Lectin immunoassays using antibody fragments to detect glycoforms of human chorionic gonadotropin secreted by choriocarcinoma cells.
Immobilized antibodies are commonly used to recognize and bind proteins of interest from heterogeneous samples; however, subsequent probing of the glycan(s) of captured glycoproteins with lectins is limited by interference due to the competing oligosaccharides inherently present on antibodies. To prepare capture antibodies with significantly reduced binding of any lectin, the glycosylated protein domains (F(c)) of two anti-human chorionic gonadotropin antibodies were proteolytically removed. Depending on the individual antibody, usable fragments were generated either directly or effectively separated after cleavage through partial reduction and thiol coupling to an appropriate matrix. Importantly, neither method required additional purification of the antibody fragments before immobilization. Binding of a variety of lectins to the functional fragments was reduced by approximately 90% compared with intact immunoglobulin G in both an enzyme-linked immunosorbent assay and a biosensor format. These carbohydrate-free antibody fragments were used to bind the glycoprotein hormone, human chorionic gonadotropin, produced during normal pregnancy and that secreted by three human choriocarcinoma cell lines. Lectins bound to the unpurified gonadotropin glycoforms in distinct patterns consistent with glycan structures previously elucidated by others on hormone samples purified from the urine of pregnant women and of patients with choriocarcinoma. The methods described in this article are applicable for generating capture reagents universally suitable for lectin immunoassays of glycoproteins.
Kelly
Lisa S
LS
Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA 30602, USA.
Kozak
Marina
M
Walker
Tiffany
T
Pierce
Michael
M
Puett
David
D
D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000378:metabolism; D014408:Biomarkers, Tumor / Q000032:analysis* / Q000276:immunology; D015374:Biosensing Techniques; D045744:Cell Line, Tumor; D002822:Choriocarcinoma / Q000737:chemistry* / Q000276:immunology; D006063:Chorionic Gonadotropin / Q000032:analysis* / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006023:Glycoproteins / Q000032:analysis / Q000276:immunology; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000276:immunology; D007141:Immunoglobulin Fc Fragments / Q000737:chemistry; D037102:Lectins / Q000737:chemistry*; D011247:Pregnancy; D014594:Uterine Neoplasms / Q000737:chemistry* / Q000276:immunology
D000911:Antibodies, Monoclonal; D014408:Biomarkers, Tumor; D006063:Chorionic Gonadotropin; D006023:Glycoproteins; D007140:Immunoglobulin Fab Fragments; D007141:Immunoglobulin Fc Fragments; D037102:Lectins