Polyclonal antibodies against the polypeptide and carbohydrate epitopes of recombinant human choriogonadotropin β-subunit

Abstract

In our previous paper (Chen et al. (1991) J. Biol. Chem. 266, 4081–4087) we reported the preparation and characterization of recombinant human choriogonadotropin β subunit (hCGβ) using the baculovirus-insect cell expression system. The rhCGβ was found to contain high mannose type N-linked carbohydrates and 3–4 serine-linked disaccharide chains. Despite the carbohydrate structural variation, the rhCGβ was similar to hCGβ in in vitro immunological and biological properties. In order to evaluate its in vivo immunological properties, rabbit antiserum against rhCGβ was produced. The antiserum was found to be almost identical to anti-hCGβ in binding to hCGβ as well as in its crossreactivity with human lutropin (hLH), hCG and human follitropin (hFSH) as indicated by radioimmunoassays using 125I-hCGβ as a tracer. Further characterization of the anti-rhCGβ antiserum revealed that there are three types of antibodies in terms of antigenic specificity present in the anti-rhCGβ 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 rhCGβ, rhCG and thyroglobulin and failed to react with transferrin, α 1,-acid glycoprotein and hCGα, all containing complex type carbohydrates. This was further supported by the fact that the recombinant unglycosylated hCG or periodate oxidized rhCGβ also did not show any reactivity with the carbohydrate specific antibodies. Two types of peptide epitopes seemed to be present in rhCGβ since when the flowthrough fraction from the ovalbumin-glycopeptide-affinity column was passed through the hCGβ-Sepharose column, the antibodies in the flowthrough from the latter column were specific to the unique antigenic determinants present only in the rhCGβ and not in hCGβ. The eluate from the hCGβ-Sepharose column contained the third type of antibodies, being the predominant ones, directed to the common epitopes between rhCGβ and hCGβ. 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. The antibodies unique to rhCG polypeptide probably were against those epitopes which were either masked or were present in a different conformation in the native hCGβ indicating that the high mannose type carbohydrates might have a different steric or subtle conformational effect than the complex type carbohydrate on the rhCGβ polypeptide chain.