免疫金银染色法快速检测水产品中孔雀石绿残留的研究

Rapid detection of malachite green residues in aquatic products with immunogold-silver staining method

中文关键词:  孔雀石绿  食品安全快速检测  渔药残留  免疫层析  免疫金银染色法

英文关键词:malachite green  rapid detection of food safety  fishery drugs residue  immunochromatography  immunogold-silver staining

中文摘要:

      目的 研究一种快速、稳定地检测水产中孔雀石绿残留的方法。方法 基于胶体金免疫层析技术, 采用竞争法, 以硝酸纤维素(nitrocellulose, NC)膜为载体, 以孔雀石绿单克隆抗体为标记蛋白, 制备一种金标免疫层析试纸条用于渔药残留快速检测, 同时用免疫金银染色法提高试纸条的灵敏度。结果 该检测系统选用20 nm直径的胶体金用于标记抗体, 最适pH为9.0, 最适蛋白结合量为每1 mL胶体金结合15 μg抗体蛋白。标记成功的抗体以1:10比例稀释并取20 μL固定到用PBS (0.01 mol/L, 10%蔗糖, 0.05% 吐温-20)预处理过的玻纤膜上, 作为金标垫。用于包被NC膜的抗原与二抗浓度分别为3 mg/mL和0.7 mg/mL。用免疫金银染色法放大显色信号, 制备的试纸条检出限为0.2 ng/mL, 并应用到实际鱼样的检测中。整个测试时间约为10 min。结论 本方法重现性良好, 且置于室温环境下保存90 d后仍稳定可靠, 为水产品中孔雀石绿残留的快速检测提供了一条可行途径。

英文摘要:

      Objective To develop a rapid, steady detection method of malachite green residues in the aquatic product. Methods A colloidal gold immunochromatographic assay strip was developed for rapid detection of malachite green (MG) residues in aquatic products and immunogold-silver staining method was employed to improve the sensitivity of the assay strip. This method was based on colloidal gold immunochroma-tography technology which involved the competition method and used nitrocellulose (NC) membrane as the carrier, malachite green monoclonal antibody as the marker protein. Results The colloidal gold nanoparticles with diameter 20 nm were selected to mark the antibody. The optimum pH was 9.0 and the optimum combination condition was 1 mL of colloidal gold combining with 15 μg of antibody. The successfully marked antibody was diluted in 1:10 proportion and 20 μL of the antibody was taken out and fixed on the glass fiber membrane pretreated by PBS (0.01 mol/L, 10% sugar, 0.05% Tween-20) to form the colloidal gold pad. The concentration of antigen and secondary antibody was 3 mg/mL and 0.7 mg/mL, respectively. The limit of detection (LOD) of this strip for MG was 0.2 ng/mL by conducting a silver-enhanced colloidal gold assay to enhance the original signal and this approach was applied to the practical detection of fish samples. The detection time of the whole procedure was approximately 10 min. Conclusion This method has a good reproducibility and keeps stable after storage for 90 d at room temperature. Therefore, this method provides a potential strategy for rapid detection of MG residue in aquatic products.