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目的:制备抗人ILT4分子单克隆抗体(mAb)并进行初步鉴定。方法:应用淋巴细胞杂交瘤技术,制备小鼠源性抗人ILT4 mAb。用间接ELISA法测定腹水mAb的效价。以Western blot测定mAb的抗原结合活性。通过流式细胞术(FCM)对mAb结合转染细胞表面及天然细胞表面ILT4分子的活性进行鉴定。结果:获得7株分泌抗ILT4 mAb的杂交瘤细胞株。ELISA法测定腹水mAb的效价均达1×10-6,7株mAb均为IgG1(κ)。Western blot结果显示,3株mAb与人ILT4有良好的结合活性。用转染细胞及U937细胞做FCM分析发现另外4株mAb可结合真核表达的及天然的ILT4分子。结论:获得7株能特异性识别ILT4分子的mAb,为研究ILT4分子的组织分布和功能研究提供了可靠的实验手段。
Objective: To prepare anti-human ILT4 monoclonal antibody (mAb) and preliminary identification. Methods: Mouse-derived anti-human ILT4 mAb was prepared using lymphocyte hybridoma technique. Indirect ELISA was used to determine the titer of ascites mAb. The mAb antigen binding activity was determined by Western blot. The activity of mAb binding to ILT4 molecules on the surface of the transfected cells and native cell surface was identified by flow cytometry (FCM). Results: Seven hybridoma cell lines secreting anti-ILT4 mAb were obtained. The titer of ascites mAb measured by ELISA reached 1 × 10-6, 7 mAbs were IgG1 (κ). Western blot results showed that the three mAbs had good binding activity with human ILT4. FCM analysis of transfected cells and U937 cells revealed that the other 4 mAbs could bind to eukaryotic expressed and native ILT4 molecules. CONCLUSION: Seven mAbs that specifically recognize ILT4 are obtained and provide reliable experimental methods for studying the tissue distribution and function of ILT4.