Radioimmunoassay of the serum free beta-subunit of human chorionic gonadotropin in trophoblastic disease

We developed a RIA specific for the free beta hCG employing anti-beta hCG monoclonal antibody 1D12. This RIA was highly sensitive to free beta hCG; the minimum detectable concentration was 0.4 ng/ml. alpha hCG, LH, beta LH, and FSH had little effect in the assay; the cross-reactivity of hCG was about 4%. Using this RIA, we measured serum free beta hCG concentrations in 38 normal pregnant women and 72 untreated patients with 3 types of trophoblastic disease: hydatidiform mole (n = 15), invasive mole (n = 29), and choriocarcinoma (n = 28). All of these samples were simultaneously assayed for hCG by RIA. In normal pregnant women, serum hCG changed as pregnancy progressed, but serum free beta hCG was not detected at any time. In contrast, serum free beta hCG was measurable in the majority of patients with trophoblastic disease. Strong correlations were found between the concentration of free beta hCG and that of hCG in each type of trophoblastic diseases. The mean free beta hCG to hCG ratio was lowest for hydatidiform mole and highest for choriocarcinoma, and the difference between the ratios in these 2 groups was statistically significant. Serial measurements in 7 patients with trophoblastic disease failed to reveal remarkable changes in the free beta hCG to hCG ratio throughout their clinical course. We conclude that the production of free beta hCG increases with the immaturity of the trophoblastic cell, and the degree of differentiation of trophoblastic cells may be reflected by the free beta hCG to hCG ratio.     

Occurrence of fragmentation of free and combined forms of the beta-subunit of human chorionic gonadotropin.

In an attempt to further study various fragments of free and combined forms of hCG beta present in biological fluids, we performed one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by Western immunoblotting using antipeptide antibodies directed to the hCG beta-(111-116) portion (monoclonal antibody FB12) antiserum to the hCG beta(8-16) portion or antiserum which was specific for fragments ending at residue 47. Results observed in a crude preparation of urinary hCG demonstrated that in addition to the carboxyl-terminal part of the reduced hCG beta nicked subunit (beta NS) [hCG beta-(48-145)], three other fragments of mol wt 18,000 (F1), 16,500 (F2), and 12,000 (F3) were detectable after cleavage of disulfide bonds. Both the immunoreactivity pattern and peptide sequencing revealed that the F1 fragment was constituted of the hCG beta-(1-47) sequence, whereas the F2 fragment comprised the 6-47 portion. We then studied the beta NS in urine from either pregnant women or four patients with choriocarcinomas. Results showed that both hCG and the free beta-subunit contained beta NS. Furthermore, free hCG beta present in those urine samples appeared to be extensively, if not totally, nicked. Results observed in urine were confirmed using separation of hCG from its beta-subunit by a two-step chromatography procedure, identification of hCG and hCG beta immunoreactive peaks by specific monoclonal immunoradiometric assay, and analysis of resulting preparations by one-dimensional electrophoresis under reducing conditions, followed by Western immunoblotting with FB12. This latter protocol was also used to investigate the presence of beta NS in sera of four patients with choriocarcinoma tumors. In those sera, hCG appeared to be nicked. This study demonstrates that the beta-subunit of hCG is modified by multiple fragmentations.     

Structure of the human chorionic gonadotropin beta-subunit fragment from pregnancy urine

A major portion of the hCG immunoreactivity detectable in pregnancy urine is derived from a fragment of hCG beta. This lacks the COOH-terminal portion of hCG beta, but retains immunoreactivity with most antibodies raised against the beta-subunit of hCG. To improve clinical measurements of hCG and assess the importance of such fragments in human urine, we have isolated and determined the structure of this molecule. The hCG beta fragment was isolated from a partially purified commercial preparation of hCG (Organon) by gel filtration and immunoaffinity chromatography using monoclonal antibodies. It was found to consist of two polypeptide chains composed of residues beta-(6-40) disulfide-bridged to residues beta-(55-92). It also differs from the beta-subunit of hCG in its carbohydrate structure, lacking sialic acid and having a low but variable amount of galactose. A beta-fragment containing the same two NH2-terminal sequences was also isolated from a single pregnant woman's urine. The two major polypeptides comprising the beta-fragment contain a total of nine half-cystine residues, raising the possibility that a free thiol may exist or that a third undetected disulfide-bridged peptide is present in the intact fragment. However, tests for the presence of a free thiol have been negative. Another intrinsic characteristic of the beta-fragment is the formation of a variable amount of dimer in solutions of neutral pH. beta-fragment will not combine with intact alpha-subunit. Despite the absence of regions beta-(1-5), beta-(41-54), and beta-(93-145), the beta fragment is recognized by the SB-6 antibody and most monoclonal antibodies elicited to the beta-subunit, thus excluding half of the amino acids of the beta-subunit from the epitope(s) where these antibodies bind.     

Glycosylation changes in human chorionic gonadotropin and free alpha subunit as gestation progresses.

Carbohydrate is important to the structure, function, and circulatory survival of the glycoprotein hormones. Human CG (hCG) and the related free alpha-molecule of pregnancy contain four and two asparagine-linked oligosaccharides, respectively. The present study analyzes changes in the glycosylation patterns of hCG and free alpha in early vs. late gestation. Five volunteers provided 24-h urine samples, weekly, throughout their pregnancies. Extracts of early pregnancy (weeks 7-12) and late pregnancy (weeks 28-32) urines were pooled. Early and late samples from each patient were subjected to gel filtration to separate hCG and free alpha, and the populations thus obtained were analyzed by lectin affinity chromatography on Concanavalin A-Sepharose (Con A) and Lens culinaris-agarose (Lch). Using Con A, free alpha and hCG were separated into an unbound fraction (eluted with Con A buffer), a weakly bound fraction (eluted with 10 mmol alpha-methyl-D-glucoside) and a tightly bound fraction (eluted with 500 mmol alpha-methyl-D-mannoside). For free alpha-molecule, a significant decrease in tightly bound Con A forms, was noted from early to late pregnancy with a mean difference of 17.0 +/- 2.4% (P less than 0.01). Concomitantly, in late pregnancy, an increase in Con A unbound forms of free alpha was noted with mean difference of 12.5 +/- 1.7% (P less than 0.01). These changes indicate the presence of more highly branched oligosaccharides on free alpha as gestation advances. No changes were noted in the Con A binding of intact hCG; nearly all hCG bound in both early and late pregnancy. Using Lch, free alpha and hCG were separated into an unbound fraction (eluted with Lch buffer) and a bound fraction (eluted with 500 mmol alpha-methyl-D-mannose). Both free alpha and intact hCG in late pregnancy exhibited increased binding to Lch, with mean differences from early to late pregnancy of 30.2 +/- 4.8% (P less than 0.01) and 11.4 +/- 4.5% (P less than 0.05), respectively. These data indicate increased incorporation of fucose into the carbohydrate moieties in late pregnancy. Taken together, these data derived by analysis using lectin specificity imply the presence of more highly branched, fucosylated oligosaccharides as gestation progresses.     

Fragmentation of the beta-subunit of human chorionic gonadotropin produced by choriocarcinoma

When human chorionic gonadotropin (hCG) was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions with dithiothreitol (DTT), a smaller weight material (CTP'), in addition to the beta-subunit, could be detected by Western blot analysis using antiserum for hCG beta-carboxy-terminal peptide (CTP). The CTP' band was much more apparent with urinary hCG from a patient with choriocarcinoma than with that from normal pregnant women. Second-dimensional electrophoresis of the choriocarcinoma hCG (c-hCG) after reduction with DTT indicated that the CTP', Mr 25,000, was released from the beta-subunit. The carbohydrate structure of the CTP' was analyzed by affinity with lectin-peroxidase on a nitrocellulose membrane. The CTP' did not interact with Concanavalin A, but exhibited strong interaction with both RCA120 and Arachis hypogaea after removal of sialic acid, indicating that it was released as a fragment containing an O-linked sugar chain as was found in the hCG beta carboxy-terminal portion. Western blot analysis using the antisera for hCG alpha, hCG beta, and hCG beta-CTP showed that the CTP' contains not only the carboxy-terminal portion but also a part of the internal (core) portion of the beta-subunit molecule. This dissociation of the c-hCG beta was further supported by the presence of a faster moving component (FMC) which may correspond to the NH2-terminal side counterpart. The desialylated FMC could be detected by Concanavalin A and RCA120 but not by Arachis hypogaea, indicating that it contains N-linked rather than O-linked sugar chains. The FMC does not contain any of the epitopes for the antisera examined in Western blot. These results indicate that the beta-subunit of the choriocarcinoma urine hCG has an unusual site which is dissociated into two components of Mr 25,000 (CTP') and Mr 18,000 (FMC) by DTT reduction.     

A monoclonal antibody against a synthetic peptide is specific for the free native human chorionic gonadotropin beta-subunit

A totally synthetic molecule (109-145 peptide) analogous to the beta-subunit carboxyl terminus was used as an antigen in the development of antibodies by the hybridoma technique. A monoclonal antibody (702 D7) specifically recognized the free native beta-human CG (beta hCG). 702 D7 was of the immunoglobulin G1 subclass and was directed against an antigenic site localized in a 10-amino acid sequence (109-118) or less. The recognition of an epitope located in the 109-118 region could explain the specific recognition of beta hCG observed with 702 D7, in contrast to monoclonal antibodies directed against a 118-145 region with a recognition of both beta hCG and whole hCG, as observed with a second monoclonal antibody (1032) to synthetic peptide. Immunohistochemical results and preliminary data obtained from the immunoradiometric assay show that 702 D7 provides a clinical tool for the detection of free beta-subunit secretion even at low concentrations, and could allow the study of this subunit or its metabolites produced by normal and tumoral cells.     

Evidence for the presence of human chorionic gonadotropin (hCG) and free beta-subunit of hCG in the human pituitary.

Previous studies have indicated that the pituitary gland may produce free alpha-subunit and small quantities of hCG in addition to other glycoprotein hormones. Since synthesis of holo-hCG requires the presence of both subunits, we have investigated the occurrence in human pituitary of free beta-subunit of hCG, in addition to intact holo-hCG. We processed a pituitary extract by fractionated ammonium sulfate precipitation followed by sequential chromatography on Sephadex G-100 and Ultrogel AcA 44. The fractions obtained were assessed for their reactivities with a panel of polyclonal and monoclonal antibodies specific for holo-hCG, beta-subunit of hCG, alpha-subunit, or hCG/LH. In addition to the expected LH and alpha-subunit, we detected materials which eluted from the column in positions very similar to those of cochromatographed 125I-hCG tracer and hCG-beta (NIH CR123-beta), and which showed immunoreactivity in specific immunoradiometric assays for holo-hCG and hCG-beta, respectively. Holo-hCG and hCG-beta material derived from the urine of a postmenopausal woman showed behaviors on the column similar to the pituitary forms. Both the pituitary holo-hCG- and free hCG-beta-subunit activity could be enriched (approximately 500 times) by affinity chromatography on an hCG antibody-coupled Sepharose column. When subjected to isoelectric focusing in granulated gel holo-hCG and hCG-beta-subunit of pituitary origin were focused in the pI-range of pregnancy hCG and pregnancy hCG-beta-subunit, respectively. Like pregnancy hCG, most (75%) of the pituitary hCG was bound to a column of Con A-Sepharose; however, the Con A-nonbinding hCG fraction (approximately 25%) was much higher than that found in pregnancy hCG. On the basis of immunoreactivities, the content of holo-hCG in our pituitary extract was estimated to be 60 micrograms/g, and that of free beta-subunit 45 micrograms/g; for comparison, LH was approximately 20 mg/g, and free alpha-subunit 1.6 mg/g. In addition, we could demonstrate the presence of both holo-hCG- and free hCG-beta-subunit-like immunoreactivity in NaCl-extracts from single pituitaries of two postmenopausal women. In these studies a second hCG-beta-immunoreactive material eluting far behind the hCG-beta-position was found. Chromatography of purified LH-beta-subunit, which crossreacts 1.56% in the hCG-beta IRMA, yielded an elution pattern clearly distinguishable from that of the hCG-beta-immunoreactive substances.(ABSTRACT TRUNCATED AT 400 WORDS)     

A radioimmunoassay for the core fragment of the human chorionic gonadotropin beta-subunit

We developed a RIA for the beta-core fragment of hCG that is excreted in the urine of pregnant women and some patients with cancer. We purified beta-core from crude commercial preparations of hCG (of which beta-core is a major constituent) derived from pregnancy urine and used this purified beta-core material to immunize rabbits. One antiserum (RW25) was particularly useful in that a RIA with purified beta-core as both radioligand and reference preparation had high sensitivity for beta-core detection and low cross-reactivity with other hCG-related molecules and glycoprotein hormones. The cross-reactivities of purified hCG (CR125), hCG beta (CR125), and hCG alpha (CR125) preparations were in each instance less than 3 x 10(-3) (wt/wt). The cross-reactivities of purified pituitary glycoprotein hormones were in each instance less than 2 x 10(-4) (wt/wt). Using the RW25 RIA, virtually all beta-core immunoreactivity in pregnancy urine eluted from Sephadex G-100 in a position coincident with that of purified beta-core. Urine from men and nonpregnant women contained very low levels of beta-core immunoreactivity (less than 6.5 micrograms/L), while urine from pregnant women and patients with testicular cancer or other neoplasms had levels of beta-core immunoreactivity ranging as high as 26,000 micrograms/L. We conclude that the improved specificity of our beta-core RIA will facilitate studies of the physiology and cancer biology of beta-core molecules.     

Isoimmunization against human chorionic gonadotropin with conjugates of processed beta-subunit of the hormone and tetanus toxoid.

The immunogenicity of the conjugate prepared from "processed" beta-subunit of human chorionic gonadotropin (choriogonadotropin, HCG) and tetanus toxoid has been studied in animals and a human subject. The conjugate elicited the formation of high-affinity (Ka = 10(9)-10(11) M-1) anti-HCG and anti-tetanus antibodies. On primary immunization, the antibody, response lasted for several months. Repeat injection of the conjugate in the declining phase of antibody titers produced a booster response without a lag period. The antibodies reacted with the beta-subunit of HCG and the complete HCG molecule but were devois of significant crossreactivity with human growth hormone, placental lactogen, follicle-stimulating hormone, thyroid-stimulating hormone, and luteinizing hormone at tonic and surge levels. The antibodies were competent for neutralizing the biological activity of HCG in the mouse uterine weight gain assay, the ventral prostate weight gain assay, and the radioligand assay for binding of 125I-labeled HCG to receptors on corpus luteum. HCG (5000 international units) administered to an immunized subject was completely bound by circulating antibodies. Administration of HCG (in contrast to conjugate) was without booster effect on anti-HCG titers.     

Human chorionic gonadotropin beta-subunit affects the folding and glycosylation of alpha-cys mutants

Human chorionic gonadotropin (hCG) is a member of a family of heterodimeric glycoprotein hormones that contain a common alpha-subunit but differ in their hormone-specific beta-subunits. Both subunits have five and six disulfide bonds, respectively, which consist of cystine knot structure. It is evident from numerous studies that the structure of beta-subunits is rigid, whereas that of alpha-subunit is flexible and can be molded by a beta-subunit. Previously, we reported that secreted forms of a mutants where either cysteine residue in the disulfide bond 7-31 or 59-87 was converted to alanine contained a disulfide-linked homodimer in addition to a monomer. To study whether the hCGbeta-subunit affects the conformations of alpha mutants, alpha-subunits lacking either the 7-31 or 59-87 disulfide bond were expressed with wild-type (WT) hCGbeta in Chinese hamster ovary cells, and homodimer formation and glycosylation of dimerized alpha-subunit were assessed by continuous labeling with [35S]methionine/cysteine, immunoprecipitation with anti-alpha or -hCGbeta serum, digestion with endoglycosidase-H or -F, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis in a non-reducing condition. Our data showed that a homodimer was not observed in the half-Cys mutants except one, where cysteine at position 7 was converted to alanine, in the presence of beta-subunit. This finding indicated that hCGbeta-subunit rescued the a half-Cys mutants from the formation of intermolecular disulfide-linked homodimer by preferentially combining with the alpha mutants. In both free WT and all mutants treated with endoglycosidase-H, no or faint bands were recognized as the same migration as seen in endoglycosidase-F treatment. Even in the endoglycosidase-H sensitive cases, the amount of sensitive alpha-subunits was less than 5% of total alpha-subunits. In contrast to free alpha-subunits, distinct endoglycosidase-H sensitive bands were seen in both WT and mutants, although the ratio was various. We concluded that hCGbeta-subunit affects the folding and glycosylation of the alpha-subunit mutants.     

Structural and functional studies of the tryptic core of the human chorionic gonadotropin beta-subunit

The beta-subunit of hCG was digested with trypsin to produce a modified form of the subunit for structure-function and immunological studies. After digestion of hCG beta with trypsin, the residual disulfide-linked core was isolated and found to be lacking the carboxy-terminal peptide (residues 115-145) and to contain bond cleavages between residues 2-3, 43-44, 74-75, and 95-96. The locations of these bond cleavages within the disulfide-bridged core were identified by isolation of the following peptides after reduction and S-carboxymethylation of the trypsin beta-core: beta 1-43, beta 3-43, beta 44-74, beta 44-95, beta 75-95, and beta 96-114. The circular dichroic spectrum of the tryptic beta-core over the wavelength region of about 200-320 nm was similar to that of the native subunit. In addition, the tryptic beta-core retained nearly full immunopotency in both polyclonal and monoclonal competitive RIAs and could combine with complementary native alpha-subunit. The hybrid, composed of the tryptic beta-core and native alpha, was purified and displayed a molar potency of about 0.1% relative to intact hCG in both a radioreceptor assay and an adenylate cyclase assay. Thus, the hybrid retained little biological activity. Although the extensive bond cleavages in the tryptic beta-core did not appear to change its secondary and tertiary structure sufficiently to significantly alter the circular dichroic spectrum, the immunoreactivity, or the capability to combine with its alpha-subunit complement, the biological functional integrity of the tryptic beta-core-containing hybrid was essentially abolished. Hence, the tryptic beta-core provides a useful derivative for detailed structure-function studies aimed at defining the necessary determinants for subunit association, receptor binding, and subsequent biological actions.     

The cell-free synthesis of the alpha subunit of human chorionic gonadotropin.

The synthesis of the alpha subunit of human chorionic gonadotropin (hCG) was demonstrated in a cell-free system composed of polysomes derived from first trimester placenta and cell sap prepared from ascites tumor cells. The in vitro synthesized proteins labeled with [35S]methionine were shown to have at least 4 tryptic peptides that co-migrated with the same peptides from authentic hCG. Sodium dodecylsulfate-polyacrylamide gel electrophoresis revealed the synthesis of a discrete protein, migrating with an apparent molecular weight of about 17,000, which contained methionine-labeled tryptic peptides found in the alpha subunit. The level of radioactivity in these tryptic peptides was five times greater with polysomes from first trimester placentae than with those from term placentae. The efficiency of total protein synthesis in both cases was about the same. These data strongly suggest that the decrease in blood levels of hCG after the first trimester is caused by a selective decrease in the rate of synthesis of the hormone.     

Monoclonal antibodies to the free beta-subunit of human chorionic gonadotropin define three distinct antigenic domains and distinguish between intact and nicked molecules.

The present study was designed to characterize monoclonal antibodies (mAbs) specific for the free beta-subunit of hCG (free hCG beta), to develop two-site immunoradiometric assays (m-IRMAs) specific for free hCG beta, and to study the reactivities of various molecular forms of hCG beta in these assays. We attempted first to delineate the antigenic regions present specifically on the free hCG beta by studying the binding pattern of seven mAbs directed preferentially to hCG beta, designated FBT11, P8E, P10F, HB2, P5D, P5H, and INN-64. Competitive inhibition experiments performed by RIA demonstrated the specificity of these mAbs for the free hCG beta as noncross-reacting with the beta-subunit of human (h) LH beta. m-IRMAs were used to analyze the arrangement of epitopes on hCG beta. Experiments performed with the seven mAbs used either as capture antibodies or radiolabeled indicators confirmed the specificity of the seven mAbs for the free hCG beta, and that mAbs FBT11, P8E, and P10F bound to equine LH beta (eLH beta), but did not bind to a fragment of hCG beta called the beta-core fragment (beta CF). These antibodies defined an antigenic domain identified as A. In contrast, mAb HB2 bound neither to eLH beta (e beta) nor to beta CF and was directed to domain B (e beta negative, beta CF negative). Finally, mAbs P5D, P5H, and INN-64 bound to beta CF, but did not bind to eLH beta, and defined a third domain identified as C (e beta negative, beta CF positive). Collectively, these results demonstrate that at least six antigenic domains are present on the free hCG beta and that a limited set of amino acids was shared among these domains; domains A, B, and C are present only on the free beta-subunit, while three other domains recognized by mAbs FB19, FBT10, and 518B7 are present on both free hCG beta and hCG. Thus, most of the surface of the hCG beta appears to be antigenic and accessible to antibody binding. Three different m-IRMAs specific for free hCG beta were then constructed using either mAb FBT11 (domain A) or HB2 (domain B). The study of the reactivities of various molecular forms of hCG beta in these assays demonstrated that the recognition of hCG beta forms nicked at position 43 (beta 43), 44, and/or 51 (beta 44/51) varied between assays.(ABSTRACT TRUNCATED AT 400 WORDS)     

Immunological properties of the beta-subunit of human chorionic gonadotropin. I. Effect of chemical and enzymatic modifications

The beta-subunit of hCG (hCG beta) displays immunological cross-reactivity with human LH (hLH). A detailed study was undertaken to investigate the effect of chemical and enzymatic modifications on the immunological behavior of hCG beta, particularly on its cross-reactivity with hLH, with a view to obtaining a highly hCG-specific antigen. hCG beta was modified in both the carbohydrate and protein parts of the molecule. The carbohydrate part was modified enzymatically by sequential cleavage of monosaccharides by specific glycosidases, and the protein portion was modified chemically in the amino and carboxyl groups and in the cystinyl, tyrosyl, histidyl, and arginyl residues. These derivatives were immunologically evaluated by RIAs; their hCG beta activities were measured in the [125I]hCG beta-anti-hCG beta system, and the hLH cross-reactivity in the [125I]hLH-anti-hLH system. The present studies have led to the following conclusions. 1) The carbohydrate does not play a significant role in the immunological activity of hCG beta. 2) The antigenic determinants of hCG beta reside primarily in the polypeptide chain and are conformational rather than sequential in nature. 3) hCG beta has two types of antigenic determinants, those which are unique to hCG and those which are common to both hCG and hLH. 4) Finally, it is possible to destroy preferentially one or the other type of determinants. The controlled reduction and alkylation of hCG beta yielded derivatives which retain significant immunological activity in the hCG beta system, but have no or reduced cross-reactivity in the hLH system. These derivatives are much more specific antigens than hCG beta and, therefore, are of potential importance for the development of a specific RIA for hCG and possibly for use as contraceptive agents.     

Conformational intermediates in the production of the combinable form of the beta-subunit of chorionic gonadotropin

Human trophoblastic cells synthesize and secrete hCG as well as uncombined forms of the alpha- and beta-subunits of hCG. We have previously reported that the rate-limiting step in alpha beta-dimer assembly in cultured JAR choriocarcinoma cells is a conformational change in beta-subunit accompanied by the formation of intramolecular disulfide bonds. We now report on the intermediate steps in the acquisition of this combinable conformation by the beta-subunit. The earliest biosynthetically labeled form of beta detected in JAR cells is a precursor termed p beta 1 that lacks at least one of the intramolecular disulfide bonds found in mature beta-subunit, that does not combine with alpha-subunit, and that does not react with a monoclonal antibody specific for free beta. The p beta 1 precursor rapidly assumes (within 5 min) a new conformation termed p beta 2 that, in contrast to p beta 1, migrates more slowly on nonreduced sodium dodecyl sulfate-polyacrylamide gels, combines with alpha to form the hCG dimer, and reacts with the monoclonal anti-free beta antibody. Pulse-chase kinetic experiments support the following sequence of events: p beta 1----uncombined p beta 2----combined p beta 2. The transition of p beta 1 to uncombined p beta 2 involves the formation of at least one intramolecular disulfide bond coincident with the conformational shift of the p beta molecule. Furthermore, treatment of the nonreduced subunits with trypsin releases a [35S]cysteine-labeled peptide from p beta 1, but not from either form of p beta 2. This peptide presumably contains one of the two crucial cysteine residues that participate in forming the disulfide bond that distinguishes p beta 1 from the p beta 2 forms. Dimer p beta 2 differs from both p beta 1 and uncombined p beta 2 in that it contains an O-linked N-acetylgalactosamine, which represents the first step in the formation of the O-linked glycans of beta-subunit. Dimer p beta 2 is, therefore, the most fully processed and kinetically the latest of the three p beta forms that appear in JAR cell lysates. We conclude that formation of an appropriate array of intramolecular S-S bonds accompanies the acquisition of a combinable conformation of beta-subunit, and we have identified intermediate steps in the pathway leading to this conformational change. The data suggest that it is the achievement of this conformation by beta-subunit that limits the alpha beta combination reaction rather than the amount or conformation of alpha-subunit.     

Preparation and characterization of antibodies to the urinary fragment of the human chorionic gonadotropin beta-subunit

hCG is a glycoprotein hormone composed of two dissimilar subunits (alpha and beta) and is normally synthesized by trophoblastic tissue. Its measurement by immunoassay is widely employed as a test for pregnancy, but can be complicated by cross-reactivity with human (h) LH. Immunoassays based on the beta-subunit of hCG have been employed to decrease this cross-reactivity with hLH, but when these assays are used with urine specimens, the antibodies employed also detect a fragment of hCG beta, which can lead to significant differences in measurement. To overcome these problems, we have developed a series of monoclonal antibodies to the beta fragment of hCG recovered from pregnancy urine. Some of the antibodies that bind to this beta fragment are directed to a region of hCG beta that is different from the epitopes recognized by antibodies raised against the intact beta-subunit. The new epitopes available in the hCG beta fragment form the basis for novel immunoassays. These beta fragment antibodies are used in conjunction with other antibodies, directed to different epitopes of the hormone, to produce a series of immunoradiometric assays that can discriminate among intact hormone, free hCG beta, and hCG beta fragment. The hCG beta fragment antibodies described herein have affinities between 10(9) and 10(11) M-1 for the beta fragment and exhibit varying degrees of discrimination between the hCG beta fragment, the beta-subunits of hCG and hLH, and intact hCG and hLH.     

Differential occurrence of free beta and free alpha subunits of human chorionic gonadotropin (hCG) in pregnancy sera

Levels of hCG alpha beta dimer, free alpha-subunit and free beta-subunit were measured in pregnancy sera. Dimer and free alpha were quantitated by radioimmunoassays (RIAs) using specific polyclonal antisera. Free beta was quantitated both by monoclonal anti-beta RIA and by polyclonal anti-beta RIA following the complete adsorption of cross-reacting hCG by immobilized alpha-antisera. Consistent with the findings of other laboratories, hCG levels in pregnancy sera peaked at around 10 weeks after the last menstrual period (post LMP), and declined thereafter. Free alpha levels rose as hCG levels declined and accounted for 30-40% of total serum alpha in the third trimester. Although free beta accounted for only a small proportion of the total beta-subunit at the time of the hCG peak and thereafter (2.4-3.6%), in early pregnancy serum samples, 4-6 weeks post LMP, when hCG was generally first detected, an average of 16% free beta was detected. At this time, the higher the hCG level (20-2000 ng/ml), the lower the percent free beta (54-3%). Thus, the free beta portion started high and declined prior to the hCG peak; the free alpha portion increased thereafter. To explain these findings, we propose a two phase regulation of hCG dimer formation. Up to the time of the hCG peak, supplies of alpha-subunit are limiting (hence the presence of free beta). Thereafter, beta-subunit levels drop, restricting dimer formation and leaving uncombined alpha.