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目的:制备抗神经细胞突起生长诱向因子(β-Netrin)的多克隆抗体(pAb)和单克隆抗体(mAb),并对其特异性进行鉴定。方法:应用GoldKey软件分析人βNetrinC末端结构域氨基酸的序列(共114个氨基酸),确定抗原表位并人工合成多肽。然后采用碳化二亚胺法,将合成肽与载体蛋白牛血清白蛋白(BSA)偶联作为抗原,免疫BALB/c小鼠。取免疫小鼠脾细胞与Sp2/0骨髓瘤细胞常规融合,依次进行HAT选择培养,间接ELISA法筛选阳性的杂交瘤细胞并克隆化。对分泌的mAb的效价、Ig亚类(型)及特异性,分别用间接ELISA和Westernblot进行鉴定。同时,通过免疫细胞化学染色鉴定抗体的特异性。另外,以偶联的分子免疫新西兰白兔,制备抗β-Netrin的pAb,用Westernblot鉴定其特异性。结果:以确定的此16个氨基酸的序列NH2-FRGKRT-LYPESWTDRG-COOH作为抗原表位,以人工合成多肽与BSA偶联作为免疫原,获得3株可稳定分泌特异性抗β-Netrin mAb的杂交瘤细胞。免疫细胞化学染色结果表明,这3株抗体均能特异性地识别细胞中的抗原。另外,制备了高效价的抗β-Netrin的pAb,并能特异性地识别原核表达的β-Netrin蛋白。结论:采用人工合成多肽作为半抗原成功地制备出抗β-Netrin的pAb和mAb。
OBJECTIVE: To prepare polyclonal antibodies (pAb) and monoclonal antibodies (mAb) against neurite outgrowth-inducing factor (β-Netrin), and to identify their specificity. Methods: The sequence of human βNetrinC terminal domain amino acids (total 114 amino acids) was analyzed by GoldKey software to identify epitopes and to synthesize peptides. Then, the carbodiimide method was used to conjugate the synthetic peptide with carrier protein bovine serum albumin (BSA) as antigen to immunize BALB / c mice. The splenocytes of immunized mice were routinely fused with Sp2 / 0 myeloma cells, followed by selective culture of HAT cells. The positive hybridoma cells were screened by indirect ELISA and cloned. The titer, Ig subclass (type) and specificity of the secreted mAb were identified by indirect ELISA and Western blot, respectively. At the same time, the specificity of the antibody was identified by immunocytochemical staining. In addition, New Zealand white rabbits were immunized with conjugated molecules to prepare pAb against β-Netrin, and its specificity was confirmed by Western blot. Results: The 16 amino acid sequences of NH2-FRGKRT-LYPESWTDRG-COOH were identified as epitopes and the conjugates of synthetic peptides with BSA were used as immunogens to obtain three hybrids that stably secreted specific anti-β-Netrin mAb Tumor cells. Immunocytochemical staining results showed that all three antibodies could specifically recognize the antigens in the cells. In addition, pAb with high titers of anti-β-Netrin was prepared and could specifically recognize prokaryotic expressed β-Netrin protein. CONCLUSION: pAb and mAb against β-Netrin were successfully prepared using synthetic peptides as haptens.