Specificity of human anti-carbohydrate IgG antibodies as probed with polyacrylamide-based glycoconjugates.
The TF, Tn, and SiaTn glycotopes are frequently expressed in cancer-associated mucins. Antibodies to these glycotopes were found in human serum. A set of polyacrylamide (PAA)--based glycoconjugates was applied to the direct and competitive enzyme-linked immunosorbent assays (ELISA) to characterize the specificity of serum IgG antibodies. The anti-TF, -Tn and -SiaTn IgG were affinity purified from serum of cancer patients and characterized using PAA-conjugates and free saccharides. The anti-TF and -Tn antibodies were shown to be specific. The anti-TF IgG bound both Galbeta1-3GalNAcalpha- and Galbeta1-3GalNAcbeta-PAA, the latter was three-four times more effective inhibitor of antibody binding. The anti-Tn IgG reacted only with GalNAcalpha-PAA. The anti-SiaTn IgG cross-reacted with Tn-PAA but SiaTn-PAA was five-six times more effective inhibitor in a competitive assay. The IC50 values for PAA-conjugates with the corresponding antibodies typically ranged from 2 to 5 x 10(-8) M. The antibodies display a low specificity to mucin-type glycoconjugates in comparison with PAA-conjugates as was shown for mucins isolated from human malignant tumor tissues, ovine submaxillary mucin (OSM) and asialo-OSM. The unusual IgG-antibody specificity to GalNAcbeta and GalNAcbeta1-3GalNAcbeta ligands was found in human serum.
Institute of Experimental & Clinical Medicine, Hiiu 42, 11619 Tallinn, Estonia. smorodin@ekmi.ee
E P
EP
Smorodin
O A
OA
Kurtenkov
B L
BL
Sergeyev
G V
GV
Pazynina
N V
NV
Bovin
D000180:Acrylic Resins / Q000737:chemistry; D000918:Antibody Specificity / Q000276:immunology*; D002241:Carbohydrates / Q000276:immunology*; D000939:Epitopes / Q000276:immunology; D006001:Glycoconjugates / Q000737:chemistry / Q000276:immunology*; D006801:Humans; D007074:Immunoglobulin G / Q000097:blood / Q000276:immunology*; D015335:Molecular Probes; D009077:Mucins / Q000276:immunology; D044967:Serum / Q000276:immunology; D000099166:Polyacrylamides
D000180:Acrylic Resins; D002241:Carbohydrates; D000939:Epitopes; D006001:Glycoconjugates; D007074:Immunoglobulin G; D015335:Molecular Probes; D009077:Mucins; D000099166:Polyacrylamides