HtrA3 as an Early Marker for Preeclampsia: Specific Monoclonal Antibodies and Sensitive High-Throughput Assays for Serum Screening

Abstract

Mammalian HtrA3 (high temperature requirement A3) is a serine protease of the HtrA family. It has two isoforms [long (HtrA3-L) and short (HtrA3-S)] and is important for placental development and cancer progression. Recently, HtrA3 was identified as a potential diagnostic marker for early detection of preeclampsia, a life-threatening pregnancy-specific disorder. Currently there are no high-throughput assays available to detect HtrA3 in human serum. In this study we generated and fully tested a panel of five HtrA3 mouse monoclonal antibodies (mAbs). Three mAbs recognised both HtrA3-L and HtrA3-S and the other two detected HtrA3-L only. All five mAbs were highly specific to HtrA3 and applicable in western blotting and immunohistochemical analysis of endogenous HtrA3 proteins in the mouse and human tissues. Amplified luminescent proximity homogeneous assays-linked immunosorbent assays (AlphaLISAs), were developed to detect HtrA3 isoforms in picomolar levels in serum. The HtrA3 AlphaLISA detected significantly higher serum levels of HtrA3 in women at 13–14 weeks of gestation who subsequently developed preeclampsia compared to gestational-age matched controls. These HtrA3 mAbs are valuable for the development of immunoassays and characterisation of HtrA3 isoform-specific biology. The newly developed HtrA3 AlphaLISA assays are suitable for large scale screening of human serum.

Functional Antagonism between High Temperature Requirement Protein A (HtrA) Family Members Regulates Trophoblast Invasion

“…Clinically, elevated HtrA1 and HtrA3 expression has been detected in the serum or placental tissues of patients with preeclampsia (15,22,23). Our previous study has demonstrated that HtrA4 expression is decreased in EVTs of preeclamptic placenta (7).…”

Structural insights into the activation mechanisms of human HtrA serine proteases

“…In normal pregnancy, a high level of HtrA3 in placenta and maternal peripheral blood is observed in the first trimester but drops significantly afterwards. An elevated level of HtrA3 in maternal serum at 13e14 week of gestation has been correlated with subsequently developed preeclampsia [86,87]. However, Inagaki et al [88] reported elevated levels of HtrA4, but not HtrA3, in preeclampsia.…”

Serum HtrA1 is differentially regulated between early-onset and late-onset preeclampsia

“…The HtrA2 antibody (goat polyclonal) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), the HtrA4 antibody (rabbit polyclonal) was from Abcam (Cambridge, England, UK), and the HtrA3 antibody (mouse monoclonal) was produced as previously described [33].…”