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目的:研究二硫键稳定的抗CD3/抗Pgp微型双功能抗体(Diabody)在体外和体内的稳定性。方法:将抗CD3/抗Pgp Diabody置于37℃含0.2%人血清白蛋白(human serumalbumin,HSB)的PBS中,孵育不同时间后,用流式细胞术(FACS)检测其结合活性。建立K562/A02裸鼠移植瘤模型,用Cy5荧光标记试剂盒标记抗CD3/抗Pgp Diabody,通过尾静脉注射给荷瘤裸鼠,在小动物活体成像系统上动态观察荧光信号。结果:二硫键稳定的抗CD3/抗Pgp Diabody(Ds-diabdoy)体外孵育72小时后活性无明显下降,而改造前Diabody孵育1小时后活性即开始下降,24小时后活性完全丧失。Ds-diabdoy注射后72小时仍能在肿瘤部位检测到荧光信号,而改造前Diabody在注射后24小时肿瘤部位荧光信号消失。结论:Ds-diabdoy较改造前Diabody稳定性大大提高。
AIM: To investigate the in vitro and in vivo stability of disulfide-stabilized anti-CD3 / anti-Pgp diabody. Methods: The anti-CD3 / anti-Pgp Diabody was placed in PBS containing 0.2% human serum albumin (HSB) at 37 ℃ for different time. The binding activity was detected by flow cytometry (FACS). A K562 / A02 xenograft model in nude mice was established. The anti-CD3 / anti-Pgp Diabody was labeled with Cy5 fluorescent labeling kit and the tumor-bearing nude mice were injected via the tail vein to observe the fluorescence signal dynamically on live animal imaging system. RESULTS: The activity of disulfide-stabilized anti-CD3 / anti-Pgp Diabody (Ds-diabdoy) did not decrease significantly after incubation for 72 hours in vitro. However, the activity began to decline after 1 hour incubation with Diabody and the activity was completely lost after 24 hours. Ds-diabdoy 72 hours after injection can still detect the fluorescence signal at the tumor site, while the transformation of Diabody 24 hours after injection of tumor fluorescence signal disappeared. Conclusion: Ds-diabdoy is much more stable than Diabody before modification.