Enzyme Immunoassay for the Rapid Quantitative Determination of hCG Concentration in Human Serum
Introduction
Human chorionic gonadotropin (hCG) is a glycoprotein hormone produced by the placenta during pregnancy. It consists of two subunits: alpha and beta. The beta subunit, with a molecular weight of approximately 30,000 Daltons, provides biological and immunological specificity due to its unique amino acid sequence. The alpha subunit, with a molecular weight of approximately 18,000 Daltons, is structurally similar to the alpha subunit of other pituitary glycoprotein hormones such as luteinizing hormone (LH), follicle-stimulating hormone (FSH), and thyroid-stimulating hormone (TSH) (Braunstein et al., 1976).
HCG appears in serum or urine shortly after conception, making it a reliable biomarker for pregnancy detection. However, elevated hCG levels may also be associated with trophoblastic and non-trophoblastic neoplasms, requiring differential diagnosis before confirming pregnancy (Bagshawe, 1984). Immunoassays that target the beta subunit of hCG provide high specificity and sensitivity, allowing early pregnancy detection around the first missed menstrual period.hCG concentrations vary with gestational age and are typically higher in multiple pregnancies due to increased placental mass. Conversely, conditions such as placental insufficiency, threatened abortion, and ectopic pregnancy are linked to lower hCG levels (Stenman et al., 2004).
Clinical Significance
HCG measurement is crucial not only for pregnancy detection but also for clinical assessments in oncology. Elevated hCG levels can indicate gestational trophoblastic diseases, including hydatidiform mole and choriocarcinoma, as well as some testicular and ovarian tumors. Clinicians must consider these differential diagnoses when interpreting test results (Cole, 2009). Furthermore, the rapid quantitative assessment of hCG helps in monitoring early pregnancy complications and treatment efficacy in hCG-producing malignancies (Graves & Kohorn, 2011).
Test Procedure
The hCG ELISA test follows a sandwich enzyme-linked immunosorbent assay (ELISA) format. The system uses monoclonal anti-hCG antibodies immobilized on microtiter wells, with a secondary monoclonal anti-hCG antibody conjugated to horseradish peroxidase (HRP). The test sample interacts with the solid-phase antibodies. After incubation and washing, the enzyme conjugate is added, forming a sandwich complex with HCG between the solid-phase and conjugated antibodies. Following another wash step, a chromogen-substrate solution is introduced, leading to color development. The reaction is halted with a stop solution, and absorbance is measured at 450 nm. The HCG concentration is directly proportional to the sample’s color intensity.
References
- Bagshawe, K. D. (1984). Clinical applications of hCG. Advances in Experimental Medicine and Biology, 176, 313-324.
Braunstein, G. D., Rasor, J., Adler, D., & Danzer, H. (1976). Serum human chorionic gonadotropin levels throughout normal pregnancy. American Journal of Obstetrics and Gynecology, 126(6), 678-681.
Cole, L. A. (2009). hCG, its free subunits and its metabolites in clinical practice. Clinical Laboratory Medicine, 23(3), 49-68.
Graves, C. R., & Kohorn, E. I. (2011). Clinical uses of hCG. Current Problems in Obstetrics, Gynecology and Fertility, 34(2), 69-75.
Stenman, U. H., Tiitinen, A., Alfthan, H., & Valmu, L. (2004). The classification, functions, and clinical use of different isoforms of hCG. Molecular and Cellular Endocrinology, 223(1-2), 17-24. - European Authorized Representative: JTC Diagnosemittel UG, Schulweg 8, D-34516 Voehl, Germany.