HtrA4 ELISA Assay Kit

Additional Information

Assay Background

---

Human HtrA4, the newest member of the HtrA family, an unique family of multidomain serine proteases, was initially reported as a serine protease associated with pregnancy. Polypeptide chains of human HtrA4 consist of several domains: An N-terminal insulinlike growth factor domain, followed by a Kazal-type serine protease inhibitor domain, a linker region, a trypsin-like protease domain and a PDZ domain. Two studies reported in 2012 an association between abnormal levels of HtrA4 and preeclamsia.  However, the findings are contradictory, while the study by Inagaki et al. [3] reported an elevation of HtrA4 in preeclampsia, the study by Wang et al. published that HtrA4 expression was decreased in preeclampsia. Therefore, the precise association between HtrA4 and preeclampsia was for some years unclear, till recently, when Sing et al. [4] published new data that showed that human HtrA4 expression wa  restricted to the placenta and significantly up-regulated in early-onset but not late-onset preeclampsia. The serm HtrA4 levels in normal pregnancy increased significantly between the first and second trimesters, then remained constant. Women with earlyonset but not late-onset preeclampsia showed significantly higher HtrA4 levels in serum compared with gestational age-matched controls.

Assay Principle

---

The Eagle Biosciences HtrA4 ELISA provides a highly sensitive and specific quantitative determination of HtrA4 in serum, tissue (Placenta) and cell culture supernatants. The calibration curve covers the range from 80 ng/ml to 1.25 ng/ml.

This quantitative assay is based on a two site sandwich format. A highly specific monoclonal antibody against HtrA4 is immobilised on the plate. HtrA4 will be bound to the wells, other components of the sample are removed by discarding/drying by tapping and washing of the plate. The analyte is detected in two steps using a secondary biotin-labeled monoclonal antibody and a highly polymerised streptavidin-peroxidase conjugate. Any excess is removed by discarding/drying by tapping and washing after each detection step. The amount of peroxidase bound to each well is determined by the addition of TMB Substrate. The reaction is stopped by adding the Stop Solution and the yellow colour is read in a microtiter plate reader at 450 nm. The concentration of HtrA4 in a sample is determined by interpolation from the standard curve. One kit contains reagents for 96 determinations, thus allowing the measurement of one standard curve and 40 samples in duplicate.