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.
U 364 INSERM, Faculté de Médecine, Nice, France.
C
C
Akhoundi
N
N
Rochet
B
B
Ferrua
B
B
Rossi
D000818:Animals; D000906:Antibodies / Q000276:immunology / Q000378:metabolism; D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000378:metabolism; D000917:Antibody Formation; D000918:Antibody Specificity; D001798:Blood Proteins / Q000032:analysis* / Q000276:immunology* / Q000378:metabolism; D006528:Carcinoma, Hepatocellular / Q000737:chemistry; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007536:Isomerism; D051379:Mice; D008807:Mice, Inbred BALB C; D011233:Precipitin Tests; D011817:Rabbits; D012015:Reference Standards; D012680:Sensitivity and Specificity; D014407:Tumor Cells, Cultured; D060749:alpha-2-HS-Glycoprotein
C556354:AHSG protein, human; C556357:Ahsg protein, mouse; D000906:Antibodies; D000911:Antibodies, Monoclonal; D001798:Blood Proteins; D060749:alpha-2-HS-Glycoprotein