Free Β-subunit Research Articles

False-Positive hCG Assay Results Leading to Unnecessary Surgery and Chemotherapy and Needless Occurrences of Diabetes and Coma

Much concern has been raised by the unraveling at the hCG Reference Service of six cases of persistent phantom human chorionic gonadotropin (hCG). These are false-positive hCG results, which are likely attributable to human anti-mouse IgG or to heterophilic antibodies (1)(2)(3). The hCG Reference Service, started in January 1998 to aid with the interpretation of irregular or discordant hCG immunoassay results, requests parallel serum and urine samples. Each is tested in four separate two-step microtiter plate ELISAs (assay 1 detects intact hCG, assay 2 detects nonnicked or bioactive hCG only, assay 3 detects the hCG free β-subunit only, and assay 4 detects the hCG β-core fragment only) at three different concentrations (undiluted, a 1:2 dilution, and a 1:5 dilution). From the data, inferences are made about the nature (nonnicked or nicked hCG, free β-subunit, and β-core fragment) and likely source (trophoblast disease, pituitary hCG, cancer, or phantom hCG) of the hCG immunoreactivity. …

Phantom hCG and Phantom Choriocarcinoma

Phantom hCG and phantom choriocarcinoma syndrome (pseudohypergonadotropinemia) refers to persistent mild elevations of hCG, leading physicians to treat patients with cytotoxic chemotherapy for choriocarcinoma when in reality no true hCG or trophoblast disease is present. We report here three cases of the phantom hCG and phantom choriocarcinoma syndrome referred to the hCG Reference Service. In the first case, low levels of hCG were detected in serum (49 to 89 IU/liter) 11 months after the patient had a miscarriage. The presumptive diagnosis of choriocarcinoma was made. After two courses of chemotherapy and a hysterectomy low levels of hCG were still detected. Samples were sent to the hCG Reference Service. While low levels of hCG were detected in serum by three different assays (17, 22, and 9.2 IU/ml), no hCG was detected in the urine. When serum was diluted, levels did not decrease parallel to the dilution. The lack of dilutional parallelism and absence of urine reactivity indicated that the molecule measured was a pseudogonadotropin or phantom hCG, an interfering substance in hCG tests. Therapy was halted. In the second case, a positive serum pregnancy test was recorded 7 years after a normal pregnancy. A pelvic ultrasound and a laparoscopy revealed no pregnancy. Blood hCG levels stayed between 48 and 74 IU/liter over a 3-month period. Samples were sent to the hCG Reference Service. Low levels of hCG, free β-subunit, and β-core fragment were detected in serum using four specific assays. No hCG immunoreactivity was found in the urine sample. None of the four assay results declined parallel to dilution. Again, phantom hCG was diagnosed. In the third case, a positive serum pregnancy test was recorded 1 year after the patient had a normal pregnancy. A pelvic ultrasound revealed no fetal sac. Low levels of hCG (51–135 IU/liter) persisted for 3 months. A preumptive diagnosis of choriocarcinoma was again made. After three cycles of chemotherapy, low levels of hCG were still detected. Samples were sent to the hCG Reference Service. Low levels of hCG immunoreactivity were detected in serum in just one of three hCG assays (13 IU/liter). No immunoreactivity was detected in the urine sample. Again, phantom hCG was diagnosed, and all therapy was halted. Care is needed in interpreting persistent low levels of hCG in patients with no history of trophoblast disease. It is important for the laboratory to show dilutional parallelism in the hCG results and presence of hCG in serum and urine samples in order to exclude phantom hCG before diagnosing choriocarcinoma.

Evaluation of the Abbott system in maternal serum screening for Down syndrome: the AxSym analyzer, AFP and hCG reagents and Maciel Prenatal Interpretive Software

Multi-marker maternal serum screening for Down syndrome in the second trimester is now part of routine care in many centers. Most protocols use a combination of hCG (or its free β-subunit) and α-fetoprotein (AFP) or unconjugated estriol (UE3), or both. Risk calculation is based on these maternal serum marker values combined with maternal age and trisomy 21 maternal age-related risk. Computer programs are therefore necessary. Both technical and statistical efficiency are included in the final risk evaluation. We studied the Abbott system, comprising AxSym analyzer, AFP and hCG kits and Prenatal Interpretive Software (Maciel). Median values were established between 14 and 18 weeks of amenorrhea in a population of 1822 patients and in twin pregnancies in 157 cases. Forty maternal sera from trisomy 21 affected pregnancies were analyzed. Software was evaluated in a population of 429 patients and in 124 cases of trisomy 21. We conclude that this system constitutes an accurate and efficient method of maternal serum screening for Down syndrome.

Comment on the Nature and Specificity of Bayer Corporation and Chiron Diagnostics hCG Immunoassays

We wish to update information in the recent hCG review article (1) , regarding the specificities and antibody configurations used in the Chiron Diagnostics and Bayer Corporation hCG immunoassays. The review article depicted the diverse nature of the human chorionic gonadotropin (hCG) molecule. As described, a non-nicked hCG (the hormone), a nicked hCG, a free α-subunit, and a free β-subunit are present in blood samples, and the same molecules plus a β-core fragment occur in urine samples. As described in the review article (1) , commercial hCG assays sold in the United States use a variety of antibodies directed to different sites on hCG and related molecules. Some assays …

HCG and the free β‐subunit as screening tests for Down syndrome

Published studies have reached varying conclusions as to the benefit of replacing human chorionic gonadotropin (hCG) measurements with the free β-subunit of hCG (the free β-subunit) for Down syndrome screening. One study reports 14 per cent higher detection for the free β-subunit, while another finds an actual loss in detection. To explore this issue further, we directly compared the screening performance of hCG and the free β-subunit, alone and in combination with other serum markers, using banked sera obtained prior to amniocentesis and karyotyping. Altogether, 52 Down syndrome and 5065 unaffected pregnancies were studied. Sera were thawed and assayed for hCG and the free β-subunit over 1 year. At a 5 per cent false-positive rate, the detection rate for hCG in combination with maternal age and alpha-fetoprotein was higher than when the free β-subunit was substituted (62 versus 57 per cent). Ultrasound dating and adding unconjugated oestriol both increased detection. The present findings, along with those from six case–control studies (our re-analysis), indicate that the screening performances of hCG and the free β-subunit are similar (median change in detection 0, range −8 to +3 per cent). Under optimal sample collection and transportation conditions, laboratories can expect to achieve similar screening performance using either hCG or the free β-subunit measurements. © 1998 John Wiley & Sons, Ltd.

HCG and the free β-subunit as screening tests for Down syndrome

Published studies have reached varying conclusions as to the benefit of replacing human chorionic gonadotropin (hCG) measurements with the free beta-subunit of hCG (the free beta-subunit) for Down syndrome screening. One study reports 14 per cent higher detection for the free beta-subunit, while another finds an actual loss in detection. To explore this issue further, we directly compared the screening performance of hCG and the free beta-subunit, alone and in combination with other serum markers, using banked sera obtained prior to amniocentesis and karyotyping. Altogether, 52 Down syndrome and 5065 unaffected pregnancies were studied. Sera were thawed and assayed for hCG and the free beta-subunit over 1 year. At a 5 per cent false-positive rate, the detection rate for hCG in combination with maternal age and alpha-fetoprotein was higher than when the free beta-subunit was substituted (62 versus 57 per cent). Ultrasound dating and adding unconjugated oestriol both increased detection. The present findings, along with those from six case control studies (our re-analysis), indicate that the screening performances of hCG and the free beta-subunit are similar (median change in detection 0, range -8 to +3 per cent). Under optimal sample collection and transportation conditions, laboratories can expect to achieve similar screening performance using either hCG or the free beta-subunit measurements.

Screening for Down syndrome using urine hCG free β-subunit in the second trimester of pregnancy

Human chorionic gonadotropin (hCG) free beta-subunit levels were determined in 709 control and 13 Down syndrome urine samples from the second trimester of pregnancy. Results were normalized to urine creatinine concentration and converted to multiples of the unaffected pregnancy medium (MOM). The concentration of free beta-subunit in Down syndrome cases was 3.9 MOM. Seven of 13 Down syndrome pregnancies (54 per cent) had free beta-subunit levels at or above the 95th centile of unaffected pregnancies. Urine free beta-subunit may potentially be useful as a screening test for Down syndrome.

Immunoassay of human chorionic gonadotropin, its free subunits, and metabolites

Multiple hCG-related molecules are present in pregnancy serum and urine samples. These include non-nicked hCG (the hormone), nicked hCG, hyper- and hypoglycosylated hCG, hCG missing the C-terminal extension, free alpha-subunit, large free alpha-subunit, free beta-subunit, nicked free beta-subunit, and beta-core fragment. Over 100 immunoassays are sold for quantifying hCG-related molecules in serum or urine. Each measures nonnicked hCG and one of seven combinations of the other hCG-related molecules. This is the source of interassay discordance in hCG determinations. Whereas minor variations are noted in different kit results in normal pregnancy samples (more than twofold variation), much larger variations may be found in two immunoassay results in irregular gestations (spontaneous abortion, aneuploidy, preeclampsia, cancers, and trophoblast disease). Care is needed in choosing an immunoassay. What the assay measures may be more important than its cost or speed. This article reviews the structure of hCG and related molecules. It examines the stability and degradation of hCG, and recognition of hCG-related molecules by different types of immunoassay. Also reviewed are new assays for specifically detecting these other hCG-related molecules.

Precise gaussian distribution functions of maternal serum α-fetoprotein and free β-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 α-fetoprotein and free β-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 R 2 to the frequency distributions of the data. For normal cases values were as follows (in mean, SD, and R 2, respectively): log α-fetoprotein –0.07199, 0.15681, and 0.9970; log β-human chorionic gonadotropin –0.15203, 0.24284, and 0.9987. For trisomy 21 cases the values were (in mean, SD, and R 2, respectively) for log α-fetoprotein –0.19303, 0.15802, and 0.9828 and for log β-human chorionic gonadotropin 0.19996, 0.29760, and 0.9669. Distributions reconstructed with use of statistical means and SDs generated goodness of fit R 2 from 0.585 to 0.914. Use of means and SDs derived from distribution functions increased the R 2 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 α-fetoprotein and normality of log transforms of β-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. (Am J Obstet Gynecol 1997;177:882-6.)

Dual-label time-resolved immunofluorometric assay for simultaneous determination of pregnancy-associated plasma protein A and free β-subunit of human chorionic gonadotrophin

Using time-resolved fluorometry, a simple one-step dual-label immunometric assay has been developed, which allows simultaneous determination of pregnancy-associated plasma protein A (PAPP-A) and free β-subunit of human chorionic gonadotrophin (β-hCG) in first-trimester maternal serum samples. Two monoclonal antibodies were biotinylated and immobilized onto the surface of streptavidin-coated microtitration plates, and used to capture PAPP-A and β-hCG, respectively. Europium (Eu) and Samarium (Sm) chelates were conjugated to two additional monoclonal antibodies acting as detection antibodies for PAPP-A and β-hCG. The assay was performed using a 4-h one-step format. The within-run precision with buffer-based calibrators was below 8% over the working range of PAPP-A (40–10 000 mIU/l) and β-hCG (7.3–525 μg/l) and no hook effect was observed. The intra- and inter-assay coefficients of variation were below 7.1% for serum samples. PAPP-A and β-hCG concentrations measured by the dual assay in 39 first-trimester serum samples correlated excellently with those obtained by DELFIA single-label PAPP-A ( r=0.997) and the β-hCG part ( r=0.993) of the DELFIA AFP/βhCG dual-label assay.

Regulation of the DNA binding of p53 by its interaction with protein kinase CK2

Some of the numerous functions of the growth suppressor protein p53 are regulated by its interaction with viral and cellular proteins. C-terminal sequences of p53 are implicated in binding to the regulatory β-subunit of protein kinase CK2. Using a p53-specific DNA binding element we found that the β-subunit of CK2 inhibited the DNA binding of p53 whereas the α-subunit had no influence. The CK2 holoenzyme consisting of two α- and two β-subunits led to a supershift in DNA binding of p53 similar to the p53-specific monoclonal antibody PAb421 as well as the C-terminus of p53. Thus, our results showed an individual role of the free β-subunit of CK2 on the DNA binding activity of p53.

The beta-core fragment of human chorionic gonadotrophin inhibits growth of Kaposi's sarcoma-derived cells and a new immortalized Kaposi's sarcoma cell line.

Kaposi's sarcoma (KS), a condition often associated with HIV infection, is more common in men than in women; pregnancy and sex hormones could be involved. Urinary human chorionic gonadotrophin (hCG) has been reported to inhibit the growth of KS cell lines, with great variability among preparations. Urinary hCG often contains free forms of the hCG subunits and a fragment of the free beta-subunit, the beta-core, which may have biological activity. We compared the effect of the beta-core fragment, the beta-subunit, recombinant and urinary hCG on KS immortal and spindle cells. A new immortal KS cell line was phenotypically and karyotypically characterized. The effects on growth of this cell line and of primary KS spindle cells by hCG and its purified derivatives were tested. Induction of apoptosis was demonstrated using acridine orange/ethidium bromide staining. The beta-core fragment harboured the most potent growth inhibitory activity on a molar basis. After 72 h of treatment with the beta-core, 60-70% of KS cells show apoptotic nuclei. No effects were observed on endothelial cells. The beta-core fragment of hCG proved to be the most effective part of the hCG molecule, inducing growth inhibition and apoptosis of KS cells. Thus, the beta-core could be the most appropriate hCG derivative for the therapy of KS.

Stability of hCG free β-subunit and β-core fragment in urine

Stability of hCG free β-subunit and β-core fragment in urine

Pathophysiological importance of various molecular forms of human choriogonadotropin

Human chorionic gonadotropin (hCG), its subunits and fragments are widely used for diagnostic purposes. In addition to the diagnosis of pregnancy and pregnancy related disorders, hCG determinations are used for diagnosis of trophoblastic and recently also nontrophoblastic tumors. The use for diagnosis of nontrophoblastic tumors requires highly specific and ultrasensitive assays. With these, it is possible to measure the concentrations of both hCG, the free β-subunit and the so called β-core fragment in healthy subjects. Therefore it is important to establish reference values for these and also to be aware of the influence of physiological factors on the serum and urine concentrations. Improved standardization of the assay methods is also essential for these novel applications of hCG determinations to become useful.

Urinary analysis for Down's syndrome: is the measurement of urinary β-core the future of biochemical screening for Down's syndrome

Most Down's Screening protocols have concentrated on the analysis of oncofetal antigen levels present in serum. Urine has largely been ignored, but mass screening based on a urine text has several logistical advantages. We have examined the levels of urinary β-core as a marker of Down's syndrome. (β-core is a major immunoreactive degradation product of hCG and in particular its β-subunit). Elevated maternal serum hCG is the single most effective biochemical marker of Down's syndrome and levels are more than doubled. Measurement of elevated free β-subunit has been shown to be a superior discriminator than measurement of intact or total-hCG. This was shown by an increase in comparative serum levels from 2.04 to 2.41 multiples of the control population medium (MoM). The median MoM value of β-core for Down's cases in studies has varied between 4.38 and 6.28. The proportion of true Down's cases having a β-core value greater than the 95th centile of the controls varied from 61 to 93%. This is far superior to any single serum marker. If other complimentary urinary markers can be found urinary screening could replace serum screening not only because of the logistical advantages but increased sensitivity.

Characterization of a monoclonal antibody reacting with the free human luteinizing hormone beta-subunit.

Measurement of serum luteinizing hormone (hLH) is important for the detection and follow-up of patients with pathological processes of the reproductive axis. Detection of the uncombined form of the beta-subunit of hLH, or free hLH beta, has proved to be of clinical interest in the recognition of gonadotroph adenomas. As no monoclonal antibody specific for the free hLH beta is at present available, we elicited monoclonal antibodies using free hLH beta as an immunogen. An antibody, named BLH01, was selected for its specific binding to the free beta-subunit, and its antibody-binding site was characterized at the molecular level, emphasizing the importance of amino acid residues located between the disulfide-bonded Cys93 and Cys100. This region has been demonstrated as being particularly critical for the specific binding of lutropic hormones to their receptors. Topographic assignment of the epitope recognized by BLH01 was then achieved by cross-matching studies based on a library of antibodies directed to hLH beta, and the location of some epitopes on the three-dimensional model of the beta-subunit is proposed. A two-site immunoassay based on BLH01 as capture antibody was then developed. This assay, using BLH01, may constitute a simple, sensitive and highly specific procedure for assessing the clinical usefulness of measuring free hLH beta, particularly for the diagnosis and follow-up of patients presenting with pituitary adenomas.

A new series of mycobacterial expression vectors for the development of live recombinant vaccines

Recombinant BCG (bacillus Calmette-Guérin) is a promising candidate as a live vaccine delivery system. Thus far, however, only autoreplicative plasmids carrying the heterologous genes to be expressed in BCG, together with antibiotic-resistance genes, have been successfully used. This could potentially lead to the spreading of antibiotic resistance among other bacteria, and might therefore be unsafe for the environment. In this study, we present a series of three Escherichia coli-Mycobacteria shuttle vectors which enable expression and secretion of antigens without the use of antibiotic-resistance markers. All these plasmids confer mercury resistance to the host bacteria as the only selectable marker and contain a unique restriction site to allow for single-step in-frame cloning of open reading frames downstream from the Mycobacterium tuberculosis 85A antigen promoter and export signal. The system was used to express the free β-subunit of human chorionic gonadotropin (hCGβ), a potential target of an immunotherapeutic vaccine.

Nicked free β-subunit of human chorionic gonadotropin: A potential new marker for Down syndrome screening

OBJECTIVE: Our purpose was to investigate maternal serum levels of nicked free β-subunit in normal and Down syndrome pregnancies. STUDY DESIGN: Serum specimens were obtained from 64 karyotypically normal and 6 Down syndrome pregnancies before amniocentesis. Two immunoenzymometric assays for the β-subunit of human chorionic gonadotropin were used to determine the level of free β-subunits with peptide linkage breaks (”nicks”) between residues 43 to 48. RESULTS: The mean level of nicked free β-subunit in Down syndrome was 4.76 multiples of the median, which was significantly elevated compared with normal controls (107 multiples of the median, p < 0.05). In 5 of 6 cases levels were ≥2 multiples of the median, with a range 1.96 to 15.43 multiples of the median, which was 2- to 3-fold higher than the regular free β-subunit assay (mean level 1.53 multiples of the median, range 0.70 to 3.10 multiples of the median). In one case no nicked free- β subunits were detectable. CONCLUSION: Our preliminary data indicate that nicked free β-subunit of human chorionic gonadotropin might be a sensitive marker for Down syndrome screening. (A M J O BSTET G YNECOL 1996;174:609-11.)

Serum levels of β-subunit of chorionic gonadotropin in patients with pituitary tumors

Many studies have shown that normal and tumoral pituitary is able to synthesize chorionic gonadotropin (CG). The aim of the present work was to investigate the circulating levels of free beta-subunit of CG (CG-beta) in a large number of patients with pituitary tumors in basal conditions and after thyrotropin-releasing hormone (TRH) injection. The study includes 27 healthy subjects, 23 patients with prolactinoma, 20 with growth hormone-secreting adenoma and 77 with non-functioning pituitary adenoma (NFPA). The CG-beta was evaluated using a new one-step immunometric assay employing two monoclonal antibodies directed against epitopes present only on the free CG-beta and showing a detection limit of 0.04 U/l and a cross-reactivity with complete CG < 0.01%. In basal conditions, serum CG-beta was undetectable in healthy subjects and in the majority of patients, while in seven patients with NFPA and four with prolactinoma the CG-beta values ranged between 0.05 and 0.72 U/l. In these 11 patients serum levels of intact CG were found within the normal range (normal range < 5 U/l), while two patients with NFPA and one with prolactinoma had levels of free alpha-subunit inappropriately high with respect to gonadotropins and thyrotropin. Injection of TRH caused CG-beta to increase in two out of 16 patients with NFPA, whereas it was ineffective in 12 healthy subjects and 10 patients with prolactinoma. The present data indicate that detectable level of CG-beta not associated with hypersecretion of the intact CG molecule may be observed in about 10% of patients with NFPA or prolactinoma, while abnormal CG-beta responses to TRH are observed infrequently in individual patients with NFPA.

Formation of A hCG variant with truncated peptide moiety as a biosynthetic product in vitro

Summary In serum of tumor patients variants of human chorionic gonadotropin (hCG) or of the free β-subunit with lower molecular weights may be present as well as proteolytically nicked forms. The origin of these variants, secreted by cells or formed in the periphery, is unknown. In the present study, brefeldin A (BFA) was used to block transiently the anterograde transport from the ER to the Golgi apparatus in JAr cells to increase the residency of hCG in the ER and thus to amplify possible proteolytic processes taking place in this cellular compartment. In JAr cells, BFA (2 hours, 0.05-0.4 μg/ml) diminished the amount of secreted free β-subunit (up to 90%) in a dose-dependent and reversible manner; hCG secretion was less affected. In the BFA cultures, the molar ratio hCG/free was increased up to five-fold. Pulse-chase experiments, using [ 3 H]-Leu for labeling of newly synthesized protein, showed the presence of lower molecular weight variants of the hCG-α and β subunits when hCG was arrested in the ER in presence of BFA. This could be observed most clearly in 8-bromo-cAMP (1 mM) stimulated cultures. In BFA treated cultures, the free β-subunit as well as the β-subunit contained in hCG showed and about 3 kD lower apparent molecular weight in comparison to the β-subunits of controls (34 kD). The lower molecular weight variants of the β-subunits still contained the carboxy-terminal peptide (CTP) since they were bound to anti-CTP-Sepharose. The carbohydrate part could also not account for the difference in the apparent molecular weight since it was not abolished by removal of the N-linked carbohydrate residues. The results demonstrate the capability of the ER to produce truncated variants of the free β-subunit and a truncated β-subunit contained in hCG which are secreted in vitro . During a transient arrest of the de novo synthesized proteins in the ER, the free β-subunit as well as hCG may be most probably proteolytic cleaved in the amino-terminal domain of the β-subunit or in the core part of the molecule.