[关键词]
[摘要]
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(McAb)与其半抗原(IteAH)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,ITeAH对McAb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,ITeAH与McAb的结合常数分别为1.9×106、1.6×106、1.4×106 L/moL,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pH下,由ELISA结果可推测其影响了ITeAH与抗体结合。同步荧光光谱显示ITeAH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ITeAH与McAb相互作用后,抗体的二级结构发生明显变化。
[Key word]
[Abstract]
The interaction between iso-tenuazonic acid (ITeAH) hapten and monoclonal antibody (McAb) against ITeAH was studied by ultraviolet-visible (UV-vis) spectroscopy, fluorescence spectroscopy, and circular dichroism (CD). Fluorescence results were verified using enzyme-linked immunosorbent assay (ELISA). The results suggested that fluorescence intensity of McAb was quenched by ITeAH, while the quenching mechanism was based on static quenching and nonradioactive energy-transfer theory. The binding constants (Ka) at 298, 310, and 318 K were calculated as 1.9 × 106, 1.6 × 106, and 1.4 × 106 L/moL, respectively, consistent with the values determined by ELISA. The binding point (n) number was about 1, while the average binding distance (r) was 3.35 nm. Furthermore, thermodynamic parameters evaluated during the binding process showed that the binding force between ITeAH and McAb constituted of mainly electrostatic attraction, which affected the extent of binding between ITeAH and McAb, when studied under different pH conditions by using ELISA. The synchronous fluorescence spectra indicated that the addition of ITeAH did not change the microenvironment of tyrosine and tryptophan residues in the McAb, and that the binding site for ITeAH was more inclined to tyrosine residues. CD analysis showed significant changes in the secondary structure of McAb after interaction with ITeAH.
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[基金项目]
国家自然科学基金(30871755;31271866);国家科技支撑计划(2012BAD31B0302);广州市科技计划项目(2013J4100053;2013J2200080;2014J4100185);广东省自然科学基金项目(S2012010010323)