抗人NRP1单克隆抗体的活性鉴定及初步应用

Characterization and application of monoclonal antibodies against human NRP1

目的鉴定抗人神经菌毛素1(neuropilin1,NRP1)单克隆抗体的生物活性并利用该抗体检测常见肿瘤细胞株NRP1蛋白的表达情况。方法腹水法制备4株抗NRP1单克隆抗体并用rProtein A亲和柱纯化抗体,间接ELISA检测4株抗体的滴度水平,Western blot分析4株抗体结合NRP1蛋白的特异性,流式细胞术分析4株抗体结合NRP1蛋白的亲和力,共聚焦免疫荧光实验进一步验证抗体结合NRP1蛋白的能力;细胞免疫组织化学染色检测14株肿瘤细胞表达NRP1蛋白的情况。结果 4株抗体均能特异结合NRP1蛋白,其中A6结合NRP1能力最强,能结合在表达NRP1蛋白的细胞膜表面。细胞免疫组织化学染色结果显示NRP1在HepG2、C6、HEK293、BEL-7402、MDA-MB-453呈高水平表达,在U87、MGC803、MCF7、MDA-MB-231中表达水平相对较弱,而在U251、BGC823、H6、HT-29的表达介于两者之间,在SMMC-7721细胞中未检测到表达。结论 4株抗NRP1单克隆抗体均能特异结合NRP1蛋白,多种肿瘤细胞株均不同程度表达NRP1蛋白,为进一步探讨NRP1蛋白与肿瘤发生发展的关系奠定了基础。

With the aim of exploring the roles of neuropilin-1 （NRP1） protein in tumor,we have established 4 anti-NRP1 monoclonal antibodies （mAbs） in our previous work, and will characterize these mAbs in the present study. Indirect ELISA, Western blot, flow cytometer and confocal immunofluorescence have been applied to determine the binding specificity and affinity of the mAbs with NRP1 on MDA-MB-453 cells. The results showed that the mAbs could specifically bind with NRP1 and the A6 mAb had the highest affinity with NRP1. Then the A6 mAb was applied to investigate the expression of NRP1 on 14 cancer cell lines in immunocytoehemical detection. Results indicated that NRP1 was highly expressed on HepG2, C6, HEK293, BEL-7402, MDA-MB-453, moderately expressed on U251, BGC823, H6, HT-29, lowly expressed on U87, MGC803, MCF7, MDA-MB-231, and little expressed on SMMC-7721. The common expression of NRP1 in cancer cell lines would help to elucidate the correlation ofluding drug exploita the NRP1 expression to oncogenesis and these mAbs against NRP1 may play a vital role in anticancer therapy inction.