抗人RANTES分子单克隆抗体的制备及特性鉴定

目的:制备抗人RANTES分子单克隆抗体(mAb)并进行初步鉴定,为研究RANTES分子的组织分布和功能提供实验手段。方法:应用淋巴细胞杂交瘤技术,制备小鼠源性抗人RANTESmAb。用ELISA法鉴定腹水mAb的效价。用Q FastFlow阴离子交换柱纯化mAb。用Westernblot鉴定mAb的抗原结合活性。用免疫组化染色法,对RANTES分子在进行小肠移植术的大鼠移植肠组织中的分布进行鉴定。结果:获得4株分泌抗RANTESmAb的杂交瘤细胞株。间接ELISA法测定腹水mAb的效价均达1×10-6,3株mAb为IgG1亚类(κ),1株为IgG2b(κ)。Westernblot的结果显示,3株mAb与人RANTES均有良好的结合活性。用3株mAb进行免疫组化染色的结果显示,RANTES分子在进行小肠移植术的大鼠小肠腺上皮细胞胞质中呈高表达。结论:获得4株能特异性识别天然RANTES分子的mAb。大鼠小肠移植后其肠上皮细胞中可高水平地表达RANTES。

Preparation and characterization of monoclonal antibodies against human RANTES molecule

AIM: To prepare monoclonal antibodies against human RANTES molecule and identify the expression of RANTES in rat small intestine after small bowel transplantation. METHODS: Murine mAbs were prepared by B lymphocyte hybridoma technique. The expression of RANTES in rat small intestine after small bowel transplantation was detected by immunohistochemistry. RESULTS: Four hybridoma cell lines secreting monoclonal antibodies to human RANTES, FMU-RANTES 1, FMU-RANTES 2, FMU-RANTES 3 and FMU-RANTES 4, were established. The titers of a scetic mAbs reached to 1 x 10(-6) and the Ig subclass of FMU-RANTES 1, FMU-RANTES 3 and FMU-RANTES 4 was IgG1(kappa) and that of FMU-RANTES 2 was IgG2b(kappa). Among these mAbs, FMU-RANTES 1, FMU-RANTES 2 and FMU-RANTES 3 could bind human RANTES protein in Western bolt. FMU-RANTES 1, FMU-RANTES 2 and FMU-RANTES 4 could be used in immunohistochemistry staining. Rat RANTES molecule could be detected in the cyto plasm of epithelial cells in rat small intestine after small bowel transplantation. CONCLUSION: Four mAbs against RANTES molecule were prepared, which can provide a useful tool in research on the structure and function of RANTES molecule. High expression of RANTES may be involved in the rejection of allogeneic graft.