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Technical Briefs |
1 Quest Diagnostics, Nichols Institute, 33608, Ortega Highway, San Juan Capistrano, CA 92690;
2 Nichols Institute Diagnostics, San Clemente, CA 92673;
aauthor for correspondence: fax 949-728-4990, e-mail pandianr{at}questdiagnostics.com
Hyperglycosylated human chorionic gonadotropin (HhCG) is a glycoprotein hormone secreted during embryonic implantation and trophoblast invasion of the uterine wall and is an early marker of pregnancy (1). Relative to hCG, HhCG has a higher molecular mass (38.5–40 kDa, depending on the amount of carbohydrate) and a higher number of asparagine (N)-linked triantennary carbohydrates and serine (O)-linked tetrasaccharide core structures in the ß-subunit (2). Although both are secreted from the placenta and choriocarcinoma, HhCG is produced by mononucleated cytotrophoblasts, and hCG is produced by syncytiotrophoblast cells (3)(4)(5)(6). Because the cytotrophoblasts are primitive and invasive in nature, HhCG is also called invasive trophoblast antigen (ITA) (5).
Birken et al. (7) described a monoclonal antibody (B152) specific for the ß-subunit C-terminal peptide and the O-linked oligosaccharide of HhCG. Although the epitope for this antibody does not require sialic acid, the presence of the O-linked tetrasaccharide core structure is essential (1).
Using IRMAs and ELISAs, investigators showed that (a) HhCG rapidly increases in early pregnancy, attaining substantially higher concentrations and decreasing earlier than hCG (1)(8); (b) HhCG is increased in Down syndrome-affected pregnancies in both the first and second trimesters (9)(10)(11); and (c) the HhCG:hCG ratio appears to be higher in those with invasive vs noninvasive trophoblastic disease (12).
The above HhCG assays were performed manually using large sample volumes (200 µL) and long incubation times (turnaround time, 1–2 days). We therefore developed an automated immunochemiluminometric assay (ICMA) that uses two monoclonal antibodies: the HhCG-specific B152 antibody described above and a hCG ß-subunit-specific antibody (B207). Both antibodies were purified from cell lines provided by Dr. O’Connor (Columbia University, New York, NY). B152 was biotinylated with long-chain NHS-biotin (13), and B207 was conjugated with acridinium ester (14).
The Nichols Institute Diagnostics Advantage® instrument automatically pipetted 15 µL of sample into a cuvette, followed by 25 µL of streptavidin-coated magnetic particles (4 g/L Dynal M-270), 70 µL of capture antibody (6 mg/L B152), and 260 µL of buffer [0.1 mol/L phosphate-buffered saline (PBS), pH 8.2, containing 50 g/L bovine serum albumin (BSA)]. During a 30-min incubation at 37 °C, HhCG in the sample bound to the B152 capture antibody, which in turn bound to the magnetic particles. The magnetic particles were automatically washed three times to remove unbound materials. Detection antibody [300 µL of 1 mg/L B207 in 0.5 mol/L PBS (pH 7.4) with 5 g/L protease-free BSA, 60 mL/L normal mouse serum, and 1 g/L mouse 
-globulin] was then added to the washed magnetic particles. During this 10-min incubation at 37 °C, the B207 antibody bound to a hCG-shared epitope on the HhCG molecule, forming a sandwich complex. After another three washes, the magnetic particle-containing wells were transferred to the on-board luminometer. Hydrogen peroxide (3.25 mL/L)- and sodium hydroxide (0.25 mol/L)-containing solutions were automatically injected into the wells, initiating the chemiluminescence reaction. The generated "flash" of light was quantified and expressed as relative light units (RLU). The RLU are directly proportional to the concentration of HhCG in the sample. The relationship was linear up to 7500 µg/L, at which concentration the curve plateaued (Fig. 1
). No hook effect was observed with concentrations as high as 30 000 µg/L [HhCG from choriocarcinoma, prepared in 0.5 mol/L PBS (pH 7.4) containing 1 g/L protease-free BSA].
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The calibration curve (Fig. 1
, inset) was stored by the instrument, so calibrators did not have to be included every time the assay was performed. The limit of detection, based on the mean RLU for 20 replicates of the zero calibrator plus 2 SD, was 0.1 µg/L. The limit of quantification, based on assays of serial dilutions of a sample (HhCG = 2 µg/L) with the CV calculated from 10 observations at each dilution, was 0.2 µg/L (CV <20%). On the basis of results from three serum pools and one urine pool, the intraassay CV for 20 replicates was <3.5% and the interassay CV was <7.5%. Thus, the assay could be performed in singlicate.
The assay had <0.1% cross-reactivity with all glycoprotein hormones except hCG, which had a cross-reactivity <3.5% (Table 1
). The dose–response of each hCG preparation (total, free ß, nicked, and nicked free ß-hCG) was parallel to that of HhCG. Although hCG cross-reactivity was minimal, it is not clear whether such cross-reactivity was attributable to native hCG itself or to contamination of hCG preparations with HhCG. The latter is highly likely because of (a) the different cross-reactivities reported for various hCG preparations [Table 1
and Refs. (5)(7)]; (b) the similarities between dose–response curves for HhCG and the cross-reactants; and (c) the 0.9% cross-reactivity observed when recombinant murine hCG was tested (Table 1
). Because the reported hCG cross-reactivity was small, HhCG could be measured in the presence of hCG. The HhCG:hCG ratio may be clinically useful (1)(15)(16).
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To determine the suitability of various sample types, we assayed undiluted and diluted serum, EDTA plasma, citrated plasma, urine, and amniotic fluid. The recovery, relative to the undiluted sample concentration, was 90–110%. In addition, stability studies using urine and serum samples from women with Down syndrome-unaffected pregnancies showed that HhCG was stable for 3 days at room temperature and 7 days in the refrigerator, as well as after two rapid freeze-thaw cycles. HhCG was stable in pooled urine and serum for a minimum of 10 months at -70 °C. More studies are required to determine the effect of additional rapid freeze-thaw cycles, slow freeze-thaw cycles, and storage at -20 °C.
The correlation (r) between the previously described ELISA and our ICMA was 0.94 (Deming regression: y = 1.01x - 3.64 µg/L), based on urine samples from 84 pregnant women. When we assayed urine samples from Down syndrome-affected and -unaffected pregnancies that were previously assayed by Cole et al. (9) with the ELISA, we observed a similar increase in HhCG concentrations in Down syndrome patients. Thus, the ICMA compares well with the ELISA and can be applied clinically as described previously (5)(9)(10).
In conclusion, we have developed an automated ICMA for the measurement of HhCG (ITA). This assay may facilitate exploration of HhCG utility for Down syndrome screening, early pregnancy detection, and differentiation of invasive from noninvasive trophoblastic disease.
Acknowledgments
We sincerely thank the following individuals for their contributions: Dr. Steve Birken (Columbia University, New York, NY) supplied various hormone preparations. Dr. Laurence Cole (University of New Mexico, Albuquerque, NM) provided valuable urine and serum samples. Esther Carlton (Quest Diagnostics Nichols Institute, San Juan Capistrano, CA) kindly provided samples from pregnant women and assisted with the statistical analysis. Phil Miller, Darren Carns, and Dr. Delbert A. Fisher also provided invaluable assistance. We gratefully acknowledge Patricia M. Vendely for assistance in editing this manuscript.
References
- and ß-subunits of human chorionic gonadotropin from normal and aberrant pregnancy and choriocarcinoma. Endocrine 1997;7:15-32.[ISI][Medline]
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The following articles in journals at HighWire Press have cited this article:
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  M. R Rubin, J. P Bilezikian, S. Birken, and S. J Silverberg Human chorionic gonadotropin measurements in parathyroid carcinoma Eur. J. Endocrinol., October 1, 2008; 159(4): 469 - 474. [Abstract] [Full Text] [PDF]
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 G. E. Palomaki, G. J. Knight, L. M. Neveux, R. Pandian, and J. E. Haddow Maternal Serum Invasive Trophoblast Antigen and First-Trimester Down Syndrome Screening Clin. Chem., August 1, 2005; 51(8): 1499 - 1504. [Abstract] [Full Text] [PDF]
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M. J.N. Weinans, U. Sancken, R. Pandian, J. M.W. van de Ouweland, H. W.A. de Bruijn, J. P. Holm, and A. Mantingh Invasive Trophoblast Antigen (Hyperglycosylated Human Chorionic Gonadotropin) as a First-Trimester Serum Marker for Down Syndrome Clin. Chem., July 1, 2005; 51(7): 1276 - 1279. [Full Text] [PDF]
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G. E. Palomaki, L. M. Neveux, G. J. Knight, J. E. Haddow, and R. Pandian Maternal Serum Invasive Trophoblast Antigen (Hyperglycosylated hCG) as a Screening Marker for Down Syndrome during the Second Trimester Clin. Chem., October 1, 2004; 50(10): 1804 - 1808. [Abstract] [Full Text] [PDF]
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R. Pandian, L. A. Cole, and G. E. Palomaki Second-Trimester Maternal Serum Invasive Trophoblast Antigen: A Marker for Down Syndrome Screening Clin. Chem., August 1, 2004; 50(8): 1433 - 1435. [Full Text] [PDF]
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L. A. Cole, J. M. Sutton, T. N. Higgins, and G. S. Cembrowski Between-Method Variation in Human Chorionic Gonadotropin Test Results Clin. Chem., May 1, 2004; 50(5): 874 - 882. [Abstract] [Full Text] [PDF]
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G. E. Palomaki, G. J. Knight, M. M. Roberson, G. C. Cunningham, J. E. Lee, C. M. Strom, and R. Pandian Invasive Trophoblast Antigen (Hyperglycosylated Human Chorionic Gonadotropin) in Second-Trimester Maternal Urine as a Marker for Down Syndrome: Preliminary Results of an Observational Study on Fresh Samples Clin. Chem., January 1, 2004; 50(1): 182 - 189. [Abstract] [Full Text] [PDF]
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