Urinary screening tests for fetal Down syndrome: II. Hyperglycosylated hCG

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...

mentioning

confidence: 90%

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

Pandian

Ossolinska-Plewnia

2003

“…Stability studies of urinary ITA have also found that, as with ␤-core fragment of hCG, losses in ITA concentrations might occur after multiple freeze-thaw cycles (31 ). The current study was designed to avoid the possible confounding effect of freezing-thawing or long-term storage by use of prompt shipment of fresh specimens directly to the laboratory for immediate assay.…”

Section: Discussionmentioning

confidence: 99%

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

Palomaki

Knight

Roberson³

et al. 2004

Background: Down syndrome screening is commonly performed in the US using maternal age and three or four second-trimester maternal serum markers that can identify up to 75% of affected pregnancies by offering diagnostic studies to 5% of women. Invasive trophoblast antigen [ITA; hyperglycosylated human chorionic gonadotropin (hCG)] is a promising marker that can be measured in urine or serum in the first or second trimester. We report preliminary results for urinary ITA in an ongoing observational study. Methods: Women undergoing second-trimester amniocentesis for reasons not associated with biochemical testing provided consent and a urine (and possibly serum) sample that was tested within a few days. Demographic and pregnancy-related information was collected, along with karyotype. Screening performance was modeled for ITA alone and in combination with serum markers Results: Twelve recruitment centers collected urine from 2055 women with singleton pregnancies between 15 and 20 weeks of gestation (2023 unaffected, 28 Down syndrome, and 4 pregnancies with other chromosome abnormalities). After correction for gestational age, urine concentration, and maternal race and weight, the ITA measurements were higher in women with a Down

“…Hyperglycosylated human chorionic gonadotropin (hCG-h) 1 is the major form of hCG occurring in early pregnancy and trophoblastic disease, whereas the glycans are smaller in hCG produced later in pregnancy (1,2 ). hCG-h can be measured by immunoassays based on monoclonal antibody B152, which reacts with a type 2 core 1 Nonstandard abbreviations: hCG-h, hyperglycosylated human chorionic gonadotropin; hCG␤, ␤ subunit of hCG; DELFIA, dissociation-enhanced lanthanide fluorescence immunoassay.…”

Section: To the Editormentioning

confidence: 99%

“…hCG-h can be measured by immunoassays based on monoclonal antibody B152, which reacts with a type 2 core 1 Nonstandard abbreviations: hCG-h, hyperglycosylated human chorionic gonadotropin; hCG␤, ␤ subunit of hCG; DELFIA, dissociation-enhanced lanthanide fluorescence immunoassay. O-glycan attached to Ser132 and surrounding peptide structures but not with hCG lacking this glycan or having a type 1 core O-glycan in this position (3 ).…”

Section: To the Editormentioning

confidence: 99%

“…O-glycan attached to Ser132 and surrounding peptide structures but not with hCG lacking this glycan or having a type 1 core O-glycan in this position (3 ). In early pregnancy, reduced hCG-h concentrations in urine have been associated with early pregnancy loss (2 ), whereas increased concentrations at 9 to 12 weeks have been associated with Down syndrome (1 ). A high proportion of hCG as hCG-h is useful in diagnosing malignant trophoblastic disease (4 ).…”

Section: To the Editormentioning

confidence: 99%

See 1 more Smart Citation

Elimination of Complement Interference in Immunoassay of Hyperglycosylated Human Chorionic Gonadotropin

Stenman¹,

Birken

Lempiäinen³

et al. 2011

by a specific method, such as LC-MS. It is also possible that differences in the calibration of the 2 assays could contribute to this apparent improvement.These results confirm that although immunoassay-measured DHEAS interferes less in the Roche Testosterone II assay than in the Testosterone assay, the Testosterone II assay still appears susceptible to interference from DHEAS or by other steroids detected by the DHEAS immunoassay. This work highlights residual specificity problems with the Roche Testosterone II assay and provides further evidence of the problems in developing direct testosterone immunoassays with a specificity adequate for measuring testosterone in women.