针对牛和猪前列环素合酶产生的单克隆抗体的特性鉴定及牛前列环素合酶的定量分析。

Characterization of monoclonal antibodies generated against bovine and porcine prostacyclin synthase and quantitation of bovine prostacyclin synthase.

作者信息

Siegle I, Nüsing R, Brugger R, Sprenger R, Zecher R, Ullrich V

机构信息

Faculty of Biology, University of Konstanz, Germany.

出版信息

FEBS Lett. 1994 Jun 27;347(2-3):221-5. doi: 10.1016/0014-5793(94)00504-4.

DOI:10.1016/0014-5793(94)00504-4

PMID:8034007

Abstract

Monoclonal antibodies were raised against prostacyclin synthases purified from bovine and porcine aortae, respectively. Two monoclonal antibodies, RS1 and RS2, were purified and characterized. As shown by enzyme activity precipitation and Western blot analysis, in solubilized bovine and porcine aortae microsomes the monoclonal antibodies reacted only with prostacyclin synthase. The monoclonal antibody RS1 cross-reacts with partially purified prostacyclin synthase from human umbilical veins in an ELISA-based assay. None of the antibodies inhibited the enzyme activity. By combination of the monoclonal antibody RS2 with a polyclonal antibody we established an enzyme-linked immunosorbent assay (ELISA) for quantitation of bovine prostacyclin synthase. ELISA data were confirmed by Western blot analysis. Among different bovine tissues, aortae with 1665 +/- 200 ng/mg microsomal protein showed the highest content of PGIS. Significant lower concentrations were observed in tongue, lung, kidney and thymus ranging from 49 +/- 13.4 to 2.7 +/- 0.9 ng/mg protein. The monoclonal antibody RS1 binds to endothelial cells and vascular smooth muscle cells in human liver tissue.

摘要

分别制备了针对从牛主动脉和猪主动脉中纯化的前列环素合酶的单克隆抗体。纯化并鉴定了两种单克隆抗体RS1和RS2。如酶活性沉淀和蛋白质印迹分析所示，在溶解的牛主动脉和猪主动脉微粒体中，单克隆抗体仅与前列环素合酶发生反应。在基于酶联免疫吸附测定（ELISA）中，单克隆抗体RS1与人脐静脉中部分纯化的前列环素合酶发生交叉反应。这些抗体均未抑制酶活性。通过将单克隆抗体RS2与多克隆抗体结合，我们建立了一种用于定量牛前列环素合酶的酶联免疫吸附测定（ELISA）方法。ELISA数据通过蛋白质印迹分析得到证实。在不同的牛组织中，每毫克微粒体蛋白含1665±200纳克前列环素合酶的主动脉中PGIS含量最高。在舌、肺、肾和胸腺中观察到的浓度显著较低，范围为每毫克蛋白49±13.4至2.7±0.9纳克。单克隆抗体RS1与人肝组织中的内皮细胞和血管平滑肌细胞结合。