论文部分内容阅读
目的探究抗人死亡受体5(DR5)单链抗体(aDR5ScFv)的特异性。方法 ELISA检测aDR5ScFv的效价、亲和力和表位分析。Western blotting检测单链抗体的特异性。MTT检测aDR5ScFv对结肠癌细胞SW480的生长抑制。结果 ELISA显示抗人死亡受体5单链抗体抗体效价为1.2×104。通过间接ELISA证实aDR5ScFv与aDR5mAb识别DR5胞外段(eDR5,系本实验室自己构建)的同一个位点。aDR5ScFv的亲和力常数为2.12×10-2。eDR5与aDR5ScFv能够特异结合。MTT检测结果显示,aDR5ScFv抑制SW480细胞生长呈剂量依赖性,aDR5ScFv终质量浓度分别为0.225 mg/ml、0.45 mg/ml、0.9 mg/ml、1.2 mg/ml。200μl时,SW480细胞的存活率分别为97.7%,70.9%、70.1%、23.6%。结论 aDR5ScFv能与eDR5特异性结合,并有较强的活性。
Objective To investigate the specificity of anti-human death receptor 5 (DR5) single chain antibody (aDR5ScFv). Methods ELISA, titers, affinity and epitope analysis of aDR5ScFv were performed. Western blotting detection of single chain antibody specificity. Growth Inhibition of aDR5ScFv on Colon Cancer Cell SW480 by MTT. Results ELISA showed that the anti-human death receptor 5 single chain antibody antibody titer was 1.2 × 104. Confirmation by indirect ELISA that aDR5 ScFv and aDR5 mAb recognize the same site of the extracellular segment of DR5 (eDR5, which is self-constructed by our laboratory). The affinity constant of aDR5ScFv was 2.12 × 10-2. eDR5 and aDR5ScFv specifically bind. MTT assay showed that aDR5ScFv inhibited the growth of SW480 cells in a dose-dependent manner. The final concentrations of aDR5ScFv were 0.225 mg / ml, 0.45 mg / ml, 0.9 mg / ml and 1.2 mg / ml, respectively. The survival rates of SW480 cells were 97.7%, 70.9%, 70.1% and 23.6% at 200μl respectively. Conclusion aDR5ScFv specifically binds to eDR5 and has strong activity.