Fully Automated Chemiluminometric Assay for Hyperglycosylated Human Chorionic Gonadotropin (Invasive Trophoblast Antigen)

Abstract

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 ␤-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 ␤-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 -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 L) and long incubation times (turnaround time, 1-2 days). We therefore developed an automated immunochemiluminometric assay (ICMA) that uses two monoclonal antibodies: the HhCGspecific B152 antibody described above and a hCG ␤-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 L of sample into a cuvette, followed by 25 L of streptavidin-coated magnetic particles (4 g/L Dynal M-270), 70 L of capture antibody (6 mg/L B152), and 260 L 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 L 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 perox...

Maternal Serum Invasive Trophoblast Antigen (Hyperglycosylated hCG) as a Screening Marker for Down Syndrome during the Second Trimester

“…Using standard methods of data analysis that have been shown to be robust in the past, we found that ITA performance was essentially equivalent to hCG. For example, among 533 serum ITA measurements collected as part of another study (14 ), there was no such deviation from expected at high ITA concentrations among the unaffected pregnancies, as found in the current study (Fig. 2).…”

“…The monoclonal antibody (B152) specific for ITA used in this assay has minimal cross-reactivity with hCG and is applicable to many sample types, including serum (14 ). Singleton samples were assayed.…”

Determination of hyperglycosylated human chorionic gonadotropin produced by malignant gestational trophoblastic neoplasias and male germ cell tumors using a lectin-based immunoassay and surface plasmon resonance

“…This antibody was raised against a choriocarcinoma-derived hCG, and its increased binding may indicate trophoblastic disease (Birken et al 2001), but it may also be useful in detecting risk of pregnancy complications such as early pregnancy loss (Kovalevskaya et al 2002b) and Down syndrome , Pandian et al 2003. A lectin immunoassay using WGA binding on hCG in serum (Abushoufa et al 2000) was not an effective additional marker in screening for Down syndrome in the first trimester (Spencer et al 2002), but it was not tested with urine specimens.…”

Elimination of Complement Interference in Immunoassay of Hyperglycosylated Human Chorionic Gonadotropin

“…A high proportion of hCG as hCG-h is useful in diagnosing malignant trophoblastic disease (4 ). These results need to be confirmed, but an earlier-version assay for hCG-h is not presently available (5 ). We developed a time-resolved immunofluorometric assay that uses B152 and found that serum contains factors that interfere with the assay to cause falsely low results.…”