ELISA抑制法检测动物组织中α1,3-Gal抗原

目的: 用人工合成的Gal/BSA抗原对骨髓瘤细胞的Gal抗原表位数进行赋值;再用骨髓瘤细胞作为参照品,建立并优化单克隆特异性抗体M86检测动物组织中α1,3-Gal(Gal)抗原的ELISA抑制法。应用优化后的方法,检测猪和小鼠组织Gal抗原表位数。方法: 将待测物抗原(Gal-BSA系列稀释液、SP2/0细胞及待检组织样品)与M86特异性抗体反应后离心,取含有M86剩余抗体的上清液;再与Gal-BSA包被的固相抗原反应,计算出待测样品和标准曲线样品的M86结合抑制率;之后绘制相应的质量浓度-结合抑制率曲线,再根据曲线拟合公式计算其50%结合抑制时的质量浓度;进而计算Gal抗原表位数。结果: 各结合抑制曲线相关性较好,R²> 0.95。SP2/0骨髓瘤细胞Gal抗原数为每个细胞6.19×10⁸个;利用优化了的方法检测猪的冻干组织抗原含量趋势为:肺> 肾> 脾> 心> 腹膜> 心包膜> 肝组织> 皮肤;小鼠的新鲜湿组织抗原含量趋势为:心> 肺> 脾> 肾> 肝。结论: 应用骨髓瘤细胞作为参照品,所建立的M86抗体ELISA抑制法检测动物组织的Gal抗原含量具有较好的敏感性和特异性,为进一步建立该方法规范性的行业标准奠定了基础。

Objective: To determine Gal antigen expression of myeloma cells with artificial antigen Gal-BSA as a standard material by using a refined ELISA inhibition method of M86 binding, and then by using myeloma cells as reference materials to detect the α1, 3-Gal (Gal) epitopes in porcine and mouse tissues. Methods: The antigen in the test samples (series of dilutions of Gal-BSA, SP2/0 myeloma cells and tissue samples) was reacted with the specific M86 antibody, and then centrifuged. The supernatant containing the M86-binding inhibition rates of the test samples and the standard materials. According to the corresponding concentration-binding inhibition curve, the 50% inhibition concentration (IC₅₀) was calculated. Subsequently, the Gal antigen epitopes were calculated. Results: Each curve had good correlation, R²>0.95. And the number of α1, 3-Gal expressed in SP2/0 myeloma cells was 6.19×10⁸/cell. The tendency of Gal expression in porcine freeze-dried tissues was lung > kidney > spleen > heart > peritoneum > pericardial > liver > skin. The amount of Gal expression in fresh wet tissues of mice was in the following order: heart > lung > spleen > kidney > liver. Conclusion: The ELISA inhibition method of M86 binding established in this study by using SP2/0 as a reference material has good sensitivity and specificity. This experimental method provides a scientific support for further developing industry standard to detect the remnant Gal antigen contents of animal tissues or animal tissue-derived biomaterials.