Immunoprocedures for detecting human chorionic gonadotropin: clinical aspects and doping control (original) (raw)
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Journal of Pharmaceutical and Biomedical Analysis, 2021
The determination of Human Chorionic Gonadotropin (HCG) in biological fluids is of great interest in the early pregnancy diagnostics, the evaluation of pregnancy disorders, as a tumor marker, as a screening procedure for anti-doping control, and many other purposes. A simple sandwich-type UltraMicro Enzyme-Linked ImmunoSorbent Assay (UMELISA) has been developed for the measurement of HCG in serum and urine samples. Strips coated with a high affinity MAb directed against HCG are used as solid phase, to ensure the specificity of the assay. The HCG assay was completed in 1.5 h, with a measuring range of 0.76-400 mIU/mL. The intra-and inter-assay coefficients of variation were lower than 10 %, depending on the HCG concentrations evaluated. Recovery percentages were 96.43-97.16 % (serum) and 98.10-99.04 % (urine). The assay detected intact HCG, nicked HCG, HCG , and nicked HCG , and did not recognize any of the interfering molecules tested. Regression analysis showed a good correlation with Elecsys in serum
Human chorionic gonadotropin in pregnancy diagnostics
Clinica Chimica Acta, 2011
Human chorionic gonadotropin (hCG) is a 237 aminoacid glycoprotein hormone composed of two dissimilar α and β subunits noncovalently linked by charge interactions, which are both required for the biological activity of the hormone. Due to structural heterogeneity, hCG exists in biological fluids as a mixture of different isoforms, i.e., intact active hormone (hCG), nicked hCG (hCGn), free β subunits (hCGβ), free α subunit (hCGα), β-core fragment (hCGβcf, predominantly detected in urine and containing amino acids 6-40 and 55-92 linked by disulphide bridges) and nicked free β-subunit (hCGβn). Although the measurement of hCG might be useful in a kaleidoscope of clinical conditions, such as diagnosis, monitoring and follow-up of pregnancy-related disorders, prenatal screening and gynecological cancers, the leading application is still the diagnosis of pregnancy, where it can be measured quantitatively either in serum or urine, in the latter case also using qualitative and rapid immunoassays. Since there is still debate as to whether serum or urine tests are to be preferred for establishing a diagnosis of pregnancy, we discuss here the main analytical and clinical aspects of hCG measurement for the diagnosis of pregnancy, highlighting the advantages and limitations of assessing hCG in urine and serum.
Bioanalytical Method Development and Validation of HCG (Human Chorionic Gonadotropin)
International Journal of Pharmacy and Pharmaceutical Sciences, 2015
Objective: To develop and validate simple, rapid, specific, accurate and precise bioanalytical method for determination of the HCG (Human Chorionic Gonadotropin) by RP-HPLC method by using human urine. Methods: The chromatographic separation was performed using Phenom enex C18 (250 x 4.6 mm, 5μ, 300 °A) column. Mobile phase composed of sodium phosphate buffer (pH 7.0, 0.05M) and acetonitrile (87.5:12.5 % v/v) at a flow rate of 1 ml/min. Detection was carried out using UV detector at 215 nm. Bovine serum albumin (BSA) was used as an internal standard (ISTD) and extraction was carried out using protein precipitation method. The method was validated as per USFDA guidelines. Results: The method was linear over the concentration range of 0.37 to 48.4 µg/ml. and correlation coefficient (R 2 ) was found to be 0.9983 and the Lower limit of quantitation (LLOQ) was 0.37 µg/ml. Recovery was found more than 94.0% for HCG. The % CV for interday and intraday precision was found to be less than ˂1...
2015
Objective: To develop and validate simple, rapid, specific, accurate and precise bioanalytical method for determination of the HCG (Human Chorionic Gonadotropin) by RP-HPLC method by using human urine. Methods: The chromatographic separation was performed using Phenom enex C18 (250 x 4.6 mm, 5μ, 300 °A) column. Mobile phase composed of sodium phosphate buffer (pH 7.0, 0.05M) and acetonitrile (87.5:12.5 % v/v) at a flow rate of 1 ml/min. Detection was carried out using UV detector at 215 nm. Bovine serum albumin (BSA) was used as an internal standard (ISTD) and extraction was carried out using protein precipitation method. The method was validated as per USFDA guidelines. Results: The method was linear over the concentration range of 0.37 to 48.4 µg/ml. and correlation coefficient (R2 Conclusion: A simple, rapid, specific, accurate and precise analytical method was developed and validated by using human urine. ) was found to be 0.9983 and the Lower limit of quantitation (LLOQ) was 0....
Between-Method Variation in Human Chorionic Gonadotropin Test Results
Background: Results on sera and calibrators vary 1.4-to 2.3-fold among commercial human chorionic gonadotropin (hCG) assays. The relative contributions of calibrators, standards, hCG charge isoforms, and major structural variants to this variation have not been quantified. Methods: Purified hCG was separated by isoelectric focusing into four fractions with pI ranges of 3-4, 4 -5, 5-6, and 6 -7. These four fractions together with pure hCG, hyperglycosylated hCG, hCG -subunit (hCGb), nicked hCG, and hCGb core fragment (hCGbcf) were tested in nine commonly used commercial serum assays for hCG. The compositions of pure hCG preparations, standards, and commercial hCG preparations were determined by immunoassay. Results: The three pure hCG preparations and the four hCG charge isoforms each showed parallel responses in the nine commercial hCG assays. Although wide variations were found in the detection of hCG structural variants by the nine assays (hyperglycosylated hCG, hCGb, nicked hCG, and hCGbcf), this did not correlate with the between-method variation observed in results for the three pure hCG preparations. Commercial preparations of hCG and calibrators showed great variation in their content of hCG structural variants. Conclusions: Intermethod differences in hCG results were not explained by changes in responses attributable to hCG charge isoforms or to hCG structural variants, but wide variation was observed in concentrations of hCG structural variants in calibrators and in detection of these structural variants. Differences in assay specificity and in composition of the calibrators are the most likely sources of between-method variation.
Clinica Chimica Acta, 2010
Background: Human chorionic gonadotropin (hCG) is a heterodimeric glycoprotein hormone with considerable molecular heterogeneity. There is uncertainty regarding which hCG variants are detected by different hCG assays. The analytical specificity of 8 hCG assays was investigated. Methods: WHO International Reference Reagents for hCG, nicked hCG (hCGn), beta subunit (hCGβ), nicked beta subunit (hCGβn), and beta core fragment (hCGβcf) were individually added to hCG-free human serum. Specimens were analyzed with 8 commercially available hCG assays. Equimolar detection of hCG variants was defined as a recovery of 90-110%. Results: All assays detected hCG and hCGn with mean recoveries of 98.3 and 94.6%, respectively. Seven assays detected hCGβ (mean recovery 103.8%) but with high variation, and equimolar detection was observed only in four. The mean recovery of hCGβn was 85.5% but was highly variable with only two assays showing equimolar detection. With a mean recovery of 53.4%, two assays detected hCGβcf and both underestimated it considerably. Information provided by the assay manufacturer regarding hCG variant analytical specificity was inadequate or unclear in 75% of the assays. Conclusions: hCG assays vary considerably in their ability to detect different hCG variants. Manufacturers of hCG assays should clearly indicate the hCG variant specificity of their reagent systems.
Bioanalytical Method Development and Validation of HCG (Human Chorionic
2015
Methods: The chromatographic separation was performed using Phenom enex C18 (250 x 4.6 mm, 5μ, 300 °A) column. Mobile phase composed of sodium phosphate buffer (pH 7.0, 0.05M) and acetonitrile (87.5:12.5 % v/v) at a flow rate of 1 ml/min. Detection was carried out using UV detector at 215 nm. Bovine serum albumin (BSA) was used as an internal standard (ISTD) and extraction was carried out using protein precipitation method. The method was validated as per USFDA guidelines.
Clinical Chemistry, 2009
Background: The 1st WHO International Reference Reagents (IRRs) for 6 human chorionic gonadotropin (hCG)-related molecular variants, highly purified and calibrated in substance concentrations by the IFCC Working Group for hCG, permit experimental elucidation of what commercially available hCG methods measure in molar terms and enable assessment of their fitness for clinical purposes. Methods: Pools containing known amounts of the IRRs spiked into normal human serum were issued to participants through the UK National External Quality Assessment Service for hCG for a period of 7 years. Among 16 assays used, 4 recognized only hCG, whereas 6 recognized hCG and its free β-subunit (hCGβ), and 6 recognized hCG, hCGβ, and the beta core fragment. Results: Differences in calibration of current hCG assays are moderate. Mean recovery of the current International Standard (IS), hCG IS 75/589, was 107% (range 93% to 126%), whereas that of the IRR 99/688 for hCG was 139% (range 109%–164%). Between...
Clinical Chemistry, 2010
BACKGROUND Earlier studies have shown that increased concentrations of certain human chorionic gonadotropin (hCG) variants can cause false-negative results in some qualitative hCG devices. The objective of this study was to determine if increased concentrations of hCGβ and hCGβ core fragment (hCGβcf) cause falsely decreased results on 9 commercially available quantitative hCG assays. METHODS Several concentrations of purified hCGβ and hCGβcf were added to 2 sets of 6 serum samples with and without a fixed concentration of intact hCG. We examined 9 widely used immunoassays to measure immunoreactive hCG. Falsely decreased results were defined as those in which the measured hCG concentration was ≤50% of expected. RESULTS High concentrations of hCGβ (≥240 000 pmol/L) produced falsely decreased hCG measurements in 2 assays known to detect this variant. Similarly, high concentrations of hCGβcf (≥63 000 pmol/L) produced falsely decreased hCG measurements in 3 assays that do not detect puri...