Thr Cys Tyr Tyr F?ii Lys Ser. Oligosaccharide side chains are attached at residues 52 and 78. In the preparations studied approximately 10 and 30% of the chains lack the initial 2 and 3 NH,-terminal residues, respectively. This sequence is almost identical with that of human luteinizing hormone (Sairam, M. R., Papkoff, H., and Li, C. H. (1972) Biochem. Biophys. Res. Commun. 48, 530-537). The

Human chorionic gonadotropin (hCG) exists in blood and urine as a variety of isoforms one of which contains peptide bond cleavages within its β-subunit loop 2 and is referred to as nicked hCG (hCGn). This hCG isoform appears to be more prevalent in the urine of patients with certain malignancies and possibly in some disorders of pregnancy. Until now, only indirect immunoassays could be used to quantify hCGn. We report the development of two monoclonal antibodies (MAbs) to a form of hCGn isolated from a choriocarcinoma patient. This hCG isoform was not only 100% nicked, but also contained 100% tetrasaccharide-core O-linked carbohydrate moieties in its β COOH-terminal region. Two-site immunometric assays have been developedusing these new antibodies, B151 and B152. The former exhibits good specificity for hCGn independent of the source of the hCGn, the form excreted by choriocarcinoma patients or the form of hCGn from normal pregnancies. The latter antibody, B152, is sensitive to the carbohydrate moieties and possibly other differences in hCG isoforms, but is not for nicking of the β-subunit. These two immunometric assays provide potential novel diagnostic tools for direct measurement of hCG isoforms which could not be accurately quantified earlier before development of the assays using these newly generated antibodies.

Most secreted eukaryotic proteins are modified by glycosylation, and it has been difficult to solve their structures by crystallographic or NMR techniques because of problems posed by the presence of the carbohydrate. The structure of a chemically deglycosylated form of the human pregnancy hormone, human chorionic gonadotropin (hCG), has been solved by crystallographic methods. Since chemical deglycosylation may have induced changes in the structure, and since it is known that deglycosylated hCG is biologically inactive, the crystallographic structure requires confirmation by NMR techniques. Also, it has not been possible to determine the structures of the isolated subunits, nor the nature of interactions between the carbohydrate side chains and the protein back bone by crystallographic methods. Structural information via NMR techniques can be obtained from proteins in solution if they can be uniformly labeled with 13C and 15N isotopes. We report the first such uniform labeling of a glycoprotein using a universal 13C-and 15N-labeling medium to express 13C, 15N-labeled hCG, suitable for solving the structure in solution of the native, biologically active form of hCG as well as that of its free subunits. The 13C, 15N-labeled recombinant hCG and its separated subunits are shown to be nearly identical to urinary hCG reference preparations on the basis of protein chemical studies, immunochemistry, biological activity, and the capability of isolated hormone subunits to recombine to form biologically active hormone. Mass spectrometric analysis and preliminary NMR studies indicate that the isotopic labeling is uniform and greater than 90% after only two growth passages in the labeling media. One unexpected finding during subunit purification was that lyophilization of glycoproteins from trifluoroacetic acid HPLC buffers may result in the loss of a significant portion of sialic acid.

The conformational properties in solution of the glycans on the alpha subunit of recombinant human chorionic gonadotropin are described, using high-resolution multinuclear NMR studies on uniformly 13C, 15N-enriched recombinant glycoprotein expressed in CHO cells. The glycan important for full biological activity of hCG, namely, that at Asn 52, appears to extend into solution both in the isolated alpha subunit and in complex with the beta subunit. The disposition of this glycan with respect to the protein backbone suggests that glycosylation maintains full biological activity of hCG either by interacting with a lectin-like region of the hCG receptor or by reducing the affinity of the hormone for the hCG receptor and preventing its down-regulation.

This chapter focuses on some clinical applications of human chorionic gonadotropin (hCG) assays, especially to detect early pregnancy loss. The collection and storage of urine samples for assay is an easy and painless procedure, so urinary measurements of hCG, rather than serum measurements, have been utilized for most epidemiologic studies of human reproduction.

In humans, maintenance of pregnancy requires extending the lifetime of the corpus luteum, which produces essential steroids. The signal for this maintenance is provided by human chorionic gonadotropin (hCG), a dimeric glycoprotein hormone produced by the trophoblast cells of the early embryo. The absence of hCG would result in rapid termination of early pregnancy.

Abstract At least 95 per cent of the menstrual cycles of healthy, reproductive-aged women are ovulatory (Vollman, 1977). However, for the average non-contracepting couple, there is only about a 25 per cent chance that an ovum will produce a recognized pregnancy (Leridon, 1977). This gap between the number of available ova and the much smaller number of observed pregnancies presumably reflects some mixture of failure to conceive and early death of the conceptus. The rate of fertilization in humans is unknown, because we have no method to detect the event of fertilization. That, in turn, makes us unable to measure the proportion of fertilized ova lost before pregnancy becomes clinically apparent. In principle, the risk of early loss could range from zero (assuming that 25 per cent of ova are fertilized and all survive) to nearly 75 per cent (assuming that every egg is fertilized but only 25 per cent survive).

In addition to high concentrations of hCG, pregnancy urine contains even higher concentrations of a fragment of the hCG beta-subunit. This biologically inactive material complicates immunological measurement of hCG, since it cross-reacts with many polyclonal and monoclonal antibodies to the hCG beta-subunit that are employed for assays of hCG in urine. Although we and others have developed antibodies to this fragment, specific measurement of the fragment in the presence of free hCG beta has remained difficult due to intrinsic cross-reactivity of these antibodies with the intact hCG beta. Rather than attempt to increase specificity by assay optimization, we developed a new, highly specific monoclonal antibody, designated B210, which cross-reacts less than 0.1% with the free hCG beta-subunit in both liquid and solid phase immunoassay formats. We have used this new monoclonal antibody in immunoradiometric assays to measure specifically the hCG beta fragment in urine throughout pregnancy as well as in the sera of two individuals with cancers producing the hCG beta-subunit. We discovered that the hCG beta fragment can bind three monoclonal antibodies simultaneously, indicating that although the epitope for antibody B210 is a new determinant exposed on the hCG beta fragment and not on intact hCG or on free hCG beta-subunit, the hCG beta fragment retains at least two other hCG beta-related epitopes intact, i.e. those that bind monoclonal antibodies B108 and B201.

Nearly two decades ago our colleague, the late Dr. Hymie Nossel, considered the possibility that radioimmunoassays might have as much application in the field of coagulation as they had been shown to have in the field of endocrinology. He selected fibrinopeptide A as a likely candidate for measurement (1) and initiated a new era in the quantitation of peptides or proteins that are biochemical markers for various aspects of the coagulation process.

Elucidation of the primary molecular structure of hCG, coupled with monoclonal antibody technology, has permitted the construction of a partial map of hCG surfaces. Based on this information, two-site immunoradiometric assays have been developed which permit the measurement of intact hCG and its subunit molecular forms with unprecedented sensitivity and specificity. These assays have been employed in a determination of the incidence of early pregnancy loss in a normal population with the finding that 22% of all conceptions producing measurable hCG terminate before becoming clinically evident.