Polyclonal antibodies against the polypeptide and carbohydrate epitopes of recombinant human choriogonadotropin beta-subunit.
In our previous paper (Chen et al. (1991) J. Biol. Chem. 266, 4081-4087) we reported the preparation and characterization of recombinant human choriogonadotropin beta subunit (hCG beta) using the baculovirus-insect cell expression system. The rhCG beta was found to contain high mannose type N-linked carbohydrates and 3-4 serine-linked disaccharide chains. Despite the carbohydrate structural variation, the rhCG beta was similar to hCG beta in in vitro immunological and biological properties. In order to evaluate its in vivo immunological properties, rabbit antiserum against rhCG beta was produced. The antiserum was found to be almost identical to anti-hCG beta in binding to hCG beta as well as in its crossreactivity with human lutropin (hLH), hCG and human follitropin (hFSH) as indicated by radioimmunoassays using 125I-hCG beta as a tracer. Further characterization of the anti-rhCG beta antiserum revealed that there are three types of antibodies in terms of antigenic specificity present in the anti-rhCG beta antisera pool as shown by dot blot and radioimmunoassays. The carbohydrate-specific antibodies were separated by affinity chromatography using an ovalbumin-glycopeptide-Sepharose column. The antibodies held on the ovalbumin affinity adsorbent were specific for the high mannose type carbohydrates such as those present in rhCG beta, rhCG and thyroglobulin and failed to react with transferrin, alpha 1-acid glycoprotein and hCG alpha, all containing complex type carbohydrates. This was further supported by the fact that the recombinant unglycosylated hCG or periodate oxidized rhCG beta also did not show any reactivity with the carbohydrate specific antibodies. Two types of peptide epitopes seemed to be present in rhCG beta since when the flowthrough fraction from the ovalbumin-glycopeptide-affinity column was passed through the hCG beta-Sepharose column, the antibodies in the flowthrough from the latter column were specific to the unique antigenic determinants present only in the rhCG beta and not in hCG beta. The eluate from the hCG beta-Sepharose column contained the third type of antibodies, being the predominant ones, directed to the common epitopes between rhCG beta and hCG beta. The high mannose type specific antibodies are potentially useful in differentiating between the high mannose and complex type of N-linked carbohydrates present in a glycoprotein. Also, the antibody could provide an effective reagent in studying the intracellular processing of the N-linked oligosaccharides.(ABSTRACT TRUNCATED AT 400 WORDS)
Chen
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Department of Biological Sciences, State University of New York, Buffalo 14260.
Bahl
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D000818:Animals; D000906:Antibodies / Q000276:immunology* / Q000302:isolation & purification; D000918:Antibody Specificity; D002240:Carbohydrate Sequence; D002241:Carbohydrates / Q000276:immunology*; D006063:Chorionic Gonadotropin / Q000276:immunology*; D018997:Chorionic Gonadotropin, beta Subunit, Human; D002846:Chromatography, Affinity; D000939:Epitopes / Q000276:immunology*; D006023:Glycoproteins / Q000276:immunology*; D006801:Humans; D008358:Mannose / Q000276:immunology; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000276:immunology*; D010455:Peptides / Q000276:immunology*; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000276:immunology*
D000906:Antibodies; D002241:Carbohydrates; D006063:Chorionic Gonadotropin; D018997:Chorionic Gonadotropin, beta Subunit, Human; D000939:Epitopes; D006023:Glycoproteins; D010446:Peptide Fragments; D010455:Peptides; D011993:Recombinant Fusion Proteins; D008358:Mannose