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.

我们开发了一种夹心 ELISA，使用我们生产的两种抗 iNOS 抗体（抗肽和抗重组 iNOS 抗体）的组合来检测诱导型一氧化氮合酶 (iNOS)。通过蛋白质印迹法显示，针对基于大鼠 iNOS 氨基酸序列和重组小鼠 iNOS 的合成肽的抗体对 130-kDa 亚基小鼠重组 NOS 呈阳性。以小鼠重组 iNOS 为标准，ELISA 测定的检测限估计为 0.1-2 ng。该方法未检测到人重组 iNOS。测定内和测定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。由于背景较高，添加到人血清中的 iNOS 的回收率不足。另一方面，添加到 100,000 x g 大鼠肝脏匀浆上清液和 2% 牛血清白蛋白溶液中的 iNOS 以及添加到 PBS 中的 iNOS 均可以检测到。我们还检查了用脂多糖 (LPS) 处理的大鼠肝脏，脂多糖 (LPS) 强烈诱导 iNOS 表达。使用这种方法，在对照和 LPS 处理的大鼠肝脏中检测到 0.59 和 1.4 ng iNOS，相当于 1 mg 蛋白质。这些结果表明，这种夹心 ELISA 对于小鼠（也包括大鼠）iNOS 具有敏感性和特异性，并且能够检测组织匀浆中的 iNOS。