Determn. of glycogen phosphorylase isoenzyme BB - by sandwich-type enzyme immunoassay using selected monoclonal antibodies

摘要

Enzyme immunoassay for glycogen phosphorylase isoenzyme BB (GP-BB) in EDTA-contg. plasma samples is performed by: (a) coating a solid phase with monoclonal antibody VID12 (produced by hybridoma cell line ZIM-0326); (b) stabilising the antibody of incubating for several hours in a buffer soln. contg. 2-7 wt.% sugar, 0.3-0.8 wt% protective protein, 0.01-0.03 wt.% bactericide and 0.02-0.1 vol.% detergent; (c) adding the test sample, negative controls (GP-BB-free EDTA-contg. plasma) and positive controls (contg. known amts. of GP-BB in EDTA-contg. plasma) to different portions of the solid phase; (d) adding a soln. of peroxidase-labelled monoclonal antibody IIF11 (produced by hybridoma cell line ZIM-0329) in a buffer soln. contg. 5-15 vol.% normal calf serum, 0.02-0.1 vol% detergent and 2-7 mM EDTA, and incubating; (e) removing the liq. phase; (f) adding a substrate soln. contg. H2O2, a chromogen and a buffer, and incubating; (g) adding an acid to stop the colour reaction; and (h) measuring the colour intensity. USE/ADVANTAGE - The assay is useful for diagnosis of ischaemic heart disease. The antibodies exhibit no cross-reactivity with isoenzymes MM or LL. The detection limit is 0.5 mg/ml.