抗蓝载体单克隆抗体的制备及特性鉴定

Preparation and Identification of Monoclonal Antibody(mAb) Against Blue Carrier Immunogenic Protein

目的制备抗蓝载体(blue carrier,BC)单克隆抗体(mAb),为相关研究提供支持与工具。方法用人工合成肽与蓝载体交联后免疫Balb/c小鼠,取其脾细胞与NS-1融合制备杂交瘤,经筛选和3次克隆化,从制备的腹水中纯化单克隆抗体。用ELISA以及Dot-ELISA方法鉴定单克隆抗体的特性(效价、Ig亚类、特异性以及亲和力)。结果筛选到1株可稳定分泌抗蓝载体单克隆抗体的杂交瘤细胞5B5,腹水效价2×10-6,单抗亚类为IgG1,抗体亲和力为7.8×108。Dot-ELISA结果显示,在相同条件下5B5mAb只与蓝载体特异性结合,与钥孔戚血蓝素(KLH)、牛血清白蛋白(BSA)、鸡卵清白蛋白(OVA)等载体蛋白不发生交叉反应。结论获得1株能特异性识别蓝载体蛋白的mAb,为评价短肽及半抗原交联免疫效果、某些疾病诊断及治疗方法疗效提供了工具。

Objective Preparation and identification of monoclonal antibody （mAb） against blue carrier. Methods Balb/c mice were immunized with blue carrier protein which had been coupled to synthetic peptides, and the hybridoma cells were generated with mouse spleen cells and myeloma cells NS-1. After fusion and three times of cloning, the monoclonal antibody were purified from ascites and characterized by indirect ELISA and Dot-ELISA. Results One hybridoma cell line secreting anti-blue carrier mAb was obtained and named as 5B5 which belongs to isotype IgG 1. The titer of the mAb in ascites was 2×10^-6 and the Kaff was 7.8×10^8. In the Dot-ELISA assay, the mAb bound to blue carrier specifically. Conclusion One mAb with high titer, specificity and affinity was prepared. Preparation of anti-blue carrier mAb provides a useful tool for evaluation of blue carrier conjugate immunogen, some new diagnosis and therapy.