双酚A人工抗原的光谱表征及免疫鉴定研究

Study on Spectral and Immune Identification of Artificial Antigen of Bisphenol A

双酚A(bisphenol A,BPA)是一种酚类环境雌激素,即使极低剂量残留对人类健康也有危害。为了建立其简单快速并适应现场检测的免疫分析方法,必须先合成它的人工抗原并进行鉴定。通过对双酚A进行结构修饰,用碳二亚胺将结构修饰了的双酚A与牛血清蛋白相偶联,经透析得到纯的双酚A人工抗原并冷冻干燥备用。然后用紫外光谱、红外光谱和免疫方法对人工抗原进行鉴定。结果表明,合成的人工抗原紫外光谱图中具有蛋白质和双酚A的特征吸收峰;人工抗原红外光谱图中呈现双酚A和牛血清蛋白的红外特征吸收峰;通过竞争ELISA试验表明用合成的人工抗原免疫的Balb/c小鼠血清中产生了双酚A的抗体。三种方法综合鉴定结果表明双酚A人工抗原合成成功,该人工抗原可以进一步用于制备抗双酚A的单克隆抗体。

Bisphenol A（BPA） is able to mimic the effects of endogenous hormones and it influences the central nervous system in human being. In recent years, BPA has largely arisen because it is released into our environment from many kinds of the polycarbonate plastics and epoxy resins of food cans. Because trace residual amount of BPA might be harmful to human being, many instrument analysis methods have been developed for the determination of BPA. But these instrument analysis methods require complicated pre-treatment steps and do not suit rapid processing of multiple samples. Gold-labeled immunochromatography assay method can provide in situ monitoring of trace BPA in ground-water and drinking water. In order to build the simple and fast gold-labeled immunochromatography assay method, it is necessary to synthesize and identify artificial antigen of BPA firstly. The modified BPA was conjugated with the carrier of bovine serum albumin （BSA） by EDC · HCl, after dialyzed, the synthesized artificial antigen of BPA was lyophilized, and then stored at -20℃. Three methods such as UV and IR spectra scanning and immunoassay were used to determine whether artificial antigen of BPA bad been synthesized. The results showed that UV spectrum of artificial antigen of BPA has one absorbance band at 278 nanometers （nm） which is the characteristic absorbance band of the carrier of bovine serum albumin, and another absorbance band at 226 nm which is the characteristic absorbance band of BPA. The IR spectrum absorbance bands of bovine serum albumin and BPA appeared on the IR spectrum graph of artificial antigen of BPA, moreover, shrinked vibration bands at 1 000-1 300 cm^-1 showed that a C-N bond was produced and suggested that the modified BPA and BSA had been linked. The antibody of anti- BPA was raised in sera of immunized Balb/c mice by indirect competitive ELISA. So the above results showed that the artificial antigen of BPA was synthesized successfully. The artificial antigen of BPA can be used to develop monoclonal antibody of anti-BPA and also to prepare gold-labeled immunochromatography assay method for in situ monitoring of BPA in the future.