the IgG1 subclass and recognized different epitopes on calpastatin. At least two groups were distinguished; the first group was specific for muscle-type (M-) calpastatin and the second group recognized not only M-calpastatin but also erythrocyte-type (E-) calpastatin. The inhibitory effect of all monoclonal antibodies on calpastatin activity was relatively low even at high concentrations of antibodies. Enzyme immunoassay systems were developed for direct determination of calpastatin subtypes in human cells requiring no other sample treatment than the disruption of the cells. The assay methods were, in principle, based on the sandwich enzyme immunoassay using epitope-specific monoclonal antibodies. The enzyme immunoassay system for M-calpastatin was specific for M-calpastatin and could not detect E-calpastatin. The enzyme immunoassay system for total calpastatin detected not only M-calpastatin but also E-calpastatin. The sensitivity of these assay systems was 10 pmol l-1 of calpastatins. Antigenicity of calpastatins was found to be unchanged in the presence of EDTA and haemoglobin. Good reproducibilities of within-and between-assay series and excellent recovery of exogenous calpastatins from cell lysates were observed. From these results, it seems that our newly developed subtype-specific enzyme immunoassay systems for calpastatins are useful in biochemical studies and clinical testing for determination of calpastatin subtypes.
Yokota
H
H
Biotechnology Research Laboratories, Takara Shuzo Co., Ltd., Shiga, Japan.
Katayama
M
M
Hino
F
F
Kato
I
I
Takano
E
E
Maki
M
M
Hatanaka
M
M
Murachi
T
T
D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D001666:Binding Sites, Antibody; D002135:Calcium-Binding Proteins / Q000032:analysis*; D002478:Cells, Cultured / Q000737:chemistry; D000939:Epitopes / Q000276:immunology; D004912:Erythrocytes / Q000737:chemistry; D006801:Humans; D007124:Immunoenzyme Techniques; D014407:Tumor Cells, Cultured / Q000737:chemistry
D000911:Antibodies, Monoclonal; D002135:Calcium-Binding Proteins; D000939:Epitopes; C033668:calpastatin