Intact HCG and Related Molecular Forms

Introduction
Human chorionic gonadotropin (HCG), a protein hormone that stimulates progesterone secretion, appears in biological fluids as intact HCG, free alpha subunit, free beta subunit, and various fragments such as nicked HCG and nicked free beta-HCG. For more than 25 years, researchers have investigated the molecular forms of HCG in pregnancy and in different physiopathological processes, including trophoblastic diseases, testicular cancers, and nongonadal and nonplacental tumors. Immunological HCG measurements contribute much to understanding the structure, metabolism and clinical significance of the molecules, but immunoassay specificities for different forms must be identified in order to produce consistent and reliable results.¹

Figure 1. Among the several HCG forms that appear in the blood are free alpha subunit, free beta subunit, nicked free beta (with cleavage sites--between amino acids 47 and 48, 43 and 44, or 44 and 45--in blue), intact dimer, nicked dimer (includes nicked beta subunit), and beta-core fragment (amino acids 6 ‚ 40 and 55 ‚ 92).

Structure of HCG
HCG is a dimer consisting of two noncovalently associated, dissimilar subunits. Both the pituitary gland and placental tissue express the alpha subunit (92 amino acids; MW 14.5 kDa), which is immunologically indistinguishable from the alpha subunits of the other pituitary glycoprotein hormones (namely luteinizing hormone, LH; follicle stimulating hormone, FSH; and thyroid stimulating hormone, TSH). The HCG beta subunit (145 amino acids; MW 22.2 kDa) determines the biological and immunoreactive uniqueness of the intact HCG molecule. While the beta subunit closely resembles the beta subunit of LH (the molecules are identical in 97 of 121 amino acids), beta-HCG differs in its serine-rich, extended, carboxyl-terminal (C-terminal) peptide (24 amino acids). Antibodies developed against the C-terminal peptide crossreact with LH minimally or not at all. The C-terminal antibodies prove useful in measuring low HCG concentrations present at early stages in pregnancy and in ectopic pregnancies. Researchers have found increased free beta-HCG levels in patients with malignant tumors, suggesting clinical implications for the molecule.²

The other identified HCG forms have limited (< 20%) or no biological activity.^3-5 Nicked HCG has a cleavage site in the beta chain between amino acids 47 and 48 (or less commonly between positions 43 and 44 or 44 and 45). Circulating nicked HCG levels rise throughout pregnancy, increasing to about 9 percent of the total HCG by the second month and to about 21 percent at term. Nicked free beta-HCG appears in the circulation as well. The main degradation product of beta-HCG is the beta core fragment, which is measurable only in urine and which goes by a variety of names in the literature, including urinary gonadotropin peptide (UGP).

Total HCG Assay
The ratio of nicked and intact HCG concentrations differs among diseases and varies throughout the course of some diseases. Accurate diagnoses depend on total HCG measurements, including both nicked and intact forms. Total HCG assays should measure both intact and nicked forms of HCG and the free beta subunit. Many immunoassays do not detect nicked HCG forms. Dr. Van Ingen and coworkers (Rotterdam, The Netherlands) demonstrated that IMMULITE® HCG recognizes both the intact and nicked forms (abstract accepted for presentation at the 1998 annual meeting of the AACC). The researchers measured HCG concentrations in pooled serum samples using several immunoassay systems. They spiked the samples with identical mass concentrations of either HCG or nicked HCG. Table 1 lists the results, expressed as HCG concentration relative to IMMULITE HCG. While IMMULITE HCG detects nicked and intact forms, the other automated immunoassays detect only a small percentage of nicked HCG.

Table 1.

Reactivities of several immunoassay systems expressed as HCG concentrations (by mass) relative to IMMULITE® HCG results (= 100%).

DPC Assays for HCG and Free Beta-HCG
The IMMULITE HCG assay (described by Vankrieken et al.)6 has a calibration range of up to 5,000 mIU/mL (3rd IS 75/537), with a detection limit of approximately 1.1 mIU/mL. The assay exhibits no high-dose hook effect at HCG levels as high as 2,000,000 mIU/mL. FSH, LH and TSH do not crossreact. Reactivity of the beta subunit of HCG is approximately 130%. This assay therefore measures intact HCG as well as the free beta subunit of HCG. In addition, IMMULITE HCG also measures nicked HCG and nicked free beta-HCG (DPC data on file). Accurate and precise total HCG results are available on the IMMULITE analyzer in 40 minutes.

The IMMULITE Free Beta HCG assay* is a solid-phase, two-site sequential chemiluminescent immunometric assay, with a calibration range of up to 80 ng/mL (80 mIU/mL, 1st IRP 75/551), and a detection limit of approximately 0.02 ng/mL. Crossreactivities of intact HCG, free alpha-HCG, LH beta subunit and TSH beta subunit are approximately 0.089%, 0.15%, 0.4% and 0.002%, respectively. No crossreactivity has been observed with FSH, FSH beta subunit or TSH. Due to the binding sites determined by epitope mapping on the free beta chain, the IMMULITE Free Beta HCG assay fully recognizes the nicked free beta subunit of HCG.

Conclusions
HCG circulates in several forms, the proportions of which may vary with patient condition or disease. Commercial assays for HCG have different antibody specificities; these differences must be recognized to achieve consistent and reliable HCG results. The IMMULITE HCG assay measures both the intact and nicked forms of HCG as well as the free beta and nicked free beta subunit. The IMMULITE Free Beta HCG assay is specific for the beta subunit, measuring both the unnicked and nicked forms. Cole¹ advises that laboratories be aware of what an HCG assay actually measures, and underscores the importance of measuring multiple forms of HCG and beta subunit depending on the clinical context.

References

1. Cole LA. Clin Chem 1997; 43(12): 2233-43.

2. Cole LA, Kardana A. Clin Chem 1992; 38(2): 263-70.

3. Bidart JM, Puisieux A, Troalen F, Foglietti MJ, Bohuon C, Bellet D. Biochem Biophys Res Commun 1988; 154(2):626-32.

4. Cole LA, Kardana A, Andrade-Gordon P et al. Endocrinology 1991; 129(3):1559-67.

5. Cole LA, Kardana A, Park SY, Braunstein GD. J Clin Endocrinol Metab 1993; 76(3):704-10.

6. Vankrieken L, De Hertogh R. Clin Chem 1995; 41(1):36-40.

*Available outside the US

DPC also offers Coat-A-Count® HCG IRMA which recognizes intact HCG and nicked HCG.