Commercial hCG Research Articles

Generation of epitope-specific hCG aptamers through a novel targeted selection approach.

Human chorionic gonadotropin (hCG) is a glycoprotein hormone used as a biomarker for several medical conditions, including pregnancy, trophoblastic and nontrophoblastic cancers. Most commercial hCG tests rely on a combination of antibodies, one of which is usually specific to the C-terminal peptide of the β-subunit. However, cleavage of this region in many hCG degradation variants prevents rapid diagnostic tests from quantifying all hCG variants in serum and urine samples. An epitope contained within the core fragment, β1, represents an under-researched opportunity for developing immunoassays specific to most variants of hCG. In the study described here, we report on a SELEX procedure tailored towards the identification of two pools of aptamers, one specific to the β-subunit of hCG and another to the β1 epitope within it. The described SELEX procedure utilized antibody-blocked targets, which is an underutilized strategy to exert negative selection pressure and in turn direct aptamer enrichment to a specific epitope. We report on the first aptamers, designated as R4_64 and R6_5, each capable of recognising two distinct sites of the hCG molecule-the β-subunit and the (presumably) β1-epitope, respectively. This study therefore presents a new SELEX approach and the generation of novel aptamer sequences that display potential hCG-specific biorecognition.

The concentration of commercial HCG trigger is imprecise and inaccurate

The concentration of commercial HCG trigger is imprecise and inaccurate

Absence of TGF-β Receptor Activation by Highly Purified hCG Preparations.

Recently, several LH/human chorionic gonadotropin (hCG) receptor-independent activities for hCG have been described, including activation of the TGF-β receptor (TGFβR) by hyperglycosylated hCG and stimulation of trophoblast invasion. Because the hCG concentrations used in these studies have been rather high, reflecting physiological hCG levels in pregnancy, even a minor contamination with growth factors, which act at very low concentrations, may be significant. Several commercial hCG preparations have been found to contain significant amounts of epidermal growth factor (EGF), which we also confirmed here. Furthermore, we found that some hCG preparations also contain significant amounts of TGF-β1. These hCG preparations were able to activate ERK1/2 in JEG-3 choriocarcinoma cells or TGFβR in mink lung epithelial cells transfected with a reporter gene for TGFβR activation. No such activation was found with highly purified hCG or its free β-subunit (hCGβ), irrespective of whether they were hyperglycosylated or not. Taken together, our results suggest that the growth factor contaminations in the hCG preparations can cause activation of TGFβR and, at least in JEG-3 cells, MAPK signaling. This highlights the importance to carefully control for potential contaminations and that highly purified hCG preparations have to be used for biological studies.

Investigation of an Immunoreactive Chorionic Gonadotropin-Like Substance in the Placenta, Serum, and Urine of Pregnant Bottlenose Dolphins (Tursiops truncatus)

This study was designed to test the hypothesis that an immunoreactive chorionic gonadotropin (CG)like substance is present in full-term dolphin placentas and to determine if CG immunoreactivity can be detected in corresponding serum and urine samples for potential application to diagnose pregnancy. Six placentas were collected immediately after parturition from four captive bottlenose dolphins in 2003, 2007 (Experiment 1), and 2011 (Experiment 2). Serum and urine were collected during early, middle, and late pregnancy from the same dolphins. In Experiment 1, an eCG radioimmunoassay (RIA) was used to analyze dilutions of supernatants from the homogenates of each placenta for eCG immunoreactivity, and a commercial hCG Enzyme-Linked Immunosorbent Assay (ELISA) was used to analyze individual serum samples and dilutions of pools of serum and urine for hCG immunoreactivity. Specific CG immunoreactivity was not detected above assay sensitivities in any of the supernatants of respective placental homogenates, including the highest concentrate (100 mg/mL), nor in any of the individual samples and pools of serum and urine. In Experiment 2, the highest placental homogenate was increased five-fold (500 mg/mL), sensitivity of the eCG RIA was increased six-fold, and a different combination of hCG antibodies was used in an alternative commercial “sandwich”-type ELISA. Despite the optimization, specific CG immunoreactivity in placental tissue and individual serum and urine samples was not detected above assay sensitivities. In conclusion, the hypothesis that an immunoreactive CG-like substance is present in full-term dolphin placentas was not supported. In addition, non-immunoreactivity of a CG-like substance in serum and urine samples collected during various stages of pregnancy precluded the development and application of a CG-based immunoassay for diagnosing pregnancy status in dolphins.

Inappropriate Use of Commercial Human Chorionic Gonadotropin Assays

I am disappointed that the recent Q & A article in Clinical Chemistry dealing with assays for human chorionic gonadotropin (hCG) did not mention that all commercial hCG assays in the US are FDA cleared only for the early detection of pregnancy(1). The current situation is very …

The Role of Human Chorionic Gonadotropin as a Thyroid Stimulator in Normal Pregnancy

The first clinical reports of a placental thyroid stimulator arose from studies indicating that placental tumors that secrete very large amount of human chorionic gonadotropin (hCG), hydatidiform moles and choriocarcinomas, were associated with hyperthyroidism [summarized by Yoshimura and Hershman (1)]. In 1967, Burger reported that impure, commercial hCG had thyroid-stimulating activity in a mouse bioassay (2). Several years later my laboratory (3) and that of Nisula (4) reported that highly purified hCG had activity in this bioassay. We found that the molar TSH was hCG (4). Because hCG varies in its carbohydrate composition, there has been considerable controversy in regard to the potency of these variants. hCG lacking its C-terminal tail, therefore closely resembling LH, is a more potent TSH than native hCG (5). Desialylated basic variants of hCG are more potent in binding to the TSH receptor than fully sialylated acidic hCG (6). However, the acidic hCG has a longer half-life in the circulation than the more basic partially desialylated hCG, and this may contribute to greater thyrotropic potency in humans (7). These studies mainly arose from patients with placental tumors or from a few patients with gestational thyrotoxicosis associated with high hCG levels. The possibility that hCG plays a role in regulation of the thyroid gland during normal pregnancy was first considered when there were more sensitive immunoassays for TSH. Braunstein and Hershman (8) reported that there was an inverse relationship between TSH and hCG at about 10–12 wk of pregnancy, the time of peak hCG levels. Harada et al. (9) further advanced this concept by showing the inverse relationship more clearly and amassing data showing an increase of free T4 and free T3 associated with the peak hCG. We postulated that hCG caused an increase of free thyroid hormone levels, thus suppressing TSH, albeit within the physiological range of serum TSH. Glinoer et al. (10), in a landmark study using a more sensitive TSH measurement, further refined the mirror image temporal relationship between hCG and TSH in normal pregnancies. Other reports also contributed to the concept that hCG is a thyroid stimulator in the early part of pregnancy when hCG levels are very high (11). In this issue of the Journal, Haddow et al. (12) have done a complex analysis of the relationship between hCG, TSH, and free T4 during the first trimester (wk 11–13) and second trimester (wk 15–18) in 9562 women with singleton pregnancies. They found that higher hCG levels were associated with a lower body mass index. This fits with the observation of Goodwin et al. (13) that hCG is higher in women with nausea of pregnancy and hyperemesis gravidarum than in women who do not have any nausea, assuming that weight gain is restricted by nausea. More importantly, the data of Haddow et al. further strengthen the role of hCG as a thyroid stimulator in normal pregnancy. At wk 11–13, higher hCG correlates with higher free T4 levels, although the relationship is a shallow slope, confirming the data of Glinoer et al. (10) that in normal pregnancy hCG only modestly increases free T4. The relationship between hCG and TSH is more difficult to appreciate in the complex analysis by Haddow et al. (12). It shows clearly that low TSH is associated with high levels of hCG, but when TSH is in the higher deciles, there is no correlation with hCG. My interpretation is simplistic. Placental secretion of hCG is not regulated by thyroid hormones. However, when hCG levels are high, they increase thyroid hormone secretion, and the negative feedback at the level of the pituitary suppresses TSH release. When TSH levels are in the higher part of the normal range, or perhaps slightly elevated for pregnancy, the contribution of hCG to increase free T4 and T3, resulting in lower TSH levels, is not sufficient to lower serum TSH significantly. Perhaps evolution designed the hCG system as a backup for stimulating the thyroid gland to produce essential amounts of thyroid hormone, but this backup system did not fully evolve to perfection.

Analytical and clinical evaluation of a human chorionic gonadotrophin plus β (hCG + βhCG) immunoassay in germ cell tumours and gestational trophoblastic disease

Background. – In gestational trophoblastic disease (GTD) and germ cell tumours (GCT), irregular human chorionic gonadotrophin (hCG) forms may be the principal source of immunoreactivity. These hCG variants are detected in a variable manner by commercial hCG immunoassays. Methods. – We analytically evaluated the automated electrochemiluminescent Elecsys hCG+β assay (second version) from Roche Diagnostics and compared the method with a free β-hCG IRMA in 248 follow-up samples from 15 patients with GTD or GCT. Results. – Intraassay and interassay imprecision were, respectively ≤ 2% and 5%. Functional sensitivity was ≤ 0.25 IU/l. We found 19 discordant results in 248 samples (7.7%) measured with both the hCG+β and the free β-hCG assay. All 19 were ≤ to the detection limit of the free β-hCG assay and > to the normal cut-off value in the hCG+β assay. An increased half-life of hCG (resulting in delayed clearance) in comparison to free β-hCG can explain 13 of the 19 discordant results. The remaining discordant samples suggest an increased sensitivity of the Elecsys hCG+β assay in comparison to the free β-hCG IRMA, probably because of the broad reactivity of the former for clinically relevant hCG isoforms (intact, nicked, β-core and free β-hCG). Conclusion. – The Elecsys hCG+β assay is a sensitive and precise automated method and is reliable for monitoring patients with GTD and GCT.

Choriocarcinoma with negative urinary and serum beta human chorionic gonadotropin (β HCG) : A case report

This was a rare case where a patient presented clinically as a case of post abortal sepsis and ultrasound showing the picture of an intramural degenerating fibroid. Her serum and urine both were negative for beta human chorionic gonadotropin (betaHCG). Patient succumbed to choriocarcinoma 1 month later. Failure to detect urinary and serum betaHCG lead to maternal mortality due to the choriocarcinoma. The failure to detect, certain degradation products of HCG which may predominate in gestational trophoblastic neoplasia, by many common HCG testing kits lead to the error of diagnosis. Only 3 of the 7 common commercial serum HCG tests appropriately detects nicked HCG and its free betaHCG, DPC immulite assay, being the most sensitive method. Though of rare occurrence, this awareness is important for diagnosis and follow-up of gestational trophoblastic neoplasia and could have been life saving in our case.

Stimulation of Kaposi's sarcoma cell growth by urine from women in early pregnancy, the current source for clinical-grade human chorionic gonadotropin preparations.

Clinical-grade preparations of human chorionic gonadotropin (hCG) have been shown to be toxic to Kaposi's sarcoma (KS) cells. However, the results of clinical studies using commercial hCG preparations KS remain highly contradictory. More particularly, some hCG preparations could have a paradoxical growth effect on KS. Such discrepant results may be explained by the fact that the anti-KS activity is not associated with hCG itself but with one or more factors that are co-purified with the hormone. We found here that crude urine from first trimester pregnant women, the current source for commercial hCG, had a growth stimulatory effect on KS cells. By contrast, urine from last trimester pregnant women, from non-pregnant young women, from menopausal women and from men exhibited neither a growth stimulatory nor a growth inhibitory effect on KS cells. The amplitude of this pregnancy urine-associated pro-KS activity/hCG unit was higher than that achieved with clinical-grade hCG preparations. Partial co-purification of pregnancy-associated factors during the extraction procedure of commercial hCG from urine may explain the pro-KS activity achieved with some hCG preparations. We, therefore, suggest a cautious use of hCG purified from pregnancy urine for the treatment of KS.

Inhibition of septic shock in mice by an oligopeptide from the β-chain of human chorionic gonadotrophin hormone

Human chorionic gonadotrophin (hCG) is a heterodimeric placental glycoprotein hormone required in pregnancy. In human pregnancy urine and in commercial hCG preparations (c-hCG) it occurs in a variety of forms, including breakdown products. Several reports have suggested modulation of the immune system by intact hormone, but such effects of breakdown products have not been reported. In a related article (Hum Immunol 62:1315, 2001), it is reported that a 400–2000 Dalton (Da) fraction from c-hCG and from human pregnancy urine inhibits Th1-mediated diabetes in NOD mice. The active component(s) were called natural (immuno)modulatory pregnancy factor(s) (NMPF). This study reports that a single treatment with the same low molecular weight NMPF fraction up to 24-h after high dose lipopolysaccharide (LPS) injection inhibited septic shock in mice. This counteracting effect of NMPF paralleled the downregulation of the effects of LPS on the production of macrophage migration inhibitory factor (MIF) by spleen cells, on the plasma level of liver aminotransferase, and on the expression of several splenic lymphocyte and macrophage surface markers. Based on the primary structure of the β-chain of hCG a synthetic hexapeptide Valine-Leucin-Proline-Alanine-Leucine-Proline (VLPALP) was designed, which demonstrated it to have the same protective effects as the 400–2000 Da NMPF fraction. These results indicate a new strategy for the treatment of septic shock and the potential of therapeutic use of this synthetic oligopeptide.

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 …

Co-purification of a ribonuclease and human chorionic gonadotrophin β-core protein from human urine and displacement of 125I-human luteinizing hormone from Candida albicans binding sites by ribonucleases

An 18 kDa pregnancy urine protein preparation, purified to apparent electrophoretic homogeneity as judged by silver-staining of polyacrylamide gels, inhibited binding of 125I-hLH (human luteinizing hormone) to Candida albicans microsomes, reacted with monoclonal and polyclonal antibodies raised against human chorionic gonadotrophin (hCG) β-core protein and exhibited ribonuclease (RNase) activity. Eleven of the 12 amino acids at the N-terminus of a protein in this preparation were identical to those of the N-terminus of human non-secretory ribonuclease. These results indicate co-purification of hCG β-core with a RNase. An 18 kDa RNase was also purified from a commercial hCG preparation (Chorulon). However, no RNase activity was detected in a highly purified commercial preparation (Profasi). Three commercial RNase preparations displaced 125I-hLH from C. albicans binders at extremely low concentrations (<0.001 μg/ml RNase) whereas only slight displacement of 125I-hLH from sheep luteal binding sites was observed with very high concentrations of the RNases (100 μg/ml RNase). The co-purification of hCG β-core and RNase from pregnancy urine and the displacement of 125I-hLH from C. albicans binding sites by RNases may be related to the close relationship that has been identified between mammalian RNase inhibitors and the extracellular domain of gonadotrophin receptors. The presence of RNase in commercial preparations of gonadotrophins should be borne in mind during any investigations that involve impure preparations of these hormones.

Immunoprocedures for detecting human chorionic gonadotropin: clinical aspects and doping control

The pregnancy hormone human chorionic gonadotropin (hCG) is also present at low concentrations in plasma and urine of men and nonpregnant women. hCG immunoreactivity occurs in various molecular forms: Besides the intact hCG heterodimer, considerable amounts of proteolytically cleaved forms, free subunits, and fragments are found in plasma and urine. Especially in urine, proteolytic fragments constitute a major part of the hCG immunoreactivity. The different forms of hCG cross-react to various degrees in immunoassays and constitute a problem for standardization of specific hCG determinations. After injection of hCG (10,000 IU of Pregnyl; Organon), above-normal concentrations of hCG can be detected in serum and urine for 7-11 days. Most immunoassays for hCG also measure hCG beta. Quantitative hCG determinations are mainly performed on serum samples, and very few commercial hCG determinations have been validated for determination of urine samples. Considerable care must therefore be exercised when utilizing such assays to analyze urines for doping control.

Specific activity of polypyrrole nanoparticulate immunoreagents: comparison of surface chemistry and immobilization options.

Polypyrrole-based colloids with differing surface chemistries were compared with respect to the specific activity of immobilized antibody. Monoclonal antibody to the alpha subunit of human chorionic gonadotropin (hCG) was modified by incorporation of cystamine into the Fc-carbohydrate, followed by reduction with dithiothreitol resulting in the generation of 4.5 free thiols per IgG. The reduced IgG was added to clean, unmodified and surface-modified polypyrrole colloids. Functionalized colloids included carboxylate-modified polypyrrole, poly[pyrrole-co-1-(2-carboxyethyl) pyrrole]-silica composite, and amine forms of the carboxylated colloids. The amine-functionalized colloids were subsequently treated with sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate to provide thiol-reactive maleimide surface groups. Following the conjugation of IgG to the colloids, bound and soluble antibody activity was quantitated using a sequentially competitive immunoassay for hCG, based on an automated commercial hCG kit. The results indicated that all forms of polypyrrole retained the equivalence of between 12 and 33 micrograms of IgG activity/mg of colloidal solids, relative to the unmodified soluble IgG.

Specific binding sites for 125I-labelled human luteinizing hormone (hLH) in microsomal fractions from Drosophila

Microsomal fractions prepared from Drosophila flies and larvae bound 125I-labelled human luteinizing hormone (hLH) in a displaceable manner. Binding increased linearly with increasing microsome concentration, and was dependent on the pH and metal ion concentration of the medium, and on the temperature and duration of incubation. Scatchard plots of 125I-hLH binding demonstrated the presence of two distinct binding sites, one with high affinity and low capacity, the other with low affinity and high capacity. 125I-hLH binding was inhibited in a dose-dependent fashion by partially purified human gonadotropin preparations, but not by other proteins, hormones and peptides. Highly purified human chorionic gonadotropin (hCG) or hLH preparations were much less effective at inhibiting 125I-hLH binding to Drosophila microsomes than partially purified gonadotropins. This was due to the presence of a contaminant present in partially purified commercial hCG preparations. Chromatography of crude hCG preparations clearly resolved the LH-binding inhibitor from hCG. Moreover, whereas hCG (and epidermal growth factor-like factors present in crude hCG preparations) were not bound to CM-Sepharose, the Drosophila LH-binding inhibitor was adsorbed strongly. Fractionation of 125I-hLH tracer on concanavalin A-Sepharose suggested that Drosophila binding sites did not preferentially bind a subfraction of the hormone tracer. These data suggest that high affinity binding sites for hLH/hCG-like molecules are present in Drosophila microsomes.

Testicular cancer secretes intact human choriogonadotropin (hCG) and its free beta-subunit: evidence that hCG (+hCG-beta) assays are the most reliable in diagnosis and follow-up

Human choriogonadotropin (hCG) and free hCG-beta values for 934 serum samples from patients with seminomatous or nonseminomatous testicular cancer were measured by highly specific immunoradiometric assays (IRMAS). In non-seminoma samples, hCG and hCG-beta were highly correlated (r = 0.82, P less than 0.001). Of 112 "marker-positive" seminoma samples, only 46 (41.1%) showed both increased hCG and hCG-beta. In 39 cases (34.8%) only hCG-beta and in 27 cases (24.1%) only dimer-hCG was increased. This makes the determination of hCG and hCG-beta, either by two assays or by a single hCG (+hCG-beta) assay, most reliable in these patients. For all samples, hCG (+hCG-beta) was measured by a polyclonal RIA and a monoclonal IRMA, which differed in their cross-reactivities with hCG-beta (234% and 720%, respectively). The hCG (+hCG-beta) IRMA, as a result of its higher hCG-beta cross-reactivity, was superior to the hCG (+hCG-beta) RIA in detecting slightly increased hCG-beta. Additionally, 11 widely used commercial hCG kits were tested for their hCG-beta cross-reactivities and showed values between less than 3% and 264%.

Further demonstration of induction of ovulation with a hybrid human chorionic gonadotropin compound (AB1ER-CR-2XY)

Further demonstration of the ability of the hybrid human chorionic gonadotropin (hCG) compound, AB1ER-CR-2XY, to induce ovulation is presented. Nineteen patients previously treated with human menopausal gonadotropin (hMG) and commercial hCG were selected for the study. The patients received 37 courses of treatment with dosages of hMG ranging from 1200 IU (16 ampules) to 8400 IU (112 ampules), followed by the administration of 5000 or 10,000 IU of the hybrid hCG. Of the total number of courses given, 75.5% were ovulatory; serum progesterone levels at midluteal phase of the cycle were within normal range, and the cycle length was about 12 days. Seven patients became pregnant, three with twins, one with triplets, and three with aborted single fetuses. Before the hybrid hCG was administered the serum estrogen levels were less than 1200 pg/ml in 12 cycles (32.4%), and the estrogen levels ranged from 1400 to 7400 pg/ml in 25 (67.6%). However, in spite of high estrogen levels, clinical hyperstimulation, which occurred in 22.4% of previous treatments with conventional hMG-commercial hCG, did not develop when the hybrid hCG was administered, an effect which could be attributed to its short circulatory half-life. Studies are in progress to confirm whether the hybrid hCG may provide a better margin of safety than commercial hCG for ovulation induction in patients pretreated with hMG.

Recent reflections on tumor markers

The necessity for monitoring serum levels of alphafetoprotein (AFP) and human chorionic gonadotropin (hCG) in patients with testicular cancer is widely known, but a few questions remain unanswered. Cross-reactivity of commercial hCG assays with luteinizing hormone remains a problem; and as patients apparently cured of testicular cancer age, they become subject to other diseases that can elevated AFP or hCG levels, creating confusion. There still are no adequate studies of the role of markers in staging, a problem that has become acute with the introduction of expectant treatment for metastases in clinical stage A disease. Calculation of the metabolic half-life of markers can be helpful in staging postoperatively, but calculations of the actual half-life of markers during induction chemotherapy is less useful than had once been hoped. Attempts to capitalize on the molecular heterogeneity of AFP have had little success, although newer analytic methods may change this; but ultrasensitive assays for urinary hCG are beginning to show promise. Studies of radioimmunodetection with anti-marker antibodies have been both encouraging and frustrating; the newly available monoclonal anti-marker antibodies may improve the results and also offer a possible means of improving chemotherapy. Placental alkaline phosphatase continues to look like a useful marker for seminoma, and lactic dehydrogenase is often helpful in all types of testicular cancer, especially in high-stage disease.

An investigation of cellular components released from human renal cancer and foetal kidney xenografts in nude mice (nu/nu) by cross-immunization of hairy littermate relatives

The release of components from human kidney tumour xenografts (GYL) and human foetal kidney explants maintained in nude mice has been studied. The GYL tumour released antigens into the serum which could be detected by the generation of antibodies following cross-immunisation of closely related hairy litter mate (HLM) mice. The production of anti-GYL antibody was monitored by an I125 binding assay using viable GYL tumour cells. In 2/16 hairy litter mate mice, cell surface antibody binding by GYL cells was twice that found with 8 other human tumour cell lines (including 2 other kidney cancer cell lines). Absorption of these antisera with 10(7) GYL tumour cells completely abolished this response, where 50%, 38% and 25% of activity remained following absorption with; a normal kidney cell line, a homogenate of normal kidney and a mixed pool of human tumour cells. Six out of 8 GYL tumour bearing nude mice tested had elevated plasma levels of HCG. Absorption of the HLM antisera with an excess of commercial HCG abrogated I125 binding by only 15%, suggesting that antibody production was not directed primarily against ectopic HCG.

First demonstration of induction of ovulation with a hybrid human chorionic gonadotropin compound (AB1ER-CR-2XY)

This study presents the first demonstration of the ability of a hybrid human chorionic gonadotropin (hCG) compound, AB1ER-CR-2XY, to induce ovulation in humans. Thirteen patients with primary infertility were treated for 16 cycles with varying dosages of human menopausal gonadotropin (hMG), and 10,000, 5000, and 2500 IU of the hybrid hCG. Presumptive occurrence of ovulation was recorded in 15 courses of medication. Echographic studies did not indicate overt follicular or ovarian enlargement. Two singleton pregnancies occurred, one during the first treatment course and the other following the second course of hMG/hybrid hCG therapy. Due to its specific characteristics, the hybrid hCG may provide equal or greater effectiveness and a better margin of safety than commercial hCG used for ovulation induction in patients pretreated with hMG.