表面等离子共振技术快速测定蛋白A与抗人T细胞单克隆抗体结合的反应常数

Rapid Determination of the Specificity Binding Constants of Protein A to Anti-human T-cell Monoclonal Antibodies by Surface Plasmon Resonance

采用表面等离子共振(SPR)技术,建立了测定两种抗人T细胞单克隆抗体(抗人TCRγδ和抗人CD3-PE)分别与蛋白A反应的动力学和热力学参数的方法。在双通道SPR仪(SR 7500DC)上,将抗人TCRγδ或抗人CD3-PE溶液流过偶联蛋白A的传感芯片,实时监测这两种抗体与蛋白A的结合和解离过程,并对其反应模式和反应常数进行分析。两种抗体与蛋白A的结合速率常数ka、结合平衡常数Ka和活化能Ea比较接近,但焓变ΔH有些差异。SPR适宜用作蛋白A与抗人T细胞单克隆抗体结合的反应常数的测定。该方法不需对分析物进行标记、专属性强,是检测和分析抗体和蛋白质A相互作用较理想的方法之一。

A new method for the determination of the specificity binding constants of protein A to two anti-human T-cell monoclonal antibodies(anti-human TCRγδand anti-human CD3-PE)respectively was established based on surface plasmon resonance(SPR).Protein A was fixed on the sensing chip surface and the analytes(TCRγδor CD3-PE)were used as mobile phase.Binding and dissociation of protein A with TCRγδor CD3-PE were monitored in real time.The interaction mode between protein A and TCRγδor CD3-PE and the specificity binding constants were determined.The results indicate that both interaction come closer in terms of association rate constant ka,association equilibrium constant Kaand activation energy Ea,yet their enthalpy changesΔH are a little different.SPR is useful in determination of the specificity binding constants of protein A to anti-human T-cell monoclonal antibodies.This method has strong specificity without labelling the analyte and is an ideal method to identify the specific interaction between proteins A and antibodies.