牛骨骼肌肌钙蛋白T单克隆抗体的制备及其特性鉴定

制备牛骨骼肌肌钙蛋白T的单克隆抗体,评价单抗的特性。肌肉提取物经聚丙烯酰氨凝胶电泳(sodium dodecyl sulfate polyacrylamide gel electrophoresis,SDS-PAGE)后切胶,用于免疫Balb/c小鼠。经聚乙二醇(polyethylene glycol,PEG)法融合,制备稳定分泌抗体的杂交瘤细胞株。用酶联免疫吸附试验(enzyme linked immunosorbent assay,ELISA)方法对单抗的效价、亚型、特异性等进行分析。经筛选后,获得2株稳定分泌抗体的杂交瘤细胞株,其中3A8特异性好、效价高,为IgG1亚型,亲和常数为8.1×10~8L/mol。免疫印迹结果显示该单抗能与牛羊的骨骼肌提取物反应,不与鸡鸭的骨骼肌提取物反应。

Preparation and Characterization of Monoclonal Antibody Against Bovine Skeletal Muscle Troponin T

Monoclonal antibody(mAb)against bovine skeletal muscle troponin T was prepared and their properties were evaluated.Balb/c mice mice were immunized with sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE)of muscle extract.Hybridoma cell strains stably secreting m Ab were fused using polyethylene glycol(PEG).The titer,subtype and specificity of mAbs were analyzed by enzyme linked immunosorbent assay(ELISA).Two hybridoma cell strains stably secreting mAb were obtained after screening.The 3A8 with the highest titer and specificity was the IgG1 subtype,and the affinity constant was 8.1×10~8 L/mol.Western blot results showed that 3A8 reacted with skeletal muscle extracts from bovine and sheep,but not chicken and duck.