噬菌体展示纳米抗体模拟黄曲霉毒素抗原的活性表征

【目的】笔者实验室前期免疫并构建噬菌体展示纳米抗体库，应用此抗体库筛选、制备可以用作黄曲霉毒素无毒替代抗原的纳米抗体。本研究旨在探明已筛选出的噬菌体展示纳米抗体Phage 2-5用作黄曲霉毒素替代抗原的活性，为后续纳米抗体的合成以及黄曲霉毒素绿色免疫分析方法的建立奠定基础。【方法】前期，笔者实验室对羊驼免疫黄曲霉毒素抗原（AFB1-BSA），经过5次免疫后，取血，提取RNA，反转录为cDNA，并采用PCR技术对抗体可变区VHH区域进行扩增，与载体pComb3X偶联，构建噬菌体展示纳米抗体库；经过吸附-洗脱的淘选过程筛选出能够与黄曲霉毒素竞争结合抗黄曲霉毒素单克隆抗体1C11的噬菌体展示纳米抗体Phage 2-5。本研究通过将抗黄曲霉毒素单克隆抗体1C11固定在酶标板上，以噬菌体展示纳米抗体Phage2-5作为竞争抗原，与反应体系中的游离黄曲霉毒素竞争结合抗体，构建酶联免疫分析（ELISA）体系，检测游离黄曲霉毒素含量，并分别对该方法的灵敏度、交叉反应率、缓冲液和样品基质对反应体系的影响进行测定。【结果】采用棋盘法确定了抗体最佳包被浓度为1.25 mg·mL-1，噬菌体最佳使用浓度为5´1011 pfu/mL。在最优条件下建立基于噬菌体展示纳米抗体Phage2-5的ELISA法，对黄曲霉毒素B1的IC50值为0.054 ng·mL-1，对黄曲霉毒素B2、G1、G2、M1的交叉反应率分别为38.6%、70.1%、14.5%、14.6%；反应最高耐受甲醇浓度为20%；当pH值为7.0时，反应灵敏度最高，升高或降低pH对反应灵敏度均有影响；盐离子浓度对反应灵敏度影响不大，反应体系在5´PBS的环境下仍能保持较高活性，但纯水溶液不利于反应的进行；因此，该反应的最佳反应缓冲液是pH值为7.0的0.01 mol·L-1 磷酸盐缓冲液（PBS），花生、大米、玉米基质对反应体系无显著影响。【结论】噬菌体展示纳米抗体Phage2-5具备良好的模拟黄曲霉毒素抗原的生物活性，用作替代抗原建立的免疫分析方法灵敏度高、甲醇耐受能力强，并且样品基质效应不明显，具有进一步研制黄曲霉毒素模拟抗原的前景。

Charericterization of a Phage-Displayed Nanobody Imitating Aflatoxin Antigen

【Objective】A phage-displayed aflatoxin mimotope was obtained from a home-made phage-displayed nanobody library, and used to develop enzyme-linked immunoassay (ELISA) towards aflatoxins. In this research, the performance of the selected mimotope was characterized. 【Method】In previous work of author’s laboratory, an alpaca had been immunized with anti-aflatoxin MAb 1C11 mixed with Freund’s incomplete adjuvant. Total RNA was extracted from alpaca’s blood and used to synthesize first strand cDNA. The phage displayed VHH library was constructed by ligating amplified VHH genes with plasmid pComb3X. In this work, anti-aflatoxin monoclonal antibody 1C11 was coated on a 96-well microplate, phage-displayed nanobody solution was mixed with aflatoxin standard or sample extracts and added into the wells to compete binding to the antibody. The assay’s sensitivity towards aflatoxin B1, cross-reactivity towards aflatoxin B2, G1, G2 and M1 was determined. The assay buffer’s pH value, ironic strength and methanol concentration were also optimized. In order to apply this assay to agro-products, peanut, rice and corn were selected to test its matrix effects.【Result】Determined by checkerboard procedure, the optimized concentration of the coating antibody was 1.25 mg·mL-1 and phage was 5´1011 pfu/mL. Under this condition, the assay had an IC50 value of 0.054 ng·mL-1 towards aflatoxin B1. Its cross-reactivity towards aflatoxin B2, G1, G2 and M1 was 38.6%, 70.1%, 14.5% and 14.6%, respectively. Methanol concentration could be as high as 20% without interference to the assay. The assay had the highest sensitivity under pH value of 7.0. Increasing or decreasing the pH value of the assay buffer reduced its sensitivity. Ionic strength influenced ELISA performance. And the selected optimum concentration was 0.01 mol·L-1, which led to the lowest IC50 value. As a result, the optimized assay buffer was 0.01 mol·L-1 phosphate buffered saline (PBS) with a pH value of 7.0. There was no significant matrix effect when the sample extract was diluted by 4 times or higher. 【Conclusion】 The immunoassay based on phage 2-5 has a high sensitivity, high tolerance to methanol and sample matrix. Phage-displayed nanobody 2-5 can be used in immunoassays towards aflatoxins in agro-products as aflatoxin mimotope.