摘要
建立了快速检测牛奶、奶粉、饲料样品中三聚氰胺的胶体金免疫层析分析法。将三聚氰胺进行分子修饰得到两种衍生物,分别与牛血清白蛋白(BSA)和卵清白蛋白(OVA)相连制得免疫原和包被抗原。运用杂交瘤抗体制备技术得到抗三聚氰胺的单克隆抗体(mAb)。利用柠檬酸三钠还原法制得平均粒径18 nm的胶体金,将胶体金与抗三聚氰胺单克隆抗体相连,所形成的金标抗体(Au-mAb)包被在胶体金垫上,包被抗原和羊抗鼠二抗分别包被在硝酸纤维素膜(NC)上作为检测线(T线)和质控线(C线)。将胶金垫、NC膜、样品垫和吸水纸组装成免疫层析快速检测试剂条。将标准溶液(或待测液)滴加到样品垫上,10 min后可在NC膜C线和T线位置上用肉眼观察到胶体金的颜色(红色),通过比对颜色的深浅判断样品中三聚氰胺的含量。结果表明,三聚氰胺的检出限为10μg/L;选用了其它10种物质对免疫层析试剂条进行了特异性实验,免疫层析试剂条与2-氯-4,6-二氨基-1,3,5-三嗪和环丙氨嗪的交叉反应率约为1%,与其它8种物质没有交叉反应;加标样品用免疫层析试剂条和酶联免疫吸附分析法(ELISA)同时检查,结果一致。本方法适用于牛奶、奶粉、饲料样品中三聚氰胺的现场快速检测。
A colloidal gold immunochromatographic strip for rapid detection of melamine in milk, milk powder and animal feeds was presented. The molecular structure of melamine was rationally modified. Two melamine derivatives were synthesized and coupled to bovine serum albumin （ BSA ） and ovalbumin （ OVA ） for the production of immunogen and coating antigen. Monoclonal antibody （mAb） against melamine was produced by hybridoma technology. Colloidal gold nanopartieles with the average particle diameter of 18 nm were linked with mAb to form the Au-mAb conjugate. The formed Au-mAb was coated on glass fiber; while the coating antigen and goat anti-mouse IgG were separately sprayed onto the nitrocellulose （NC） membrane as the test （T） line and the control （C） line. The Au-mAb pad, NC membrane, sample pad and absorptive paper were assembled into an immunochromatographic strip. Standard （or sample） solution was added to the sample pad. After 10 min, the colloidal gold color （red） was visually observed at C line and T line, and the content of the melamine was evaluated by comparing the intensity of the color on these two lines. The results demonstrated that the limit of detection （LOD） of the melamine was 10 μg/L. Ten compounds were selected for specificity testing. The cross-reactivity （CR） of the immunochromatographic strip with 2-chloro-4,6-diamino-1,3,5- triazine （CAAT） and cyromazine was about 1%, and there was no CR of the strip with other eight compounds. The samples spiked with melamine at different concentrations were detected by immunochromatographic assay and enzyme-linked immunosorbent assay （ELISA） simultaneously. The results obtained by these two methods were in a good agreement. The proposed immunochromatographic assay can be utilized as a promising tool for rapid detection of melamine in milk, milk powder and feed samples.
出处
《分析化学》
SCIE
EI
CAS
CSCD
北大核心
2013年第7期1025-1030,共6页
Chinese Journal of Analytical Chemistry
基金
国家自然科学基金(No.21175097)资助
关键词
三聚氰胺
单克隆抗体
胶体金
免疫层析法
Melamine
Monoelonal antibody
Colloidal gold
Immunoehromatographie assay
