Mizuta T, Shimada H, Arai K, Hori H, Hattori S, Yamamoto K, Sakai T, Nagai Y
Department of Internal Medicine, Saga Medical School, Japan.
Hybridoma. 1996 Oct;15(5):373-8. doi: 10.1089/hyb.1996.15.373.
Two monoclonal antibodies (MAbs) to human placenta laminin (pl-LAM), 1D8 (IgG1) and 6G5 (IgG2b) were generated and shown by ELISA and immunoblot analysis to recognize only native pl-LAM, but not denatured, reduced pl-LAM or mouse EHS laminin. Intact pl-LAM was easily isolated and purified in large scale from human placenta by 1D8-conjugated affinity chromatography. Electrophoretic analysis of the purified pl-LAM revealed the presence of a major 750-kDa component composed of 320-, 220-, and 200-kDa polypeptides and a minor 800-kDa component composed of 320-, 240-, and 220-kDa polypeptides. Neither molecule had a 400-kDa component corresponding to the A chain. It has already been shown that the 320-kDa polypeptide is identical to the M chain of human merosin (Hori et al. J. Biochem. 1994;116:1212-1219). Electron microscopy revealed that isolated merosin was composed of three short arms and one long arm. By immunohistochemistry, MAbs showed positive staining in human adult kidney and liver. These results indicate that these MAbs recognize only native merosin and can be used to study merosin structure and function by rapid purification of native merosin and by immunohistochemical analysis.
制备了两种针对人胎盘层粘连蛋白（pl-LAM）的单克隆抗体（MAb），即1D8（IgG1）和6G5（IgG2b），通过酶联免疫吸附测定（ELISA）和免疫印迹分析表明，它们仅识别天然pl-LAM，而不识别变性、还原的pl-LAM或小鼠EHS层粘连蛋白。完整的pl-LAM可通过1D8偶联亲和色谱法从人胎盘中轻松大规模分离和纯化。对纯化的pl-LAM进行电泳分析显示，存在一个由320 kDa、220 kDa和200 kDa多肽组成的主要750 kDa成分，以及一个由320 kDa、240 kDa和220 kDa多肽组成的次要800 kDa成分。这两种分子均没有对应于A链的400 kDa成分。已经证明，320 kDa多肽与人merosin的M链相同（Hori等人，《生物化学杂志》，1994年；116:1212 - 1219）。电子显微镜显示，分离出的merosin由三条短臂和一条长臂组成。通过免疫组织化学，单克隆抗体在成人肾脏和肝脏中显示出阳性染色。这些结果表明，这些单克隆抗体仅识别天然merosin，可用于通过快速纯化天然merosin和免疫组织化学分析来研究merosin的结构和功能。
