The Preparation of a hCG-Specific Immunoadsorbent using hCGβ-Carboxyl-Terminal Peptide Antibody and its Application to Isolate HCG-Like Substances from Human Pituitary Glands

It has been difficult to distinguish hCG from hLH because the two are structurally similar. However, hCG beta subunit has a unique carboxyl terminal peptide, which is not present in hLH and other gonadotropins. Taking advantage of this unique structural feature of hCG beta subunit, hCG-specific antisera have been produced. In order to isolate hCG-like substances from human pituitary glands, the specific immunoadsorbent for hCG was prepared using the isolated monospecific antibody from these antisera. Specific IgG fraction was isolated from an antiserum by affinity chromatography using synthetic carboxyl-terminal peptides as ligands. The purified IgG was conjugated to Sepharose 4B to prepare a specific immunoadsorbent. Immunoadsorbent thus prepared was fully specific to hCG without any crossreactivity with hLH. Attempts to isolate hCG-like substances from two different human pituitary extracts were made by affinity chromatography using this hCG-specific immunoadsorbent. Elution conditions for hCG-like substances from the immunoadsorbent were studied. We found that 1 M NH4OH was a highly effective eluent in this affinity chromatographic system. An advantage of 1M NH4OH is its volatile nature, which permits further steps of purification to be performed directly or after lyophilization without extensive dialysis. The immunoaffinity procedures described in this paper may provide a convenient approach to purify hCG-like substances from both endocrine glands and biological fluids.