利用捕获法建立筛选鸭坦布苏病毒适配单抗的表面离子共振方法

摘要

ABSTRACT:Objective To establish a detection method for duck Tembusuvirsuand study affinity dynamicsbetween antigen and monoclonal antibodywith surface plasmon resonance (SPR) system as technology platform.Method Coupling the mouse antibody on the CM5 chip, different concentrations of monoclonal antibodies diluted by HBS buffer were reacted using Kinetics program. Fitting different dissociation curve,KD value was calculated and detection sensitivity limit value was determined. Specificity experiment was also performed among other 5 kinds of common poultry virus antibody.ResultsKD values of 3mabs were 1.50×10-13, 1.16×10-11, and 7.85×10-12, respectively, no positive response signal in detecting other poultry virus.The minimum limit was 0.625 nmol/L in detecting Tembusu recombinant E protein.Conclusion The research showed that the method could be used for rapid screening stable mabs of the Tembusu virusand acquire affinity data effectively to establish foundation for immune screening method.%目的：以表面离子共振(surface plasmon resonance, SPR)系统为技术平台,利用捕获法建立检测鸭坦布苏病毒适配单抗的 SPR 方法,并对抗坦布苏病毒单克隆抗体的亲和力及抗原抗体结合的动力学进行研究。方法将鼠抗抗体偶联于CM5芯片上,采用Kinetics程序将HBS缓冲液梯度稀释的不同浓度的单克隆抗体进行捕获反应,拟合解离曲线计算不同单抗的KD值并确立检测灵敏度低限并对其他不同亚型及5种常见禽类病毒抗体进行特异性检测试验。结果测得3株单抗的KD值分别为1.50×10-13、1.16×10-11、7.85×10-12,建立的表面离子共振方法在检测其他禽源病毒时无阳性响应信号,坦布苏重组 E 蛋白的检测极限达0.625 nmol/L。结论该方法可用于快速筛选检测坦布苏病毒的稳定适配单抗,并可有效获知其亲和力数据,为大规模推广建立免疫筛查方法奠定基础。