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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。
OBJECTIVE: To prepare and identify monoclonal anti-human RANTES monoclonal antibody (mAb) and provide experimental means for studying the tissue distribution and function of RANTES. Methods: Mouse-derived anti-human RANTES mAb was prepared by lymphocyte hybridoma technique. The ascites mAb titer was identified by ELISA. The mAb was purified using a Q FastFlow anion exchange column. The antigen binding activity of the mAb was identified by Western blot. Immunohistochemical staining was used to identify the distribution of RANTES molecules in the transplanted intestinal tissue of rats undergoing intestinal transplantation. Results: Four hybridoma cell lines secreting anti-RANTES mAb were obtained. Indirect ELISA assay for ascites mAb titer reached 1 × 10-6, 3 mAb IgG1 subclass (κ), a strain of IgG2b (κ). Western blot results showed that all 3 mAbs had good binding activity with human RANTES. Immunohistochemical staining with 3 mAbs showed that RANTES was highly expressed in the cytoplasm of small intestine glandular epithelial cells in small bowel transplantation. Conclusion: Four mAbs that specifically recognize the native RANTES molecule were obtained. RANTES can be expressed in intestinal epithelial cells at high level after rat intestinal transplantation.