Standardization of an enzymometric assay for apolipoprotein A-I by using mixtures of monoclonal antibodies).
For the standardization of human plasma apolipoprotein A-I assay two well characterized monoclonal antibody mixtures were used to develop a sandwich immunoenzymometric assay. The monoclonal antibody mixture 1 (A05-A17-A30) in solid phase technique was selected on the basis of its higher binding capacity of [125I]HDL (41 ng per well) compared to polyclonal antibody (23 ng per well). The epitopes recognized by monoclonal antibody mixture 1 are surface antigenic sites of apolipoprotein A-I expressed on native HDL as determined by competitive inhibition of labeled HDL. The peroxidase conjugated monoclonal antibody mixture 2 (A03-A05-A17-A51) was selected on the basis of its ability to bind to apolipoprotein A-I captured by monoclonal antibody mixture 1. For this, we used the 125I-labeled monoclonal antibodies. Under optimized assay conditions, the immunoenzymometric assay is precise (intra- and inter-assay coefficients of variations 5.4% and 9.2% respectively). It yields plasma apolipoprotein A-I values that correlate highly with those obtained with polyclonal antibody (r = 0.96). So the use of well characterized monoclonal antibody mixtures reacting only to surface antigenic sites of apolipoprotein A-I present on native lipoprotein may provide the possibility of standardization of apolipoprotein A-I measurement.
D000911:Antibodies, Monoclonal / Q000276:immunology*; D016632:Apolipoprotein A-I; D001054:Apolipoproteins A / Q000097:blood* / Q000276:immunology; D001667:Binding, Competitive; D006801:Humans; D006949:Hyperlipidemias; D007124:Immunoenzyme Techniques / Q000592:standards*; D008075:Lipoproteins, HDL / Q000097:blood / Q000276:immunology; D013696:Temperature; D013997:Time Factors
D000911:Antibodies, Monoclonal; D016632:Apolipoprotein A-I; D001054:Apolipoproteins A; D008075:Lipoproteins, HDL