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PubMed1989

The transcriptional response of the human chorionic gonadotropin beta-subunit gene to cAMP is cycloheximide sensitive and is mediated by cis-acting sequences different from that found in the alpha-subunit gene.

Cyclic AMP stimulates transcription of the genes encoding the alpha- and beta-subunits of human CG (hCG). Although the cis-acting cAMP response element (CRE) of the alpha-subunit gene has been extensively characterized, relatively little is known about the putative response element of the hCG beta gene. Here, we demonstrate that cAMP stimulation of hCG beta gene transcription requires ongoing protein synthesis, whereas cAMP stimulation of alpha-subunit gene transcription does not. These results suggest that cAMP-stimulated transcription of the alpha and hCG beta genes may involve different cis-acting elements and trans-acting factors. Accordingly, we have constructed a series of deletion mutants consisting of fragments of the hCG beta promoter-regulatory region linked to the bacterial chloramphenicol acetyl transferase gene. To potentiate detection of a CRE, we constructed vectors containing the Rous sarcoma virus enhancer to augment transcriptional activity of the hCG beta-promoter. We also used vectors designed to determine whether regions containing a putative CG beta CRE could confer cAMP responsiveness to a heterologous promoter. These constructs were evaluated for activity by transfection into choriocarcinoma (BeWo) cells. Transient expression of these vectors identifies a broad region which renders transcription of the chloramphenicol acetyl transferase gene responsive to cAMP. Surprisingly, division of the hCG beta 5'-flanking and untranslated regions into smaller fragments led to loss of cAMP responsiveness, suggesting that the hCG beta CRE may be comprised of multiple domains dispersed across the proximal 650 base pairs of CG beta 5'-flanking and untranslated sequence.(ABSTRACT TRUNCATED AT 250 WORDS)

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1995

Asparagine-linked oligosaccharides facilitate human chorionic gonadotropin beta-subunit folding but not assembly of prefolded beta with alpha.

To determine the role of asparagine (N)-linked oligosaccharide chains in protein folding and assembly, the well established hCG-beta in vitro folding and assembly assays were used to analyze how the human CG (hCG) beta-subunit devoid of one or two N-linked glycans folds and assembles under different conditions. Two approaches were used: 1) site-specific mutagenesis of hCG-beta synthesized in Chinese hamster ovary cells transfected with beta-mutants lacking the asparagine glycosylation sites; and 2) enzymatic deglycosylation of hCG-beta synthesized in JAR cells with peptide N-glycosidase F or endoglycosidase H. In both cases, [35S]cysteine-labeled beta-subunits were used as substrates to measure the conversion of the hCG-beta folding intermediate p beta 1 into p beta 2 and assembly of p beta 2 with urinary alpha. Using the mutated substrates from Chinese hamster ovary cells, it was found that 60% of wild-type p beta 1 (two N-linked glycans), 60% of p beta 1 missing the Asn13-linked glycan, 40% of p beta 1 missing the Asn30-linked glycan, and 10% of p beta 1 missing two N-linked glycans were converted to the corresponding p beta 2, respectively. With the enzymatically deglycosylated substrate from JAR cells, 90% of p beta 1 (two N-linked glycans), 70% of p beta 1(1) (one N-linked glycan), and 10% of p beta 1(0) (without N-linked glycan) folded into p beta 2 under cysteamine and cystamine redox conditions with or without protein disulfide isomerase. These data demonstrate that at least one N-linked glycan is required for efficient folding of hCG-beta and that the Asn30-linked glycan is more important than Asn13-linked glycan for hCG-beta folding. It also was shown that the composition of N-linked glycans of hCG-p beta 1 did not change protein folding, since hCG-beta substrates with high mannose oligosacharides folded as efficiently as beta-substrates containing sialylated complex oligosaccharides. Moreover, assembly of the already folded, assembly-component folding intermediate, p beta 2, was not affected by removal of one or both of the N-linked glycans of the beta-subunit. These data thus show that N-linked glycans play their most important role in the folding component of the folding and assembly pathway for hCG-beta.

Animals↗

PubMed1984

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.

Animals↗

PubMed1999

Epitope analysis and molecular modeling reveal the topography of the C-terminal peptide of the beta-subunit of human chorionic gonadotropin.

Human chorionic gonadotropin (hCG) belongs to a family of heterodimeric glycoprotein hormones that share a common alpha-subunit and a hormone-specific beta-subunit. Among the gonadotropin beta-subunits, greater than 85% homology exists between lutropin (hLH)beta and hCGbeta in their first 114 amino acid residues. However, unlike hLHbeta, hCGbeta contains a 31-amino acid hydrophilic stretch at its carboxyl end (CTPbeta: C-terminal peptide). Although the crystal structure of deglycosylated hCG has been solved, the topography of CTPbeta remains unknown. In this study, we have attempted to define the topology of CTPbeta using mAb probes. We investigated three epitopes on hCGalpha, which are hidden in the hCGalphabeta dimer. However, these epitopes are not hidden in hLH, which has a similar subunit interface to that of hCG, but lacks CTPbeta. This suggested that these epitopes are not masked at the subunit interface of hLH or hCG. Hence, we hypothesized that, in the case of hCG, these epitopes are masked by the CTPbeta. Consistent with this view, several treatments of hCG that removed CTPbeta unmasked these epitopes and enhanced their reactivity with the corresponding mAbs. In order to localise the position of CTPbeta on the alpha-subunit, we used an epitope-mapping strategy [N. Venkatesh & G. S. Murthy (1997) J. Immunol. Methods 202, 173-182] based on differential susceptibility of epitopes to covalent modifications. This enabled us to predict the possible topography of CTPbeta. Further, we were also able to build a model of CTPbeta, completely independently of the epitope-mapping studies, using a homology-based modeling approach [S. Krishnaswamy, I. Lakshminarayanan & S. Bhattacharya (1995) Protein Sci. 4 (Suppl. 2), 86-97]. Results obtained from these two different approaches (epitope analysis and homology modeling) agree with each other and indicate that portions of CTPbeta are in contact with hCGalpha in the native hCG dimer.

Animals↗

PubMed1988

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.

Animals↗

PubMed1989

Ectopic production of the beta-subunit of human chorionic gonadotropin in osteosarcoma.

Production of human chorionic gonadotropin by sarcomas is an uncommon phenomenon that has rarely been documented. A case of a 26-year-old woman with osteosarcoma of the left fibula and high levels of serum beta-human chorionic gonadotropin (beta-HCG) is described. Immunoperoxidase staining showed numerous tumor cells reacting with the beta-HCG antibody. Immunohistochemical studies performed on ten additional osteosarcomas demonstrated another case in which some tumor cells presented immunoreactivity for beta-HCG. These results indicate that beta-HCG may be used as a marker of persistent or recurrent disease in those uncommon cases of osteosarcoma in which serum levels of beta-HCG are elevated at the onset of treatment.

Adult↗

PubMed1981

Molar pregnancy in British Columbia: estimated incidence and postevacuation regression patterns of the beta subunit of human chorionic gonadotropin.

A total of 171 cases of molar pregnancy were followed by serial radioimmunoassay of the beta subunit of human chorionic gonadotropin (hCG-beta) over a 4-year period. The incidence over the study interval was 1:1,202 pregnancies in the province. In 120 women with intact uteri and measurable plasma levels of hCG-beta at the initiation of follow-up, the hormone remained detectable for up to 219 days (between 31 and 32 weeks) following evacuation, with 50% of patients remaining positive at 63 days (between 8 and 9 weeks). In five women undergoing hysterectomy, the hormone remained detectable for up to 112 days (between 15 and 16 weeks) postoperatively. In 92% of the cases, hCG-beta regressed to negative without chemotherapy. The prolonged presence of hCG in plasma after evacuation of the mole, regardless of the presence or absence of the uterus, points to persistence of molar tissue and its gradual rejection from uterine and extrauterine sites as a common phenomenon in the natural history of the uncomplicated postmolar course of this disease.

Adolescent↗

PubMed1983

Detection of the free beta subunit of human chorionic gonadotropin (HCG) in cultures of normal and malignant trophoblast cells, pregnancy sera, and sera of patients with choriocarcinoma.

Immunoaffinity adsorption techniques, utilizing specific antisera for hCG and its subunits bound to Sepharose 4B, have been employed to separate hCG alpha beta dimer and free subunits of hCG. As previously reported by this and a number of other laboratories, trophoblast cells (in vivo and in vitro) produce free alpha subunit in addition to hCG dimer. We have now shown that cultured JAr choriocarcinoma cells also secrete free beta subunit: 37% of the total beta subunit (combined and free) secreted by JAr cells is in the free form. Moreover, in pooled sera from choriocarcinoma patients 15% of total beta subunit is free, and in media from placental explant cultures and in pooled first trimester pregnancy sera 11% and 6.5%, respectively, of total beta subunit are in the free form. The free beta s are all of similar mol wt to the combined forms, and associate with urinary hCG alpha to form hCG. Free alpha s, which are larger than the combined forms, are unable to associate with urinary hCG beta to form hCG. We propose that the supply of combinable alpha subunit, rather than beta, limits dimer formation.

Choriocarcinoma↗

PubMed1995

Expression and secretion of the beta subunit of human chorionic gonadotropin by bladder carcinoma in vivo and in vitro.

Expression and secretion of the beta subunit of human chorionic gonadotropin (hCG) by bladder carcinoma cell lines were investigated in vitro and in vivo. As an in vitro study, immunoreactive hCG beta (IR-hCG beta) secreted into the culture media of two bladder transitional cell lines (KoTCC-1 and HT-1197) was analyzed using three kinds of enzyme immunoassays which were specific for intact hCG, free hCG beta, and beta core fragment (beta-CF). Both of the cell lines were determined to secrete IR-hCG beta into the media, which consisted principally of free hCG beta, but detectable levels of intact hCG and beta-CF were not present in the media. Northern blot analysis revealed that the hCG beta gene was expressed in both KoTCC-1 and HT-1197 cells where the sizes of mRNA from these cells were smaller than those from placental and NJG choriocarcinoma cells. As an in vivo study, distribution of IR-hCG beta was analyzed in the tumor tissues, sera, and urine of the mice and the rats transplanted with KoTCC-1 cells. By the immunohistochemical study, the IR-hCG beta was clearly observed in transitional cell carcinoma cells of the transplanted tumor. High levels of IR-hCG beta were detected in both the serum and urine from the animals, but there were quantitative and qualitative differences between serum and urinary IR-hCG beta. Quantitatively, the concentrations of IR-hCG beta in the urine were consistently much higher than those in the serum. Qualitatively, free hCG beta was exclusively detected in the serum whereas high levels of beta-CF in addition to free hCG beta were found in the urine. Intact hCG could not be detected in the serum and urine. These distributions of IR-hCG beta in the animals transplanted with KoTCC-1 cells were completely analogous to those in a patient with hCG beta-producing bladder carcinoma. The present study shows that the same metabolic pathway of IR-hCG beta is operating in mice and rats as in humans, indicating that IR-hCG beta found in patients with bladder carcinoma originates from the tumor and it may be recognized as a tumor marker when beta-CF is measured in the patient's urine.

Animals↗

PubMed1997

Precise gaussian distribution functions of maternal serum alpha-fetoprotein and free beta-subunit of human chorionic gonadotropin for trisomy 21 screening: improved accuracy for patient counseling.

OBJECTIVE: Gaussian equation curves are used to generate baseline curves against which a priori maternal age Down syndrome risks are adjusted to develop likelihood ratios for individual patients. We sought to evaluate the accuracy of these calculations, minimize the affects of outliers, and to make improvements. STUDY DESIGN: Gaussian distribution functions were used to investigate the best model for alpha-fetoprotein and free beta-human chorionic gonadotropin multiples of the median with use of nonlinear regressions. Parameters from distribution functions can be used to compute a more precise likelihood ratio for the decision logic for trisomy 21. A total of 58,297 normal cases and 348 cases of trisomy 21 were computed. RESULTS: Log normal distribution functions generated by nonlinear regression produced excellent but exaggerated goodness of fit R2 to the frequency distributions of the data. For normal cases values were as follows (in mean, SD, and R2, respectively): log alpha-fetoprotein -0.07199, 0.15681, and 0.9970; log beta-human chorionic gonadotropin -0.15203, 0.24284, and 0.9987. For trisomy 21 cases the values were (in mean, SD, and R2, respectively) for log alpha-fetoprotein -0.19303, 0.15802, and 0.9828 and for log beta-human chorionic gonadotropin 0.19996, 0.29760, and 0.9669. Distributions reconstructed with use of statistical means and SDs generated goodness of fit R2 from 0.585 to 0.914. Use of means and SDs derived from distribution functions increased the R2 to 0.855 and 0.999. The change in the model produces, at a 5% false-positive rate, a sensitivity of 57.18% (199/348). A 1 in 113 cutoff point risk is obtained and is tighter than the 1 in 251 without the distribution functions, as versus 1 in 270 by age calculations alone. CONCLUSIONS: Our data suggest that (1) normality of log transforms of alpha-fetoprotein and normality of log transforms of beta-human chorionic gonadotropin are reasonable models, (2) distribution functions can minimize the effect of outliers, which produces more realistic risk estimates, and (3) the effect of distribution functions versus standard mean and SDs cannot automatically be extrapolated to other parameters, which must be tested individually.

Chorionic Gonadotropin, beta Subunit, Human↗

PubMed1980

Detection and quantitation of the beta-subunit of human chorionic gonadotropin in serum by radioimmunoassay.

Qualitative and quantitative radioimmunoassays (RIA) specific for the beta-subunit of human chorionic gonadotropin (HCG) produced by Wampole Laboratories and Monitor Science Corporation were compared in a study involving the sera from 213 individuals. Qualitative assays from both manufacturers were found to be equivalent in clinical specificity (99.2%). However, the clinical sensitivity of the Wampole beta-Tec qualitative RIA (95.6%) was greater than that of the Monitor Science beta-CG qualitative RIA (89%) in detecting hCG in serum. In quantitative assays, the Wampole beta-Tec RIA gave serum values 1- to 10-fold higher than those obtained using the Monitor Science beta-hCG system when the same patient or control serum was assayed. Preliminary studies comparing two additional quantitative RIA kits (Serno hCG-beta and Bio-RIA hCG-beta rapid RIA) indicate that the results obtained with the quantitative Wampole and Monitor Science reagents are not peculiar to these two assay systems. Possible explanations of the cause of such aberrant results are presented. It is concluded that variable results can be expected when assaying the same serum sample with different RIA reagents for beta-hCG owing to the lack of an acceptable reference preparation for hCG that can be utilized by all commercial or private producers of such reagents.

Chemical Phenomena↗

PubMed1983

Structurally restricted conjugates of the beta-subunit of the human chorionic gonadotropin to produce antisera.

Structurally restricted conjugates, beta-hCG(-NH2)4(-NHC-NH-NH2)11(-C-O-TT)1-11, were synthesized by protecting the amino and guanidino functional groups of the beta-subunit of the human chorionic gonadotropin (beta-hCG) with t-Boc groups; then, the carboxyl groups of the protected intermediate were coupled by carbodiimide reagents with basic groups of tetanus toxoid (TT). After removal of the t-Boc groups, the immunogen has a total of fifteen amino and guanidino functional groups, which may be superior to carboxyl groups, for immunologic recognition. Conjugates, prepared in non-polar and aqueous systems, had ratios of beta-hCG to TT of 13:1 and 7:1, respectively. Immunization of rabbits yielded antisera which showed an inhibition of 50% for 41-62 ng/ml, beta-hCG, at final dilution ratios of up to 1:80,000.

Animals↗

PubMed1996

Silencing of the gene for the beta subunit of human chorionic gonadotropin by the embryonic transcription factor Oct-3/4.

The transcription factor Oct-3/4 may be important in maintaining embryonic cells in an undifferentiated state. It is probably down-regulated at about the time that human chorionic gonadotropin (hCG) is first expressed in embryonic trophectoderm. Here we report that Oct-3/4 strongly inhibits the hCGbeta subunit (hCGbeta) promoter in JAr choriocarcinoma cells. Oct-3/4 reduced chloramphenicol acetyltransferase (CAT) reporter expression from the -305hCGbeta promoter by about 90% in transient co-transfection assays, but had no effect on expression from the -249hCGbeta promoter. The -305/-249 hCGbeta fragment specifically bound synthetic Oct-3/4 protein as measured in electrophoretic mobility shift assays, and the Oct-3/4-binding site was localized around -270 by methylation interference footprinting. Site-directed mutagenesis of this binding site abolished Oct-3/4 repression. When stably transfected into JAr cells, Oct-3/4 reduced the amounts of both endogenous hCGbeta messenger RNA and hCG protein to less than 10% of controls. We suggest that silencing of Oct-3/4 in trophectoderm is a prerequisite for hCG up-regulation in early human embryos at the time of maternal recognition of pregnancy.

Base Sequence↗

PubMed1988

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.

Amino Acid Sequence↗

PubMed2004

Endometrial adenocarcinoma associated with elevated serum concentrations of the free beta subunit of human chorionic gonadotropin.

We report a case of a histologic grade II endometrial adenocarcinoma without trophoblastic differentiation in a 24-year-old woman with an elevated serum concentration of human chorionic gonadotropin (hCG) and with no evidence of pregnancy. Serum and urine specimens were used to study the hCG immunoreactivity. Qualitative tests performed on serum and urine using 5 different assays produced conflicting results. The hCG concentration in serum and urine was quantified using assays designed to detect different molecular forms of the molecule; analysis revealed that serum hCG immunoreactivity was due entirely to the presence of the free beta subunit. Immunohistochemical analysis performed on tissue samples showed strong cytoplasmic staining for hCG. While hCG is a well-recognized tumor marker in gynecologic malignant neoplasms, immunoreactivity most often is due to the presence of both intact molecule and the free beta subunit. To our knowledge, this is the first report of an endometrial adenocarcinoma producing only the free beta subunit of hCG.

Adult↗

PubMed1997

Carbohydrate and peptide structure of the alpha- and beta-subunits of human chorionic gonadotropin from normal and aberrant pregnancy and choriocarcinoma.

Human chorionic gonadotropin (hCG), purified from the urine of 14 individuals with normal pregnancy, diabetic pregnancy, hydatidiform mole, or choriocarcinoma, plus two hCG standard preparations, was examined for concurrent peptide-sequence and asparagine (N)- and serine (O)-linked carbohydrate heterogeneity. Protein-sequence analysis was used to measure amino-terminal heterogeneity and the "nicking" of internal peptide bonds. The use of high-pH anion-exchange chromatography coupled with the increased sensitivity of pulsed amperometric detection (HPAE/PAD) revealed that distinct proportions of both hCG alpha- and beta-subunits from normal and aberrant pregnancy are hyperglycosylated, and that it is the extent of the specific subunit hyperglycosylation that significantly increases in malignant disease. Peptide-bond nicking was restricted to a single linkage (beta 47-48) in normal and diabetic pregnancy, but occurred at two sites in standard preparations, at three sites in hydatidiform mole, and at three sites in choriocarcinoma beta-subunit. In the carbohydrate moiety, alpha-subunit from normal pregnancy hCG contained nonfucosylated, mono- and biantennary N-linked structures (49.3 and 36.7%, means); fucosylated biantennary and triantennary oligosaccharides were also identified (7.3 and 6.9%). In choriocarcinoma alpha-subunit, the level of fucosylated biantennary increased, offset by a parallel decrease in the predominant biantennary structure of normal pregnancy (P < 0.0001). The beta-subunit from normal pregnancy hCG contained fucosylated and nonfucosylated biantennary N-linked structures; however, mono- and triantennary oligosaccharides were also identified (4.6 and 13.7%). For O-linked glycans, in beta-subunit from normal pregnancy, disaccharide-core structure predominated, whereas tetrasaccharide-core structure was also detected (15.6%). A trend was demonstrated in beta-subunit: the proportions of the nonpredominating N- and O-linked oligosaccharides increased stepwise from normal pregnancy to hydatidiform mole to choriocarcinoma. The increases were: for monoantennary oligosaccharide, 4.6 to 6.8 to 11.2%; for triantennary, 13.7 to 26.7 to 51.5% and, for O-linked tetrasaccharide-core structure, 15.6 to 23.0 to 74.8%. For hCG from individual diabetic pregnancy, the principal N-linked structure (34.7%) was consistent with a biantennary oligosaccharide previously reported only in carcinoma; and sialylation of both N- and O-linked antennae was significantly decreased compared to that of normal pregnancy. Taken collectively, the distinctive patterns of subunit-specific, predominant oligosaccharides appear to reflect the steric effect of local protein structure during glycosylation processes. The evidence of alternative or "hyperbranched" glycoforms on both alpha- and beta-subunits, seen at low levels in normal pregnancy and at increased or even predominant levels in malignant disease, suggests alternative substrate accessibility for Golgi processing enzymes, alpha 1,6 fucosyltransferase and N-acetylglucosaminyltransferase IV, in distinct proportions of subunit molecules.

Amino Acid Sequence↗

PubMed1992

Polypeptide nicks cause erroneous results in assays of human chorionic gonadotropin free beta-subunit.

Pregnancy and trophoblastic disease testing laboratories measure human chorionic gonadotropin (hCG; human choriogonadotropin) free beta-subunit to screen for Down syndrome and to diagnose persistent trophoblastic disease (invasive mole and choriocarcinoma). The results from various laboratories, however, vary widely for similar groups of patients. Average concentrations of free beta-subunit reported for persistent trophoblastic disease, for instance, range from 2.6% to 37% of total hCG. On hCG and its free beta-subunit, peptide bonds can be missing between beta-subunit residues 44 and 45 or between residues 47 and 48 (nicked molecules). To explore the possibility that the disparity in the reported concentrations of free beta-subunit was due to differences in recognition of nicked molecules by different antibodies, we generated 0% and 100% nicked beta-subunit standards and investigated their recognition in three separate immunoassays of free beta-subunit. The immunoassay with antibody 1E5 did not recognize nicked beta-subunit (4% cross-reactivity with nicked beta-subunit) and thus detected only intact beta-subunit. The immunoassay with antibody FBT11 gave similar results with nicked and nonnicked thus standards (96% cross-reactivity with nicked beta-subunit), as did the assay with antibody B204 (73% cross-reactivity with nicked beta-subunit). These two immunoassays thus measured total (nicked + nonnicked) beta-subunit. We used the three immunoassays to examine sera from normal pregnancy, Down syndrome pregnancy, hydatidiform mole, and persistent trophoblastic disease, all of which contain nicked and nonnicked beta-subunit molecules. The results obtained resembled the studies with nicked beta-subunit standards. The results from the FBT11 and B204 assays (total beta-subunit) were highest, results from the 1E5 assay (intact molecules only) being as much as 10 times lower. We conclude that nicks in beta-subunit and the extent of recognition of nicked molecules by different antibodies affect the concentrations reported for free beta-subunit.

Amino Acid Sequence↗

PubMed1993

Simultaneous assay of alpha-fetoprotein and free beta subunit of human chorionic gonadotropin by dual-label time-resolved immunofluorometric assay.

We developed a simple, rapid two-step dual-label assay for the noncompetitive determination of alpha-fetoprotein (AFP) and beta subunit of human chorionic gonadotropin (hCG beta) in serum. Monoclonal antibodies to detect AFP and hCG beta were labeled with europium (Eu) and samarium (Sm), respectively. Highly fluorescent chelates were developed by using the Delfia enhancement principle. The detection limits for AFP and hCG beta were approximately 0.02 kIU/L and approximately 0.2 IU/L, respectively. The within-run precision was < 5% over the whole range of AFP (1-500 kIU/L) and hCG beta (1-200 IU/L) concentrations tested. Cross-reaction of intact hCG was < 0.03%. The AFP concentrations determined with the dual-label assay correlated well with those obtained by Delfia AFP single-label kit. The concentrations of hCG beta were in good agreement with recently published data. Storing the serum samples for 24 h or 1 week at room temperature increased the hCG beta concentration by 4% and 26%, respectively. At 35 degrees C this dissociation of hCG increased 30-40-fold. Repeated freezing and thawing had no effect on the hCG beta concentration.

Antibodies, Monoclonal↗