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PubMed2002

Monoclonal antibody against free beta-subunit of human chorionic gonadotropin.

Spleen cells from BALB/c mice immunized with free native human chorionic gonadotropin hormone beta-subunit (beta hCG) were fused with mouse myeloma cells (P3/X63-Ag8) and one hybridoma secreting monoclonal antibodies (MAbs), was obtained. This hybridoma specifically recognizes beta hCG and does not cross-react with other human glycoprotein hormones, such as luteinizing hormone (LH), follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH), and human chorionic gonadotropin (hCG). The MAb was of the IgG(1) subclass and ascitic fluid from this hybridoma was purified by affinity chromatography on Protein A-Sepharose CL-4B column to isolate the IgG(1) active fraction. The affinity constant of this MAb was 1.5 x 10(10)M(-1).

Animals↗
PubMed1988

Development of highly sensitive immunoassays to measure human chorionic gonadotropin, its beta-subunit, and beta core fragment in the urine: application to malignancies.

A variety of malignancies have been associated with the presence of human chorionic gonadotropin, hCG, its subunits, and fragments of its beta-subunit in blood and urine. The usefulness of these hCG-related tumor markers in nontrophoblastic malignancies has been inhibited by inadequate assay techniques. In order to achieve the required sensitivity and specificity, concentration steps and other procedures to remove cross-reacting human luteinizing hormone were necessary. In addition, the coexistence of a fragment of the hCG-beta or beta human luteinizing hormone subunit contributes to significant errors of measurement in urine. The importance of the hCG-beta fragment as a potential tumor marker has been recognized previously but no method was available to measure this antigen readily. We report here the development of a series of radioimmunometric, two-site assays which will accurately measure hCG, hCG-beta subunit, and the beta-subunit fragment directly in small volumes of unprocessed urine. These assays are highly specific, extremely sensitive, and not labor intensive since they employ microtiter plate procedures. Application of these assays to urine samples from patients with gynecological malignancies indicated that over 50% of all patients tested excreted the hCG-beta fragment in their urine. Also, this fragment comprised more than 50% of the moles of hCG immunoreactive components present in the specimens that were positive for hCG. This cancer marker is also demonstrable in trophoblastic malignant states such as choriocarcinoma in which the low molecular weight fragment can also be visualized directly by immunoblotting procedures. We conclude that a search for hCG immunoreactivity in the urine of patients with malignancies will be improved by the inclusion of accurate measurements of the prominent quantities of the beta fragment excreted by these individuals.

Biomarkers, Tumor↗
PubMed1992

The carbohydrate chains of the beta subunit of human chorionic gonadotropin produced by the choriocarcinoma cell line BeWo. Novel O-linked and novel bisecting-GlcNAc-containing N-linked carbohydrates.

The N-linked carbohydrate chains of the beta subunit of human chorionic gonadotropin (hCG-beta) isolated from the culture fluid of the choriocarcinoma cell line BeWo were released enzymatically by peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F. Subsequently, the O-linked oligosaccharides were split off from the N-deglycosylated protein by mild alkaline borohydride treatment. The carbohydrate chains were purified in their intact sialylated forms by FPLC anion-exchange chromatography on Mono Q, HPLC on Lichrosorb-NH2, and high-pH anion-exchange chromatography on CarboPac PA1. 1H-NMR spectroscopic analysis of the major fractions demonstrates the occurrence of the following sialylated diantennary and triantennary N-linked oligosaccharides. Residues not written in bold letters are variably present. [formula: see text] The incidence of triantennary carbohydrate chains is much higher than in normal urinary hCG-beta (26% vs 2%). The same holds for the alpha 1-6-fucosylation of the asparagine-bound GlcNAc (95% vs 42%). The presence of a bisecting GlcNAc and the occurrence of alpha 2-6-linked Neu5Ac in the most abundant N-glycans, are new features for hCG-beta. The major O-linked carbohydrate chains identified are the tetrasaccharide Neu5Ac alpha 2-3Gal beta 1-3(Neu5Ac alpha 2-6)GalNAc-ol and the hexasaccharide Neu5Ac alpha 2-3Gal beta 1-4GlcNAc beta 1-6(Neu5Ac alpha 2-3Gal beta 1-3)GalNAc-ol, both also found in normal urinary hCG. In addition, two novel O-glycans were characterized: [formula: see text]

Acetylglucosamine↗
PubMed1998

Expression of the alpha- and beta-subunits of human chorionic gonadotropin by subsets of parathyroid cells in states of hyperparathyroidism.

The alpha-subunit of human chorionic gonadotropin (hCG-alpha) has previously been found to be expressed in hyperplasias and tumours of numerous endocrine tissues including all those involved in MEN-I syndrome except the parathyroid glands. In the present immunohistochemical investigation of 86 patients with various states of hyperparathyroidism, expression of hCG-alpha by subsets of parathyroid cells was shown in 46 cases (54 per cent) including all states of hyperparathyroidism investigated: primary adenoma (n = 34, 44 per cent); uraemic secondary hyperplasia (n = 34, 53 per cent); MEN-I (n = 13, 77 per cent); MEN-II (n = 2, 100 per cent); and parathyroid carcinoma (n = 3, 100 per cent). Although the number of parathyroid cells expressing hCG-alpha was in general low, the occurrence of numerous immunoreactive cells appeared to be concentrated in primary adenoma and MEN-I (20 and 33 per cent of positive cases, respectively). No expression was found in ten normal control glands, except for very rare cells in one case. Expression of hCG-alpha was in part associated with that of hCG-beta, which appeared to be more commonly expressed than hCG-alpha in cases of secondary hyperparathyroidism. In separate experiments, Bouin fixation was found to preserve the immunoreactivity of hCG-alpha and hCG-beta better than the formalin fixation used in this study, suggesting that the figures may be underestimates. These immunohistochemical results are in agreement with a previous biochemical study showing hCG-alpha and hCG-beta in extracts of parathyroid tumours and extend to the parathyroid glands the otherwise ubiquitous finding of hCG-alpha expression in MEN-I-related neoplasms.

Adenoma↗
PubMed2006

Comparative study of three amniotic fluid markers in premature rupture of membranes: prolactin, beta subunit of human chorionic gonadotropin, and alpha-fetoprotein.

OBJECTIVE: To evaluate whether prolactin, alpha-fetoprotein (AFP) or B-human chorionic gonadotropin (BHCG) is the most effective marker in vaginal fluid for diagnosing prelabor rupture of membranes (PROM). These proteins are present in amniotic and vaginal fluid and have been reported to be potent markers of PROM, but have not been used clinically nor compared to each other. STUDY DESIGN: A total of 100 pregnant women between 28 and 37 weeks of gestation were recruited for the study. Patients were divided into 2 groups. The first group consisted of 50 pregnant women diagnosed with ruptured membranes. The second group consisted of 50 normal pregnant women seen during routine antenatal clinic visit (control) group. All women underwent speculum examination aiming to sample prolactin, BHCG and AFP in the vaginal fluid. Ultrasonographic examination for gestational age and amniotic fluid index measurement was performed. The electrochemoluminescence (ECLIA) method was used for quantitative measurement of the three proteins (the total duration of the assay was 18 min). RESULTS: Vaginal fluid concentrations of the three markers were significantly higher in the PROM group than in the control group (p < 0.001). Receiver operator curve analysis indicated that AFP had 94% specificity, sensitivity, positive and negative predictive values, and efficiency. The other two markers have lower specificity, sensitivity, positive and negative predictive values, and efficiency: 70, 76, 71.7, 74.5 and 73% for prolactin and 72, 84, 75, 81.8 and 78% for BHCG, respectively. CONCLUSION: This work demonstrates that of the three markers investigated AFP has the highest diagnostic performance. Using the ECLIA method it can be an ideal marker for diagnosing PROM particularly in equivocal cases. The technique could be introduced into laboratory tests to meet clinical needs. Basel.

Adult↗
PubMed1981

Antifertility effects of immunization of female baboons with C-terminal peptides of the beta-subunit of human chorionic gonadotropin.

Fifteen adult female baboons were immunized with a conjugate of tetanus toxoid coupled with a synthetic carboxy-terminal peptide of the beta-subunit of human chorionic gonadotropin and another 15 were immunized with tetanus toxoid. Freund's complete adjuvant was used in all immunizations. All females were placed with males of proven fertility during the third menstrual cycle after the primary immunization and those not sustaining pregnancy were mated in two additional consecutive cycles. Four pregnancies occurred following 42 matings in the conjugate-immunized group and 15 pregnancies resulted from 21 matings in the tetanus toxoid-immunized animals. Low levels of antibodies reactive to baboon chorionic gonadotropin were found in the pregnant animals in the conjugate-immunized group.

Animals↗
PubMed2006

Time-dependent folding of immunological epitopes of the human chorionic gonadotropin beta-subunit.

We have explored the possibility to use 14 different monoclonal antibodies in order to follow the formation of the respective epitopes during the biosynthesis of hCG subunits and their association in JEG-3 choriocarcinoma cells using pulse (30s to 5 min)-chase (0-180 min) experiments. We found central cystine knot epitope structures (epitope beta1) to be formed immediately and simultaneously with epitopes on the protruding hCG-beta loops 1 and 3. We found also differences in the time-dependent folding of beta2 and beta4 epitopes, which are highly overlapping structures on the loops 1+3. These differences were reinforced by decreasing the temperature during the pulse-chase experiments to 25 degrees C. Moreover, we describe for the first time an intracellular intact hCG beta-subunit form that showed the transient expression of the hCG-beta-core fragment epitope beta11 in the course of the maturation of this subunit which casts new light on the presence of hCG-beta-core fragment in Down's syndrome, tumors and pregnancy.

Antibodies, Monoclonal↗
PubMed1982

Absence of the COOH-terminal peptide on ectopic human chorionic gonadotropin beta-subunit (hCG beta).

A comparison of the physical properties and immunoreactivity of ectopic hCG beta from cultured cervical cancer cells and standard hCG beta was performed. The heterogeneity of the preparations was examined by ion-exchange chromatography, and the molecular size by analytical gel filtration. Two distinct forms of ectopic hCG beta were identified. One form was indistinguishable from standard hCG beta, while the other, although larger, lacked the characteristic COOH-terminal peptide (CTP). This was shown by the failure of antisera specific for determinants on the cTP to recognize this molecule, and by the apparent absence of the 0-linked oligosaccharides and thermolysin cleavage site normally found in this region.

Agglutinins↗
PubMed1987

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.

Adenylyl Cyclases↗
PubMed1988

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.

Adolescent↗
PubMed1994

Human chorionic gonadotropin beta-subunit nicking enzymes in pregnancy and cancer patient serum.

hCG is a dimer composed of an alpha- and a beta-subunit, joined noncovalently. In addition to the hCG dimer, uncombined alpha- and beta-subunits (free beta) and nicked hCG and free beta molecules (cleaved at 44-45 or 47-48) can be detected in the circulation. Of these circulating molecules, only the intact hCG dimer fully expresses biological activity. The pathways that dissociate, nick, and degrade hCG and beta-subunit molecules in pregnancy are unknown and could have a major role in regulating hormone levels. Immunoassays for intact (nonnicked) hCG and intact (beta-subunit (with < 1% detection of nicked molecules) and a subtractive immunoassay system for measuring nicked hCG levels have been described previously. A multiantibody scavenger assay is described here for measuring nicked beta-subunit levels (< 6% detection of intact beta-subunit). In this report we use these four assays to assess conversion of intact hCG or beta-subunit to nicked forms over time (nicking enzyme activities) in control (healthy nonpregnant), pregnant, and cancer patient serum samples. Pools of pregnancy and control sera were supplemented with intact hCG and its dissociated beta-subunit and incubated at 37 C. Intact and nicked molecule measurements were made between 0-48 h. In two different pools of control sera, no loss of intact hCG or intact beta-subunit and no significant gain in nicked hCG or nicked beta-subunit were detected over 48 h. This indicated a lack of nicking enzyme activity in control serum. In two different pools of first trimester pregnancy sera, we found no obvious loss of intact hCG or gain of nicked hCG levels over 48 h. However, we found 70% and 62% losses (pools 1 and 2) of intact beta-subunit and 51% and 39% gains of nicked beta-subunit over the same time period. We inferred that an uncombined or free beta-subunit-modifying activity was present in pregnancy serum. We repeated the pregnancy serum experiment with six different concentrations of beta-subunit (0.62-29 mg/L). A linear relationship, percent nicking against time, existed for the six concentrations for up to 6 h at 37 C (r = 0.97); after that, the rate of nicking declined. A plot of rate against concentration against revealed a classical Michaelis-Menten enzyme relationship (logarithmic regression, r = 0.96). The pregnancy serum beta-subunit nicking activity was partially purified by gel filtration. A single peak of activity emerged, eluting between the 150,000-443,000 mol wt standards.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed2005

[Metastatic tumor of spermatic cord with elevation of serum human chorionic gonadotropin beta-subunit].

A 62-year-old man was presented with a firm mass in right scrotum. Serum LDH and AFP were within normal range, but hCG-beta was elevated (2.3 ng/ml). Under the diagnosis of right testicular tumor, he underwent right radical orchiectomy. The specimen was a spermatic cord tumor with poorly differentiated adenocarcinoma. hCG-beta was still elevated postoperatively and gastric fiber revealed adenocarcinoma of the stomach. Histochemical staining for hCG-beta was positive in both tumor of the spermatic cord and stomach. Finally the tumor was diagnosed as metastatic tumor of spermatic cord from gastric cancer, causing the elevation of hCG-beta.

Adenocarcinoma↗
PubMed1996

[Expression of cDNA of human chorionic gonadotropin beta-subunit (beta-hCG) cDNA in insect cells and effect of expressed product on mouse lymphocytes in vitro].

Expression vector pVL 1393-hCG beta containing beta-hCG cDNA has been constructed using an unfused protein nuclear polyhedrosis virus (AcNPV) expression vector. The insect cells (Sf 9) were cotransfected by the expression vector and nuclear polyhedrosis virus genomic DNA, and recombinant virus AcNPV-hCG beta was screened out, beta-hCG cDNA was expressed in insect cells infected by recombinant virus and recombinant beta-hCG (r beta-hCG) was secreted into medium. The purity of r beta-hCG, purified by immuno-affinity chromatography, was about 90% and the molecular weight of r beta-hCG was 22,500 Da. Like hCG, r beta-hCG suppressed significantly proliferation of induced lymphocytes, as well as production of IL-2 to some extent, on a parallel with suppression of lymphoproliferation.

Animals↗
PubMed1989

Predicting pregnancy outcome after in vitro fertilization and embryo transfer using estradiol, progesterone, and human chorionic gonadotropin beta-subunit.

Two hundred twenty consecutive in vitro fertilization (IVF) conception cycles were studied prospectively in order to examine the predictive value of serum human chorionic gonadotropin beta-subunit (beta-hCG), estradiol (E2), and progesterone (P) in predicting pregnancy outcome between 2 and 4 weeks after oocyte collection. To examine the predictive value of each hormone in predicting clinical pregnancy outcome, each patient's result at each week was assigned a scoring system based on the 25th percentile value of the concentration of beta-hCG, E2, and P in ongoing singleton IVF pregnancies for each week of the study. All but one ectopic pregnancies had scores of 0 or 1 points between 2 and 4 weeks after oocyte collection. The scores in successful pregnancies were significantly higher than in unsuccessful pregnancies 2 weeks after oocyte collection. Ninety percent of women scoring 3 points 2 weeks after oocyte collection had ongoing IVF pregnancies. The authors conclude that determination of serum beta-hCG, E2, and P concentrations between 2 and 4 weeks after oocyte collection provides clinically useful information not only in the prediction of ectopic IVF pregnancy, but also, conversely, in the identification of IVF pregnancies that are destined to be ongoing.

Chorionic Gonadotropin↗
PubMed1997

Mass spectrometric characterization of the beta-subunit of human chorionic gonadotropin.

A high-performance liquid chromatographic/electrospray mass spectrometric (HPLC/MS) technique is described for the characterization of the beta-subunit of the glycopeptide human chorionic gonadotropin (hCG). The beta-subunit of hCG was dissociated from the alpha-subunit using 0.1% trifluoroacetic acid (TFA) and separated by reversed-phase HPLC using a 0.1% TFA-acetonitrile gradient. Although reductive alkylation with 4-vinylpyridine allowed direct observation of the intact beta-subunit of hCG by HPLC/MS due to the increase in charge, the heterogeneity of the carbohydrate fractions resulted in poor detection limits and extremely complex spectra. After reductive alkylation with either iodoacetate or 4-vinylpyridine, tryptic fragments of either the alpha- or beta-subunit can be observed using reversed-phase HPLC/MS. HPLC/MS data were consistent with the reported primary sequence, although oligosaccharide attachment sites at both 127Ser and 132Ser could not be documented. Microheterogeneity of the carbohydrate moiety on both N-glycosylation sites on the beta-subunit could be readily observed. A larger degree of heterogeneity was observed on 13Asn. Differences were also observed in the oligosaccharide distribution in three commercial preparations of hCG. Detection of the C-terminal portion of the beta-subunit required enzymatic deglycosylation prior to HPLC/MS analysis.

Chorionic Gonadotropin↗
PubMed2002

Production of human chorionic gonadotropin-beta subunit associated with an osteolytic meningioma. Case report.

An osteolytic meningioma in a 36-year-old woman was accompanied by elevated serum levels of human chorionic gonadotropin-beta subunit (beta-HCG), which returned to normal after removal of the tumor. Light microscopy examination demonstrated a transitional meningioma. Immunohistochemical analysis revealed that the tumor cells had a positive reaction for beta-HCG. This case illustrates the possibility that meningioma may be associated with clinically detectable secretion of beta-HCG. To the authors' knowledge, this is the first case in which meningioma has been shown to secrete beta-HCG. The authors believe that meningioma should be considered in the differential diagnosis of choriocarcinoma, embryonal cell tumor, germinoma, and metastatic ovarian tumor associated with elevated levels of beta-HCG.

Adult↗