Development, characterization, and application of monoclonal antibodies to the native and synthetic beta COOH-terminal portion of human chorionic gonadotropin (hCG) that distinguish between the native and desialylated forms of hCG.
Although the pregnancy hormone hCG has been extensively mapped immunochemically, few monoclonal antibodies have been produced to the unique COOH-terminal region of its beta-subunit (beta CTP). We now report the development and characterization of five such monoclonal antibodies. Three of these antibodies were developed to the synthetic peptide analog of the hCG beta-(109-145) region coupled to diphtheria toxoid, and two antibodies to a conjugate of bovine thyroglobulin and the peptide hCG beta-(115-145) prepared from hCG with its carbohydrate moieties intact. The monoclonal antibodies raised against the synthetic peptide bound hCG, desialylated hCG, and synthetic peptide to a similar extent, whereas antibodies generated to the natural hCG peptide did not bind to the synthetic peptide analog of the COOH-terminal peptide (beta CTP) region or to desialyated hCG. These new monoclonal antibodies could distinguish between native and desialyated hCG in liquid phase immunoassays as well as by Western blots. They are highly specific reagents for such Western blotting and were used for studies of a crude human pituitary gonadotropin preparation to demonstrate that it contained intact hCG beta without the internal peptide bond cleavages found in the subunit present in human blood and urine. Competition experiments using combinations of monoclonal antibodies and rabbit anti-beta CTP antiserum demonstrated that two epitopes exist within the beta-(115-145) region of hCG, one of which depends on the presence of carbohydrate. In summary, the new monoclonal hCG beta CTP antibodies reported here can 1) discriminate between native and desialylated hCG, 2) identify hCG and nicked hCG on Western blots, 3) provide an immunoaffinity purification tool for hCG, and 4) bind to two distinct epitopes on the beta CTP.
Krichevsky
A
A
Department of Medicine, Columbia University College of Physicians and Surgeons, New York, New York 10032.
Birken
S
S
O'Connor
J
J
Acevedo
H F
HF
Bikel
K
K
Lustbader
J
J
Hartree
A
A
Canfield
R E
RE
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D001666:Binding Sites, Antibody; D001667:Binding, Competitive; D006063:Chorionic Gonadotropin / Q000032:analysis / Q000276:immunology*; D018997:Chorionic Gonadotropin, beta Subunit, Human; D051379:Mice; D008807:Mice, Inbred BALB C; D019158:N-Acetylneuraminic Acid; D010446:Peptide Fragments / Q000032:analysis / Q000276:immunology*; D012794:Sialic Acids / Q000032:analysis
D000911:Antibodies, Monoclonal; D006063:Chorionic Gonadotropin; D018997:Chorionic Gonadotropin, beta Subunit, Human; C093805:HCG-beta (109-145); D010446:Peptide Fragments; D012794:Sialic Acids; D019158:N-Acetylneuraminic Acid