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20nm直径的胶体金用于标记抗体,最适p H为9.0,最适蛋白结合量为每1 m L胶体金结合15μg抗体蛋白。标记成功的抗体以1:10比例稀释并取20μL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/m L和0.7 mg/m L。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/m L,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。
Objective To develop a rapid, steady detection method of malachite green residues in the aq- uatic product. Methods A colloidal gold immunochromatographic assay strip was developed for rapid detec- tion of malachite green （MG） residues in aquatic products and immunogold-silver staining method was em- ployed 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 combina- tion 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 concentrationof 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 detec- tion of MG residue in aquatic products.
作者
付林峰
汪丽
娄越
简锦明
郭希山
朱松明
FU Lin-Feng WANG Li LOU Yue JIAN Jin-Ming GUO Xi-Shan ZHU Song-Ming(College of Biosystems Engineering and Food Science, Zhejiang University, Hangzhou 310058, China)
出处
《食品安全质量检测学报》
CAS
2017年第6期2146-2153,共8页
Journal of Food Safety and Quality
基金
浙江省重点科技创新团队项目(2011R50029)~~
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
malachite green
rapid detection of food safety
fishery drugs residue
immunochromatogra-phy
immunogold-silver staining
