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

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（1.997 ng/mL），明显低于商业抗原作为包被抗原与2C9反应时的灵敏度（0.047 ng/mL），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。

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

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 1C11, 2C9, LM13 and LM47 against aflatoxin M1. The results showed that four antibodies had high sensitivity to commercial antigens, and only 2C9 could react with the artificial antigen AFM1 -BSA and the sensitivity was 1.997 ng/mL, which was significantly lower than that of AFM1-BSA as coating antigen (the sensitivity of 0.047 ng/mL). 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.