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本文采用紫外可见光谱(UV-Vis)、荧光光谱和圆二色谱(CD)技术研究了异细交链孢菌酮酸单克隆抗体(Mc Ab)与其半抗原(Ite AH)之间的相互作用并用ELISA方法对荧光结果进行了验证。结果表明,ITe AH对Mc Ab有荧光猝灭作用,淬灭机理主要为静态猝灭且遵循非辐射能量转移理论。在298、310、318 K 3个温度下,ITe AH与Mc Ab的结合常数分别为1.9×106、1.6×106、1.4×106 L/mo L,结合位点数n≈1,结合距离r为3.35 nm,经ELISA测得的结合常数与荧光分析的结果较一致。由结合作用过程的热力学参数可知,两者之间以静电引力为主要作用力,不同p H下,由ELISA结果可推测其影响了ITe AH与抗体结合。同步荧光光谱显示ITe AH的加入并未使抗体的酪氨酸和色氨酸残基附近的微环境发生明显改变,两者的结合位点更倾向于酪氨酸残基,圆二色谱分析发现ITe AH与Mc Ab相互作用后,抗体的二级结构发生明显变化。
In this paper, UV-Vis, fluorescence and circular dichroism (CD) techniques were used to study the interaction between monoclonal antibody McAb and its hapten (Ite AH) Fluorescence results were validated by ELISA. The results show that ITe AH has a fluorescence quenching effect on Mc Ab, the quenching mechanism is mainly static quenching and follows the theory of non-radiative energy transfer. The binding constants of ITe AH and Mc Ab were 1.9 × 106, 1.6 × 106 and 1.4 × 106 L / mol respectively at 3 temperatures of 298, 310 and 318 K, the number of binding sites n≈1 and the binding distance r was 3.35 nm, the binding constants measured by ELISA are more consistent with the results of fluorescence analysis. From the thermodynamic parameters of the binding process, it can be seen that electrostatic attraction is the main force between the two. Under different p H, the binding of ITe AH to the antibody can be presumed to be influenced by ELISA results. Simultaneous fluorescence spectroscopy showed that the addition of ITe AH did not change the microenvironment near the tyrosine and tryptophan residues of the antibody, and the binding sites of both were more prone to tyrosine residues. Circular dichroism analysis revealed that After the interaction between ITe AH and Mc Ab, the secondary structure of the antibody changed significantly.