hCG Variants 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.

Hyperglycosylated-hCG: Its Role in Trophoblast Invasion and Intrauterine Growth Restriction

Human chorionic gonadotropin (hCG) is produced by the placenta and its roles have been studied for over a century, being the first known pregnancy-related protein. Although its main role is to stimulate the production of progesterone by corpus luteal cells, hCG does not represent just one biologically active molecule, but a group of at least five variants, produced by different cells and each with different functions. The hyperglycosylated variant of hCG (H-hCG) plays a key role in trophoblast invasion, placental development and fetal growth. During trophoblast invasion, H-hCG promotes extravillous cytotrophoblast cells to infiltrate the decidua, and also to colonize and remodel the spiral arteries in to low resistance, larger-diameter vessels. As fetal growth is heavily reliant on nutrient availability, impaired trophoblast invasion and remodeling of the uterine arteries, leads to a defective perfusion of the placenta and fetal growth restriction. Understanding the function of H-hCG in the evolution of the placenta might unveil new ways to manage and treat fetal growth restriction.

Variant hook effect: A cause of false negative result in point of care testing

Beta Human Chorionic Gonadotrophin (β hCG) assays are sandwich chromatographic immunoassays which give a false negative results at high hCG concentrations and also in the presence of variants of hCG like hyperglycosylated hCG and hCG core fragments. This is imperative in everyday clinical scenario as it delays the diagnosis of pregnancy and its related complications. We have discussed two such cases where in the first case the diagnosis of hydatidiform mole was delayed due hook effect whereas in the second case the diagnosis of GTD was missed because of variant hook effect.

Prediction of Placenta Accreta Using Hyperglycosylated Human Chorionic Gonadotropin

Objectives: Hyperglycosylated human chorionic gonadotropin (hCG) is a variant of hCG. In addition, it has a different oligosaccharide structure compared to the regular hCG and promotes the invasion and differentiation of peripheral cytotrophoblast. This study aimed to measure hyperglycosylated hCG as a predictor in the diagnosis of placenta accreta. Materials and Methods: In general, 90 pregnant women were involved in this case-control study among which, 30 ladies (control group) were pregnant within the gestational age of ≥36 weeks with at least one previous caesarean section and a normal sited placenta in transabdominal ultrasound (TAU). The other 60 pregnant women (case group) were within a gestational age of ≥36 weeks at least, one previous caesarean section and placenta previa with or without signs of placenta accreta in TAU. Hyperglycosylated hCG and total hCG were measured in each group and the results of the surgery were followed up. Results: Hyperglycosylated hCG showed higher serum levels in patients with placenta accreta compared to those with placenta previa and control women. Hyperglycosylated hCG with an optimal cut point of (3) IU/L predicted placenta accreta in pregnant women with 90% specificity, 76.7% sensitivity, and 81.1% accuracy. Conclusions: The high specificity of the above approach makes it a good diagnostic tool (as a single test) for confirming placenta accreta in clinical settings. When this test is added to our established workup, its high positive predictive value makes it a suitable method within the algorithm of accreta confirmation when there is a high suspicion or insufficient evidence to the diagnosis of placenta accreta.

Hyperglycosylated human chorionic gonadotropin (HCG-H) as a novel predictor of ongoing pregnancy

HCG-H, the predominant hCG variant secreted immediately following implantation, is a novel marker of trophoblast invasion (1). Our objective was to determine whether the initial serum hCG-H differs between ongoing and failed pregnancies, and to compare its performance to total serum hCG as a predictor of ongoing pregnancy. Prospective cohort study. Fresh/frozen autologous IVF cycles with a day 5 single embryo transfer (ET) during 2017 with positive serum hCG (>3 IU/L) were included (n=115). Exclusion criteria were body mass index >35 kg/m2, recurrent pregnancy loss, donor egg, gestational carrier, preimplantation genetic testing and multiple gestation. HCG levels were checked exactly 11 days after ET at a single laboratory (CV<6%). Surplus frozen serum (-80oC) was batched and shipped to Quest Laboratories, where hCG-H was measured using an electrochemiluminescence assay (CV<9.1%). Linear regression analyses with robust “sandwich” standard errors adjusted for oocyte age a priori were used. Differences in hCG and hCG-H for ongoing pregnancies (>8 weeks of gestation) and failed pregnancies (clinical pregnancy loss [CPL], biochemical and ectopic pregnancies [EP]) were reported as linear regression βs with 95% confidence intervals (CI), representing differences in mean analyte values between outcomes. EP (n=1) was included in failed pregnancies but not evaluated separately. Receiver operator curves (ROC) were generated for each analyte for the prediction of ongoing pregnancy. Eighty-five patients (73.9%) had ongoing pregnancies. Both mean hCG-H and hCG were significantly higher in ongoing versus failed pregnancies (hCG-H: 19.0 vs. 7.5 mcg/L, hCG: 422.1 vs. 187.5 IU/L, p<0.001), as well as versus biochemical pregnancies (Table). No significant differences were observed between hCG values for ongoing vs. CPL. In contrast, hCG-H values were significantly higher in ongoing vs. CPL (46% of which had fetal cardiac activity) (p=0.01, Table). The areas under the ROC curves (AUC) for HCG-H and HCG were 0.825 and 0.798.Tabled 1HCG-H and HCG among pregnancy outcome groupsOutcomeOngoing pregnancy (n=85) ReferentFailed pregnancy (n=30)Clinical pregnancy loss (n=11)Biochemical pregnancy (n=18)AnalyteMean (SD) min-maxMean (SD) min-maxLinear regression β (95% CI)Mean (SD) min-maxLinear regression β (95% CI)Mean (SD) min-maxLinear regression β (95% CI)HCG-H(mcg/L)19.0 (10.3) 3.3-47.57.5 (7.9) 0.0-31.6-11.5 (-15.0,-8.0)12.2 (8.4) 1.0-31.6-6.8 (-12.0,-1.6)4.1 (5.7) 0.0-20.3-14.9 (-18.3,-11.5)HCG(IU/L)422.1 (222.5) 56.0-1193.0187.5 (201.9) 4.0-805.0-234.6 (-319.8,-149.4)338.1 (216.3) 16.0-805.0-84.0 (-214.6,46.6)84.7 (118.0) 4.0-442.0-337.4 (-408.3,-266.6) Open table in a new tab HCG-H has exciting potential as a very early predictor for ongoing pregnancy in IVF. The AUC for hCG-H was only slightly higher than that for hCG; however, in contrast to hCG, values for hCG-H were significantly different between ongoing pregnancies and CPL, just 11 days post-ET. This observation suggests that hCG-H may be a better predictor of viable pregnancy than hCG. Further evaluation of this possibility in a broader IVF population, including those with multiple embryo transfer and multiple pregnancy, is required.

Human Chorionic Gonadotropin Assays for Testicular Tumors: Closing the Gap between Clinical and Laboratory Practice.

Clinical practice guidelines recommend the measurement of human chorionic gonadotropin (hCG) and/or hCGβ in serum for management of testicular germ cell tumors (GCTs). These guidelines, however, disregard relevant biochemical information on hCG variants to be detected for oncological application. We set out to provide a critical review of the clinical evidence together with a characterization of the selectivity of currently marketed hCG immunoassays, identifying assays suitable for management of GCTs. Evidence sources in the available literature were critically appraised. Most instances of misdiagnosis and mismanagement of testicular GCTs have been associated with hCG results. According to the clinical evidence, 36% of patients with seminoma show an exclusive hCGβ increase, and 71% of patients with nonseminomatous GCTs (NSGCTs) show an increase of intact hCG and/or hCG + hCGβ, whereas the hCGβ increase in NSGCTs is variable according to the tumor stage and histology. hCG + hCGβ assays that display an equimolar recognition of hCG and hCGβ, or at least do not overtly underestimate hCGβ, may be employed for management of testicular GCTs. Assays that underestimate hCGβ are not recommended for oncological application. In addition to the hCG + hCGβ assay in service, an additional assay with broader selectivity for other hCG variants should be considered when false-negative or false-positive results are suspected on the basis of clinical data.

Comparison of two assays measuring human chorionic gonadotrophin serum concentrations in pregnancy termination and trophoblastic or non-trophoblastic tumours

Background: Differences in human chorionic gonadotrophin (hCG) results provided by the commercial immunoassays reflect the heterogeneity of antibodies and the use of suboptimal standards. As a consequence, the principal forms of hCG and metabolites are differentially detected and the hCG tests are not suited for the same clinical applications. Conflicting results are available in the literature regarding which hCG variants are recognized by the Roche Elecsys hCG + β test. The aim of our study was to compare the hCG concentrations provided by the Siemens Immulite 2000 test and the Roche test as well as to assess the concordance between both assays.Methods: In this purpose, 152 samples obtained from women and 44 samples from men were analysed by both tests during the follow-up of pregnancy termination, gestational trophoblastic disease and malignancies. The intermediate precision of the Roche test was also investigated on a pool with a low hCG concentration.Results and conclusions: The hCG concentrations measured with the Roche test were slightly lower compared with the Siemens assay; mean biases of −34.2% and −8% were respectively obtained for hCG values ≤100 UI/L and higher than 100 UI/L. The overall agreement between both assays was 96.1% for women and 97.7% for men. By using an upper reference limit of 3.2 UI/L for women and 1.6 UI/L for men, the Roche test demonstrated a respective concordance of 98.7% and 100%. This test also yielded an excellent precision with a coefficient of variation of 2.8% at a mean hCG concentration of 7 UI/L.

Human chorionic gonadotropin (hCG) and hyperglycosylated hCG, seven semi-independent critical molecules: A review.

hCG exists in two analogous structures, as typified by the hormone hCG which binds an LH/hCG hormone receptor and the TGFß receptor binding autocrine hyperglycosylated hCG. Both share a common α-subunit and ß-subunit amino acid sequence. From the common hormone hCG and autocrine hyperglycosylated hCG structures there are seven common hCG variants with wildly varying hormone hCG or autocrine hyperglycosylated hCG functions. There is placental hormone hCG and placental autocrine hyperglycosylated hCG which together control pregnancy and start hemochorial placentation during pregnancy. There is pituitary sulfated hCG, part of the hormone hCG library which works with LH in driving ovarian steroidogenesis, ovulation and luteogenesis. Fetal hCG a fetal hormone that promotes fetal organ growth during pregnancy, and ovarian hyperglycosylated hCG which drive the final proteolytic enzyme step of ovulation. Cancer hyperglycosylated hCG and cancer hyperglycosylated hCG free ß-subunit drive malignancy in most cancers. The seven variants of hCG drive critical functions during pregnancy, cancer and other bodily functions.

Standardization of Epitopes for Human Chorionic Gonadotropin (hCG) Immunoassays.

hCG and its variants are markers for pregnancy tests, pregnancyrelated complications, trophoblastic diseases, pre-natal screening of Down's syndrome and doping controls. Strong demands are imposed on diagnostic methods by the dynamic changes in the absolute and relative levels of hCG protein backbone variants and glycosylation isoforms in serum and urine during development of pregnancy or the progression/remission of tumors. Observed differences in the results between commercial diagnostic immunoassays reflect the unequal molar recognition of the different metabolic hCG variants, in particular the hCG beta core fragment (hCGβcf), by the diagnostic antibodies (Abs), as their epitopes are not standardized, and the fact that suboptimal hCG standards are used. To rapidly characterize Abs by their epitope recognition and specificity to evaluate their suitability for diagnostic immunoassays a procedure of comparative epitope mapping has been developed using epitope-defined reference Abs. Comparative epitope mapping of diagnostic Abs will provide the basis for the standardization of diagnostic antigenic domains/epitopes and consequently for improved reliability of hCG measurements. Diagnostic first line assays likely consist of pairs of Abs that recognize specific epitopes at the top of the neighboring peptide loops 1 and 3 (Ł1+3) and the cystine knot (ck) of hCGβ, respectively. In future, significant improvements of reliability, robustness and comparability of the results of immunoassays for complex glycoproteins such as hCG will be achieved by the use (i) of standardized diagnostic Abs against welldefined epitopes and (ii) of the new International Standards for hCG and for five hCG variants established by WHO, that are calibrated in molar (SI) units.

Using a simulation model to assess risk of false negative point-of-care urinary human chorionic gonadotropin device results due to high-dose hook interference

BackgroundIt is unclear if the point-of-care (POC) Clinitest hCG device is subject to high-dose hook interference from physiological concentrations of intact human chorionic gonadotropin (hCG), β-core fragment of hCG (hCGβcf), and hCG free β-subunit (hCGβ) found in urine during pregnancy. We used a simulation model to address this question and related our findings to our institution's pregnant population in order to assess risk for potential false-negative hCG results. MethodsThe expected distribution of days relative to ovulation during routine POC hCG testing was estimated from 182 patients. Clinitest-Clinitek Status hCG device susceptibility to high-dose hook interference from hCG variants and potential risk of false-negative results as it relates to this population were evaluated by testing increasing concentrations of hCG, hCGβcf, hCGβ as well as urine simulating physiological hCG, hCGβcf and hCGβ concentrations expected during early pregnancy (≤44days post-ovulation). ResultsThe Clinitest-Clinitek Status hCG device exhibited high-dose hook interference from hCGβcf alone, but not from hCG, hCGβ, or simulated physiological urinary concentrations of combined hCG, hCGβcf and hCGβ expected during early pregnancy. The majority of our patient population had urinary hCG testing conducted during early pregnancy. ConclusionThe Clinitest-Clinitek Status hCG device is unlikely to exhibit false-negative urinary hCG results due to high-dose hook interference for women in early healthy pregnancy, although additional studies are necessary to determine potential risk in other patient populations. Visual interpretation of POC urinary hCG device results is an important failure mode to consider in risk analyses for erroneous urinary hCG device results.

Proportion hyperglycosylated hCG: a new test for discriminating gestational trophoblastic diseases.

Hyperglycosylated human chorionic gonadotropin (hCG) is a variant of hCG with large oligosaccharide side chains. Although hCG is produced by syncytiotrophoblast cells, hyperglycosylated hCG marks cytotrophoblast cell. Hyperglycosylated hCG signals placental implantation. Total hCG in serum and urine is measured by the Siemens Immulite hCG pregnancy test; the result is in milli-international unit per milliliter. Hyperglycosylated hCG is determined by the B152 microtiter plate assay; the result is in nanogram per milliliter. Hyperglycosylated hCG results can be converted to milli-international unit per milliliter equivalents by multiplying by 11. The test measures proportion hyperglycosylated hCG, hyperglycosylated hCG / total hCG. Proportion hyperglycosylated hCG marks cases intent on developing persistent hydatidiform mole (68% detection at 17% false detection). Proportion hyperglycosylated hCG also marks persistent hydatidiform mole (100% detection at 5.1% false detection). Proportion hyperglycosylated hCG distinguishes choriocarcinoma and gestational trophoblastic neoplasm cases, absolutely discriminating aggressive cases and minimally aggressive cases. Proportion hyperglycosylated hCG identifies quiescent gestational trophoblastic disease cases. It recognizes quiescent cases that become persistent disease (100% detection at 0% false positive). Proportion hyperglycosylated hCG is an invaluable test for discriminating gestational trophoblastic diseases.

Screening Method to Evaluate Point-of-Care Human Chorionic Gonadotropin (hCG) Devices for Susceptibility to the Hook Effect by hCG β Core Fragment: Evaluation of 11 Devices

The predominant hCG variant in urine, hCG β core fragment (hCGβcf), has been demonstrated to cause false-negative results in qualitative point-of-care (POC) hCG devices. This is a major concern for healthcare professionals using POC pregnancy tests. We developed a screening method to evaluate qualitative POC hCG devices for their susceptibility to inhibition by hCGβcf. Using this method, we evaluated the performance of 11 commonly used devices. A wide range of purified hCG and hCGβcf concentrations were mixed and tested on 2 POC devices. By use of those results, a screening method was defined and 9 additional POC devices were evaluated. Two solutions containing (a) 500 pmol/L (171 IU/L) intact hCG with 0 pmol/L hCGβcf and (b) 500 pmol/L intact hCG with 500 000 pmol/L hCGβcf were used to screen all POC devices. The OSOM and Cen-Med Elite devices were found to be most susceptible to false-negative results due to hCGβcf. The BC Icon 20 and the Alere were the least susceptible. The remaining 7 were moderately affected. Devices that gave the strongest signal with hCGβcf alone were those that were least likely to show a hook effect. The screening method put forth here can be used by device users and manufacturers to evaluate POC devices for inhibition by hCGβcf. Of 11 devices evaluated, only 2 have been identified that exhibit minimal to no susceptibility to hCGβcf.

Negative Pregnancy Test in Patients with Trophoblastic Diseases

Quantitative and qualitative human chorionic gonadotrophin (hCG) assays are widely used to detect pregnancy state and abnormal trophoblastic lesions. At least five different forms of hCG have been characterized and different trophoblastic diseases produce different forms of hCG in varying proportions. Because of the difference in antibody specificity in various commercial automated immunoassays of HCG, discordant results may be obtained by laboratories using different hCG assays, with a falsely low or negative result obtained if the assay does not recognize the hCG variants produced from the trophoblastic tissue. On the other hand, significantly elevated hCG concentration can paradoxically lead to false-negative results in two-site immunometric assay due to high-dose hook effect. Clinicians managing patients with trophoblastic lesions should be aware of these limitations of current hCG assays and clinical laboratories should have measures to avoid analytical false negative hCG results.

The hCG Group: the Key Molecules in Human Evolution, Human Life, and Human Death

The term hCG refers to a group of molecules with common amino acid sequence, different glycosylation, and multimeric structure. These molecules include the pregnancy hormone hCG, made by placental syncytiotrophoblast cells, and the pregnancy autocrine hyperglycosylated hCG, made by placental cytotrophoblast cells, which drives placental growth during pregnancy and implantation of the placenta. Most human cancers make hyperglycosylated hCG free β-subunit. This drives cancer malignancy by following the invasive implantation pathway. Whereas hCG functions by acting on an hCG/LH joint receptor, hyperglycosylated hCG and hyperglycosylated hCG free β-subunit function by antagonizing a TGFβ receptor. CG and hyperglycosylated hCG first evolved with early simian primates. The early simian CG and hyperglycosylated CG were nonacidic rapidly clearing molecules. With their evolution evolved a primitive form of hemochorial placentation, primitive in that it was only minimally promoted by CG and hyperglycosylated CG. With the evolution of advanced simian primates came acidic variant of hCG and hyperglycosylated CG. The more acidic CG was longer circulating and more effectively promoted the establishment and growth of hemochorial placentation. With the evolution of humans came a very acidic variant of CG and hyperglycosylated CG. This was very much longer circulating and was an effective stimulant of hemochorial placentation. Early prosimian primates had a brain of 0.07 % of body mass, early simian primates had a brain mass of 0.17 %, advanced simian primates of 0.74 %, and humans of 2.4 %. Research indicates that brain mass grew with improving CG and hyperglycosylated CG activity and improving hemochorial placentation activity. The super CG variant produced by humans plays a key role in human pregnancy implantation and in failures of pregnancy, hypertense pregnancy, and invasion by gestational trophoblastic diseases. Research today shows that hyperglycosylated CG and hyperglycosylated CG free β-subunit drive most human trophoblastic and nontrophoblastic malignancies.

Candidate epitopes for measurement of hCG and related molecules: the second ISOBM TD-7 workshop

Participants of the Second International Workshop (WS) on human chorionic gonadotropin (hCG) of the International Society of Oncology and Biomarkers Tissue Differentiation 7 (ISOBM TD-7) have characterized in detail a panel of 69 antibodies (Abs) directed against hCG and hCG-related variants that were submitted by eight companies and research groups. Specificities of the Abs were determined using the First WHO International Reference Reagents for six hCG variants, i.e., hCG, hCGn, hCGβ, hCGβn, hCGβcf, and hCGα, which are calibrated in SI units, and hLH. Molecular epitope localizations were assigned to the ISOBM-mAbs by comparing ISOBM-Ab specificity, sandwich compatibility, and mutual inhibition profiles, to those of 17 reference monoclonal (m)Abs of known molecular epitope specificities. It appeared that 48 Abs recognized hCGβ-, 8 hCGα-, and 13 αβ-heterodimer-specific epitopes. Twenty-seven mAbs were of pan hCG specificity, two thereof with no (<0.1 %; epitope β1), 12 with low (<1.0 %; epitopes β2/4), and 13 with high (>>1 %; epitopes β3/5) hLH cross-reactivity. The majority of hCGβ epitopes recognized were located in two major antigenic domains, one on the peptide chain of the tips of β-sheet loops 1 and 3 (epitopes β2–6; 27 mAbs) and the second around the cystine knot (e.g., epitopes β1, β7, and β10; 9 mAbs). Four mAbs recognized epitopes on hCGβcf-only (e.g., epitopes β11 and β13) and six mAbs epitopes on the remote hCGβ-carboxyl-terminal peptide (epitopes β8 and β9 corresponding to amino acids 135–144 and 111–116, respectively). For routine diagnostic measurements, methods are used that either detect hCG-only, hCGβ-only, or hCG together with hCGβ or hCG together with hCGβ and hCGβcf. Sandwich assays that measure hCG plus hCGβ and eventually hCGβcf should recognize the protein backbone of the analytes preferably on an equimolar basis, should not cross-react with hLH and not be susceptible to blunting of signal by nonmeasured variants like hCGβcf. Such assays can be constructed using pairs of mAbs directed against the cystine knot-associated epitope β1 (Asp10, Asp60, and Gln89) in combination with epitopes β2 or β4 located at the top of β-sheet loops 1 + 3 of hCGβ involving aa hCGβ20-25 + 68-77. In summary, the results of the First and Second ISOBM TD-7 WSs on hCG provide the basis for harmonization of specificities and epitopes of mAbs to be used in multifunctional and selective diagnostic hCG methods for different clinical purposes.Electronic supplementary materialThe online version of this article (doi:10.1007/s13277-013-0994-6) contains supplementary material, which is available to authorized users.

Epitope analysis and detection of human chorionic gonadotropin (hCG) variants by monoclonal antibodies and mass spectrometry

Human chorionic gonadotropin (hCG) is an important marker for pregnancy, pregnancy-related disorders, and various cancers. Different molecular forms of hCG occur in different clinical conditions, and these can be distinguished with immunoassays using well-characterized monoclonal antibodies. Exact knowledge of the epitopes of the antibodies used is crucial for the design of assays with desired specificity. The epitopes of many hCG antibodies have been determined by comparing their reactivity with six 1st International Reference Reagents (IRRs) for hCG, but the specificity of some antibodies remains to be exactly defined. We have therefore studied the reactivity of 30 monoclonal antibodies (mAbs) with the six 1st IRRs for hCG, and variants were investigated using immunoaffinity extraction combined with liquid chromatography-mass spectrometry (LC-MS/MS) for the detection of hCG variants by specific tryptic signature peptides. Each of the mAbs had previously been characterized with regard to epitope specificity in the 2nd Tissue Differentiation Workshop on hCG of the International Society of Oncology and BioMarkers (ISOBM). Simultaneous identification of different hCG variants by LC-MS/MS confirmed that two standards used for mAb characterization, nicked hCG (hCGn, 1st IRR 99/642) and nicked β subunit of hCG (hCGβn, 1st IRR 99/692), are heterogeneous, being composed of two major variants each: hCGn44/45 and hCGn47/48 as well as hCGβn44/45 and hCGβ47/48. Furthermore, MS revealed cross-contamination by non-nicked hCG of the 1st IRR hCGn (99/642) standard. This information enabled fine-tuning of the previous epitope classifications of mAbs specific for heterodimeric hCG (c-mAbs). LC-MS/MS confirmed that c2-mAbs and most c1-mAbs did not recognize hCGn as the observed response in radioimmunoassays obviously resulted from the contamination of hCGn with hCG. Thus, c1 and c2 epitopes are partially dependent on hCGβ peptide loop 2. c3-mAbs recognized both hCG and hCGn. It appeared that c-mAbs cannot discriminate between hCGn44/45 and hCGn47/48 as they either recognize both or neither variant. For most mAbs directed against hCGβ, epitope specificity determined by LC-MS/MS was highly concordant with that obtained using standard immunological methods. In analogy to c-mAbs, hCGβ-mAbs cannot discern between hCGβn44/45, hCGβn47/48, or intact hCGβ as all 15 mAbs recognizing hCGβ also recognized both nicked variants irrespective of which of the three major hCGβ antigenic domains their epitopes were located within: on the caps of peptide loops 1 and 3, around the cystine knot, or along the hCGβCTP. LC-MS/MS confirmed that their epitopes were not located on hCGβ peptide loop 2. Thus, LC-MS/MS provided in-depth information on hCG variant composition of hCGn (99/642) and hCGβn (99/692) and hCG variant specificity profiles and facilitated precise classification of the epitopes of anti-hCG mAbs. This has impact on the design of selective immunoassays.

Analytical and clinical validation of the Immulite 1000 hCG assay for quantitative analysis in urine

BackgroundThe Siemens Immulite hCG assay detects all major hCG variants in serum. Currently, this assay is only FDA approved for qualitative measurement of hCG in urine. MethodsComplete validation of the hCG assay in urine was performed on the Siemens Immulite 1000 immunoassay platform. Reference intervals were established for females <55y, females ≥55y, and males 20–70y. ResultsThe limit of quantitation was 2.0IU/l. The Immulite hCG assay was precise for measuring hCG in urine from pregnant patients with intra- and inter-assay imprecision of <11% CV. The assay was linear over a dynamic range of 2–2600IU/l and 2–3500IU/l for hCG and hCGβ respectively. The assay was non-linear for hCGβcf. No hook effect was observed at concentrations up to 1,200,000pmol/l, for hCGβ or hCGβcf. The reference intervals were <2.0IU/l for males, <2.2IU/l for females <55y, and <12.2IU/l for females ≥55y. ConclusionThe Immulite 1000 hCG assay can accurately quantify hCG in urine.

Analysis of human chorionic gonadotropin (hCG): Application of routine immunological methods for initial testing and confirmation analysis in doping control

Human chorionic gonadotropin (hCG) is dimeric glycoprotein produced by placenta in pregnancy and also in low levels by pituitary gland. The main clinical use for exogenous hCG-administration is typically linked to infertility. The desired effect of hCG misuse in sport is due to the enhancement of testicular production of testosterone. Therefore, hCG is listed by the World Anti-Doping Agency (WADA) as a prohibited substance in male athletes and according to the recently published WADA guideline urinary concentrations of hCG > 5 IU/L may be an indicator of doping. In this study two independent immunoassays were used to implement the new WADA guideline. The assay for initial testing (Siemens Immulite 2000 XPi hCG assay) recognizes various hCG variants (e.g. hCG and β-core fragment of hCG) whereas the confirmatory assay (PerkinElmer DELFIA Xpress hCG) is sensitive to intact and nicked hCG only. Both assays showed adequate sensitivity and were proven fit-for-purpose in routine doping control. Population-based distribution of the assays was in good agreement with results of earlier studies and supported well the current threshold of 5 IU/L.

Sports drug testing using immuno-MS: clinical study comprising administration of human chorionic gonadotropin to males

The applicability of a mass spectrometry (MS)-based method for determination of various forms of human chorionic gonadotropin (hCG) in doping analysis was demonstrated. A clinical study involving the hCG-containing pharmaceuticals Pregnyl and Ovitrelle was carried out, comprising a single injection of one pharmaceutical per participant to a total of 24 healthy male voluntaries. Hereafter, serum and urine samples were collected over a period of 14 days. The analysis of the samples using immuno-MS demonstrated elimination profiles of intact hCG for both pharmaceuticals, with last day of detection following administration at day 7 in serum, and at day 10 in urine, at limit of detections as defined by the World Anti-Doping Agency. Furthermore, the method allowed detection and differentiation of the various forms of hCG known to be present in serum and urine as a function of metabolism. For both pharmaceuticals, only the intact hCG was detected in serum, whereas in urine the injection of Pregnyl as hCG source (containing urinary hCG, i.e., most hCG variants) was shown to generate a more complex hCG variant pattern compared to Ovitrelle (contains only intact hCG). By detecting hCG using this MS-based approach in doping analysis, strong analytical evidence is provided minimizing the risk of false-positive and false-negative results.

Immuno–MS Based Targeted Proteomics: Highly Specific, Sensitive, and Reproducible Human Chorionic Gonadotropin Determination for Clinical Diagnostics and Doping Analysis

The human chorionic gonadotropin (hCG) proteins constitute a diverse group of molecules that displays biomarker value in pregnancy detection and cancer diagnostics, as well as in doping analysis. For the quantification of hCGβ and qualitative differentiation between other hCG variants in a selective, sensitive, and reproducible manner, the targeted proteomics approach based on mass spectrometric (MS) selected reaction monitoring (SRM) detection was exploited. By optimizing immunoaffinity extraction using monoclonal antibodies coated to magnetic beads, access was granted for the MS to the low-abundance target proteins, ensuring proper sensitivity with limits of detection (LODs) of 2 and 5 IU/L, respectively, for urine and serum samples. Validation according to key elements and recommendations defined by the European Medicines Agency in Guideline on Validation of Bioanalytical Methods was performed. For both matrixes this demonstrated good within-day precision results (within 20% for the lowest concentration, and within 15% for the medium and high concentration), good accuracy results (within 15% for all concentrations), and proper linearity, >0.997 for serum and of 0.999 for urine, in the concentration range up to 5000 IU/L. The method's application in clinical diagnostics was tested on samples from a pregnant woman and from patients previously diagnosed with testicular cancer. For doping analysis, samples from one man having received injection of the hCG-containing pharmaceutical Pregnyl were analyzed. The method proved to be quantitatively accurate with indisputable identification specificity, reducing risks of false positive and false negative results. The successfully validated method advocates thus for more extended use of MS in routine analysis.