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目的制备黄曲霉毒素B_1胶体金免疫层析试纸条。方法用柠檬酸三钠还原法制备胶体金溶胶,用目测法、紫外可见分光光度法及精密p H试纸对其进行鉴定表征;并采用碳二亚氨法(EDC)将黄曲霉毒素B_1(AFB_1)偶联于载体蛋白卵血清蛋白(OVA)上,合成包被抗原AFB_1-OVA。通过目测法及紫外-可见分光光度法优化标记胶体金的最佳黄曲霉毒素B_1单克隆抗体浓度和pH,最后将包被有抗原AFB_1-OVA和羊抗鼠Ig G的硝酸纤维素膜和干燥的吸附有金标抗体的玻璃纤维膜组装成试纸条,初步利用竞争性免疫层析原理检测标准品黄曲霉毒素B_1。结果制备的胶体金澄清透亮,呈酒红色,无聚集现象,最大吸收波长为518 nm,呈酸性(p H=5.8)。OVA与AFB_1偶联后,偶联物紫外扫描吸收光谱与OVA及AFB_1吸收光谱有明显区别,说明有新物质生成。进一步用新合成的AFB_1-OVA组装试纸条,检测阴性样品,两线均显深红色,为阴性结果,表明合成的人工抗原成功;胶体金标记黄曲霉毒素B_1单克隆抗体最适标记p H为1 ml胶体金溶液中添加0.1 mol/L K_2CO_3量为20μl,最适蛋白浓度为13μg/ml;初步组装试纸条并检测一定浓度的AFB_1标准品,结果表明,试纸条的检测限为200 ng/ml。结论制备的胶体金符合试纸条所用胶体金的要求;并成功制备AFB_1胶体金免疫层析法测定试纸条,但尚须进一步优化条件以提高试纸条的检测性能。
Objective To prepare aflatoxin B_1 colloidal gold immunochromatographic strip. Methods Colloidal gold sol was prepared by the method of trisodium citrate reduction and characterized by visual observation, UV-Vis spectrophotometry and precision p H test paper. The aflatoxin B1 (AFB_1 ) Was coupled to the carrier protein ovalbumin (OVA) to synthesize the coating antigen AFB_1-OVA. The best aflatoxin B 1 monoclonal antibody concentration and pH for labeling of colloidal gold were optimized by visual inspection and UV-Vis spectrophotometry. Finally, the nitrocellulose membrane coated with antigens AFB_1-OVA and goat anti-mouse Ig G and dried Of gold-labeled antibody adsorbed glass fiber membrane assembled into test strips, the initial use of competitive immunochromatography standard aflatoxin B_1 standard. Results The colloidal gold was clear and bright, showing a burgundy color with no aggregation. The maximum absorption wavelength was 518 nm, showing acidity (p H = 5.8). After the coupling of OVA with AFB_1, the UV-vis absorption spectra of the conjugates were significantly different from the absorption spectra of OVA and AFB_1, indicating that new substances were formed. The test strips were further assembled with newly synthesized AFB_1-OVA. Negative samples were detected with a dark red color on both lines, indicating that the synthetic artificial antigen was successful. The colloidal gold labeled aflatoxin B 1 monoclonal antibody was optimally labeled p H 0.1 mol / L K 2 CO 3 was added to 1 ml colloidal gold solution and the optimum concentration of protein was 13 μg / ml. The test strips were preliminarily assembled and AFB_1 standard was detected at a certain concentration. The results showed that the detection limit 200 ng / ml. Conclusion The prepared colloidal gold conforms to the requirements of colloidal gold used in test strip. The test strip of AFB_1 was successfully prepared by colloidal gold immunochromatography. However, the conditions of test strip need to be further optimized to improve the test performance.