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目的制备并鉴定人血管内皮钙黏蛋白5(VECDH5)的单克隆抗体(m Ab)。方法用重组原核人VECDH5蛋白免疫BALB/c小鼠,利用杂交瘤技术进行细胞融合;间接ELISA筛选阳性克隆,得到稳定分泌抗VECDH5抗体的杂交瘤细胞株,ELISA测定其效价;Western blot法、流式细胞术、免疫荧光细胞化学染色、免疫组织化学染色鉴定其特异性及抗原表位。结果制备并获得1株可分泌特异性抗VECDH5的杂交瘤细胞株(2C11)。ELISA测定腹水效价高达1∶10 000。多种方法均证实该抗体能特异性识别人VECDH5蛋白,并可在上述不同方法中应用。经抗原表位鉴定,研究制备的2C11识别的氨基酸序列为LDREVVPWYNLTVEA。结论成功制备了亲和力高、特异型好的抗人VECDH5的m Ab。
Objective To prepare and identify the monoclonal antibody (m Ab) of human vascular endothelial cadherin 5 (VECDH5). Methods BALB / c mice were immunized with recombinant prokaryotic human VECDH5 protein and hybridized with the hybridoma technique. The positive clones were screened by indirect ELISA, and the hybridoma cell lines secreting anti-VECDH5 antibody were obtained. The titer was determined by ELISA. Flow cytometry, immunofluorescence cytochemistry staining, immunohistochemical staining to identify its specificity and epitopes. Results A hybridoma cell line secreting specific anti-VECDH5 (2C11) was prepared and obtained. ELISA ascites titer up to 1:10 000. A variety of methods have confirmed that the antibody can specifically recognize human VECDH5 protein, and can be used in the above different methods. The antigenic epitopes identified 2C11 identified amino acid sequence of LDREVVPWYNLTVEA. Conclusion The m Ab with high affinity and specificity of anti-human VECDH5 was successfully prepared.