Production and characterization of monoclonal antibodies directed to the kringle V and protease domains of human apolipoprotein(a)

Production and use of anti-apolipoprotein(a) monoclonal antibodies (MAbs) s pecific to single copy regions in the polymorphic lipoprotein(a) (Lp(a)) ha s been emphasized to be important for the standardization of measurements o f the coronary heart disease risk factor, Lp(a). Here, mouse MAbs were prep ared against the kringle V (V) and protease (P) domains of human apolipopro tein(a) (apo(a)), which domains are present in single copy in the apo(a) mo lecule. The cDNA for apo(a)VP was cloned from human liver cDNA library, and the V-P recombinant protein overexpressed in Escherichia coli was used as an antigen for the antibody production. Two antibodies named as MAb(a)20 an d MAb(a)23 were finally produced, and they were characterized for their bin ding specificity and epitopes. The specificity of the antibodies was confir med by an immunoblotting procedure and an enzyme-linked immunoassay (ELISA) . It was shown that the antibodies had little, if any, cross-reactivity wit h human plasminogen, which is relatively abundant in human serum and is hig hly homologous (85%) with apo(a) in amino acid (aa) sequence. For epitope a nalysis, 3'-deletional series of apo(a) VP cDNA were constructed, and expre ssion products of them were analyzed for the binding MAb(a) 20 and MAb(a) 2 3 do. It has been revealed that distinct epitopes were recognized by the tw o MAbs: MAb(a)23 (gamma 2b, kappa) bound to the V region about 60 aa downst ream from the N-terminal, and MAb(a)20 (gamma1, kappa) bound to the P regio n close to the C-terminal. A one step-sandwich ELISA system for Lp(a) was d eveloped using MAb(a)20 as a capturing antibody and horseradish peroxidase (HRP)-coupled MAb(a)23 as a detecting antibody. The assay was found to be s ensitive and useful for detecting Lp(a) in the range of 4-150 mug/dL (80 pM -3 nM).