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Physiological role of human specific seminal CD10-CD13 enzyme complex

人类特异精液CD10-CD13酶复合物的生理作用

基本信息

批准号：
15590583
负责人：
ABE Sumiko
金额：
$ 1.79万
依托单位：
Fukushima Medical University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2004
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15590583/
关键词：
CD 10-CD 13 enzyme complex inducible nitric oxide synthase sandwich ELISA human semen anti-peptide antibod

项目摘要

We developed a sandwich ELISA for detection of inducible nitric oxide synthase (iNOS) using a combination of two kinds of anti-iNOS antibodies, anti-peptides and anti- recombinant iNOS antibodies that we produced. An antibodies against the synthetic peptide based on the rat iNOS amino acid sequence and the recombinant mouse iNOS were shown by Western blotting to be positive against the 130-kDa subunit mouse recombinant NOS. The detection limit of the ELISA assay was estimated to be 0.1-2 ng of mouse recombinant iNOS as a standard. Human recombinant iNOS was not detected by this method. The intra-assay and inter-assay coefficients of variation were approximately 5% and 10%, respectively. The recoveries of mouse iNOS added to various biological samples were also examined. The recovery of iNOS added to human serum was insufficient because of a high background. On the other hand, iNOS added to 100,000 x g supernatant of rat liver homogenate and 2% bovine serum albumin solution as well as that added to PBS were detectable.We also examined rat livers treated with lipopolysaccharide (LPS), which strongly induces iNOS expression. Using this method, 0.59 and 1.4 ng of iNOS, equivalent to 1 mg protein, were detected in control and LPS-treated rat livers. These results suggest that this sandwich ELISA is sensitive and specific for mouse (also rat) iNOS and that it enables detection of iNOS in tissue homogenate.

我们建立了一种检测诱导型一氧化氮合酶（iNOS）的夹心ELISA，使用我们制备的两种抗iNOS抗体，抗肽和抗重组iNOS抗体的组合。针对基于大鼠iNOS氨基酸序列的合成肽和重组小鼠iNOS的抗体通过蛋白质印迹显示为针对130-kDa亚基小鼠重组NOS呈阳性。ELISA测定的检测限估计为0.1- 2ng小鼠重组iNOS作为标准。该方法未检测到人重组iNOS。批内和批间变异系数分别约为5%和10%。还检查了添加到各种生物样品中的小鼠iNOS的回收率。由于高背景，加入人血清中的iNOS的回收率不足。另一方面，在100，000 × g大鼠肝匀浆上清液和2%牛血清白蛋白溶液以及PBS中均可检测到iNOS。我们还检测了用脂多糖（LPS）处理的大鼠肝脏，其强烈诱导iNOS表达。使用这种方法，0.59和1.4纳克的iNOS，相当于1毫克蛋白质，检测到对照组和LPS处理的大鼠肝脏。这些结果表明，该夹心ELISA对小鼠（以及大鼠）iNOS是敏感和特异的，并且它能够检测组织匀浆中的iNOS。