Lectin affinity bioassay: an assay method for glycoprotein enzyme.
A simple and sensitive chromogenic microtitre plate assay for glycoprotein enzymes is described, using melanoma tissue plasminogen activator (t-PA) as a model enzyme. The assay is based on the binding of t-PA to immobilised lectin and quantitating the bound enzyme with plasminogen, fibrinogen fragments and chromogenic substrate S-2251 on an ELISA plate reader. Seven different lectins were examined for the binding of t-PA, and of these, concanavalin A was chosen for subsequent studies. The specificity of this binding can be inhibited dose-dependently in the presence of D-mannose and methyl alpha-D-mannoside, but not by D-glucose and D-lactose. The lower limit of the sensitivity of this assay is about 0.5 IU/ml. Comparison of the dose-response curves indicates that the sensitivity of this assay method is very similar to that of bioimmunoassay using anti-t-PA IgG to capture the antigen. The applicability of this method to other glycoprotein enzymes was also evaluated using alkaline phosphatase from bovine mucosa. The specificity of this method was related to the choice of substrate and this was shown by analysis of a mixture of t-PA and alkaline phosphatase. It is suggested that this assay can be adapted for the analysis of complex glycoprotein mixtures with the appropriate choice of lectin and substrate.
Electricwala
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Division of Biotechnology, PHLS Centre for Applied Microbiology and Research, Porton Down, Salisbury, U.K.
D000469:Alkaline Phosphatase / Q000032:analysis / Q000378:metabolism; D001681:Biological Assay; D003208:Concanavalin A; D005340:Fibrinogen / Q000378:metabolism; D006023:Glycoproteins / Q000032:analysis*; D037102:Lectins; D008545:Melanoma / Q000201:enzymology; D010958:Plasminogen / Q000378:metabolism; D010959:Tissue Plasminogen Activator / Q000032:analysis* / Q000378:metabolism; D014407:Tumor Cells, Cultured
D006023:Glycoproteins; D037102:Lectins; D003208:Concanavalin A; D005340:Fibrinogen; D010958:Plasminogen; D000469:Alkaline Phosphatase; D010959:Tissue Plasminogen Activator