黄曲霉毒素抗原分子中半抗原偶联比对免疫分析灵敏度的影响  

Effect of hapten coupling ratio on aflatoxin antigen molecule in immunoassay sensitivity

作　　者：严婷婷 李培武[2,3,4,6,5] 张奇 陈永勤[1] 唐晓倩 张文[2,3,4,6,5] YAN Ting-ting;LI Pei-wu;ZHANG Qi;CHEN Yong-qin;TANG Xiao-qian;ZHANG Wen(Faculty of Life Sciences,Hubei University,Wuhan 430062,China;Oil Crops Research Institute of the Chinese Academy of Agricultural Sciences,Wuhan 430062,China;Key Laboratory of Biology and Genetic Improvement of Oil Crops,Ministry of Agriculture,Wuhan 430062,China;Key Laboratory of Detection for Mycotoxins,Ministry of Agriculture,Wuhan 430062,China;Laboratory of Risk Assessment for Oilseeds Products（Wuhan）,Ministry of Agriculture,Wuhan 430062,China;Quality Inspection and Test Center for Oilseeds Products,Ministry of Agriculture,Wuhan 430062,China)

机构地区：[1]湖北大学生命科学学院,湖北武汉430062 [2]中国农业科学院油料作物研究所,湖北武汉430062 [3]农业部油料作物生物学与遗传育种重点实验室,湖北武汉430062 [4]农业部生物毒素检测重点实验室,湖北武汉430062 [5]农业部油料及制品质量监督检验测试中心,湖北武汉430062 [6]农业部油料产品质量安全风险评估实验室,湖北武汉430062

出　　处：《中国油料作物学报》2018年第3期426-431,共6页Chinese Journal of Oil Crop Sciences

基　　金：农业科学研究专项基金(201203094-4);"科学研究"1999年度公共利益特别研究基金(201513006-02);国家自然科学基金(31101299;31171702;31401601;21205118;401402)

摘　　要：为提高黄曲霉毒素的检测灵敏度,探明黄曲霉毒素抗原分子中半抗原偶联比对免疫分析灵敏度的影响。采用黄曲霉毒素M1与羧甲氧基羟氨半盐酸盐进行肟化反应生成半抗原,再经活泼脂法,将半抗原与牛血清白蛋白(BSA)脱水反应实现偶联,生成自制人工抗原AFM1-BSA。采用竞争ELISA法、荧光与紫外光谱法鉴定,结果均证明自制人工抗原AFM1-BSA合成成功。进一步通过摩尔消光系数测算得AFM1的自制和商业抗原偶联比分别为0.7和6.3,自制抗原偶联比仅是商业抗原的1/9,二者偶联方法相同,但偶联比差异显著,为进一步研究比较提供了可能。将自制人工抗原AFM1-BSA和商业抗原AFM1-BSA分别与实验室自制的系列黄曲霉毒素单抗1C11、2C9、LM13、LM47反应,结果显示四种抗体对商业抗原均有很高的灵敏度,只有2C9对自制人工抗原有较高的灵敏度(1.997ng/m L),明显低于商业抗原作为包被抗原与2C9反应时的灵敏度(0.047ng/m L),说明针对同一种毒素AFM1制备的抗体,高偶联比抗原有助于提高免疫分析灵敏度。研究结果为AFM1高灵敏免疫检测技术研究提供了科学依据。To analyze the sensitivity of enzyme-linked immunosorbent assay（ ELISA） for aflatoxin M1,the differences of hapten coupling ratio-based indirect competitive enzyme-linked immunoassay were compared. The aflatoxin M1 and CMO were used to synthesize the hapten of AFM1,and then coupling with bovine serum albumin（ BSA） to generate artificial antigen AFM1-BSA. After identifying the conjugate by means of UV,ic-ELISA and fluorescence spectra analysis,the results showed that the artificial antigen AFM1-BSA was effective. Furthermore,the artificial antigen and commercial antigen coupling ratios were 0. 7 and 6. 3 respectively. With the same methods,the artificial antigen coupling ratio was only 1/9 as that of commercial antigen,which provided reference for further research and comparison. Two kinds of antigens were analyzed by laboratory-made antibodies 1 C11,2 C9,LM13 and LM47 against aflatoxin M1. The results showed that four antibodies had high sensitivity to commercial antigens,and only 2 C9 could react with the artificial antigen AFM1-BSA and the sensitivity was 1. 997 ng/m L,which was significantly lower than that of AFM1-BSA as coating antigen（ the sensitivity of 0. 047 ng/m L）. This study revealed that the high coupling ratio antigen could improve the sensitivity of immunoassay for the antibody prepared by the same toxin AFM1. These results provided a scientific basis to further establish a high sensitive and complete enzyme-linked immunosorbent assay system for aflatoxin M1.

关 键 词：黄曲霉毒素M1 人工抗原 抗原鉴定 偶联比 ELISA 

分 类 号：S379.7[农业科学—农产品加工]