酸性橙Ⅱ半抗原及人工抗原的合成与鉴定

Synthesis and identification of acid orangeⅡhapten and artificial antigen

目的建立酸性橙Ⅱ半抗原及人工抗原的合成及鉴定方法。方法将重氮化后的2-氨基-5-磺基苯甲酸与2-萘酚经偶氮反应合成带羧基基团酸性橙Ⅱ,通过碳二亚胺法将其与载体蛋白(BSA)偶联制备酸性橙Ⅱ人工抗原。采用核磁共振碳谱和氢谱对合成的酸性橙Ⅱ半抗原进行结构分析,并用紫外光谱扫描法和蛋白质电泳法鉴定酸性橙Ⅱ人工抗原并计算偶联比。结果通过偶氮反应及碳二亚胺法最终得到纯化后的酸性橙Ⅱ半抗原约47 mg,核磁结果显示成功合成酸性橙Ⅱ半抗原。经SDS-PAGE、N-PAGE和紫外光谱扫描结果均显示酸性橙Ⅱ人工抗原蛋白分子质量大于BSA,所带负电荷大于BSA,且合成后的酸性橙Ⅱ人工抗原具有吸收峰叠加效应,均表明酸性橙Ⅱ人工抗原合成成功,偶联比为20.9:1。结论此方法能够成功合成酸性橙Ⅱ半抗原和人工抗原,为进一步制备特异性酸性橙Ⅱ抗体和建立酸性橙Ⅱ的免疫检测方法奠定基础。

Objective To explore a method for the synthesis and identification of acid orange Ⅱhapten and artificial antigen. Methods Acid orange Ⅱ hapten was synthesized by the reaction between 2-amino-5-sulfo-benzoic acid and 2-naphthol, then acid orange Ⅱ artificial antigen was prepared by active ester method. Acid orange Ⅱ hapten was identified by mass spectrometry and acid orange Ⅱ artificial antigen was identified by UV spectrophotometric, SDS-PAGE as well as N-PAGE. Results The structure of acid orange Ⅱ hapten was consistent with the predictive structure by mass spectral analysis. By the methods of SDS-PAGE and N-PAGE, the molecule weight and negatively charged electrons of acid orange Ⅱ artificial antigen were greater than BSA. Acid orange Ⅱ artificial antigen showed the superposition phenomenon by Ultraviolet spectrum. According to the UV, acid orange Ⅱ hapten and BSA coupling ratio was 20.9:1. Conclusion This method can be applied for acid orange Ⅱ hapten and artificial antigen synthesis and provide technical basis for the preparation of acid orange Ⅱ specificity antibodies and development of acid orange Ⅱ immune detection.