HtrA3 as an early marker for preeclampsia: specific monoclonal antibodies and sensitive high-throughput assays for serum screening.
HtrA3 as an early marker for preeclampsia: specific monoclonal antibodies and sensitive high-throughput assays for serum screening.
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DOI：
10.1371/journal.pone.0045956
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发表时间：
2012
期刊：
影响因子：
3.7
通讯作者：
Nie G
中科院分区：
文献类型：
--
作者：
Dynon K;Heng S;Puryer M;Li Y;Walton K;Endo Y;Nie G
哺乳动物HtrA 3（高温要求A3）是HtrA家族的丝氨酸蛋白酶。它有两种亚型[长（HtrA 3-L）和短（HtrA 3-S）]，对胎盘发育和癌症进展很重要。最近，HtrA 3被鉴定为早期检测先兆子痫的潜在诊断标志物，先兆子痫是一种危及生命的妊娠特异性疾病。目前还没有高通量检测方法可用于检测人血清中的HtrA 3。在这项研究中，我们生成并全面测试了一组五种HtrA 3小鼠单克隆抗体（mAb）。三种单克隆抗体识别HtrA 3-L和HtrA 3-S，另外两种仅检测HtrA 3-L。所有5种mAb均对HtrA 3具有高度特异性，适用于小鼠和人组织中内源性HtrA 3蛋白的Western印迹和免疫组织化学分析。开发了扩增发光邻近均相测定-联用免疫吸附测定（AlphaLISAs）以检测血清中皮摩尔水平的HtrA 3亚型。HtrA 3 AlphaLISA在妊娠13-14周的妇女中检测到显著更高的HtrA 3血清水平，与孕龄匹配的对照相比，这些妇女随后发展为先兆子痫。这些HtrA 3单克隆抗体对于HtrA 3亚型特异性生物学的免疫测定和表征的发展是有价值的。新开发的HtrA 3 AlphaLISA检测试剂盒适用于人血清的大规模筛查。
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.
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