【摘要】 目的一株抗破伤风毒素鼠源单克隆抗体的制备与鉴定。方法从实验室前期以破伤风类毒素为抗原筛选得到的6株稳定细胞株中选取4E12进行制备及鉴定,首先将4E12细胞株扩大培养后腹腔注射BALB/c小鼠制备腹水,经Protein G柱和PD10脱盐柱两步纯化鼠源单抗;其次通过SDS-PAGE电泳检测抗体纯度并通过分型试剂盒鉴定抗体轻重链亚型,免疫印迹法鉴定抗体结合区域及表位类型,非竞争ELISA法测定抗体亲和力常数以及小鼠中和实验评价抗体中和活性。结果获得能稳定分泌抗体的杂交瘤细胞株4E12,其细胞培养上清效价为4×10³,抗体轻重链分别为κ型和IgG1型,CDR3序列分别为SQSTHVPPT和ARKGTGTGFNY;4E12腹水抗体效价为3.2×10⁴,抗体经纯化后纯度>95%,以线性表位与TeNT/Hc结构域特异性结合,两者的亲和力常数为3.6×10^-10mol/L,小鼠中和实验结果显示,4E12抗体可部分中和破伤风毒素。结论筛选到一株高亲和力的鼠源单抗,为破伤风毒素检测及中和抗体研发奠定了基础。更多还原

【Abstract】 Objective To prepare and identify a mouse monoclonal antibody against tetanus toxin. Methods 4 E12 was selected from 6 stable cell strains screened by tetanus toxoid as an antigen in the early stage of experiments for preparation and identification.First,through protein G and PD10 column chromatography,monoclonal antibodies（MAb）were purified from ascite of BALB/c mice,which were injected intraperitoneally with positive cells. Next,the purity and subtyepe of MAb were separately detected by SDS-PAGE and typing kit. Then,the binding region was identified by Western blotting and the affinity constant was determined by non-competitive ELISA. Finally,the neutralizing activity was evaluated by neutralization test. Results A hybridoma cell line 4 E12 was obtained,which could stably secrete antibodies. The titer of cell culture supernatant reached 4×10³. The antibody light and heavy chain were identified as κ and IgG1 subtypes,whose CDR3 sequences were respectively SQSTHVPPT and ARKGTGTGFNY. The ascites titer of4 E12 was 3.2×10⁴,4 E12 antibody purity was over 95%,which specifically bound to a linear epitope in the TeNT/Hc domain with an affinity constant of 3.6×10^-10 mol/L. The result of mouse neutralization assay showed that 4 E12 antibody partially neutralized tetanus toxin. Conclusion A high-affinity mouse monoclonal antibody is successfully screened,which can contribute to detection of tetanus toxin and development of neutralizing antibodies.更多还原