Production and characterization of monoclonal and polyclonal antibodies to human alpha 2-HS: development of a two-site ELISA test.

A convenient and sensitive indirect sandwich ELISA test was developed for measuring both 63 kDa human alpha 2-HS secreted by human hepatoma cell lines and the 59 kDa alpha 2-HS species present in serum/plasma. Monoclonal and rabbit antibodies to plasma alpha 2-HS were produced and selected by immunoprecipitation techniques using iodinated alpha 2-HS or 35S-labeled alpha 2-HS. Various monoclonal antibodies recognizing both forms of the protein were coated onto microtiter plates and after binding of alpha 2-HS, biotinylated monoclonal antibodies with compatible binding or biotinylated immunopurified F(ab')2 fragments from the rabbit antiserum were added and subsequently revealed with avidin-biotin peroxidase complex. Formats using a rabbit detector antibody were the most sensitive and one was selected for the whole study. The test developed was capable of detecting plasma alpha 2-HS devoid of connecting peptide and HepG2 hepatoma cell line derived alpha 2-HS at the ng/ml level. The test has been used to measure levels of alpha 2-HS in both serum and supernatants from HepG2 cell lines.