噬菌体展示纳米抗体模拟黄曲霉毒素抗原的活性表征

doi:10.3864/j.issn.0578-1752.2014.04.008

Abstract

Abstract: 【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.

Key words: aflatoxin , mimotope , phage-displayed nanobody

WANG Yan-Ru-1, 2 , 4 , LI Pei-Wu-1, 2 , 3 , 4 , 5 , ZHANG Qi-1, 3 , 4 , DING Xiao-Xia-1, 4 , 5 , ZHANG Wen-1, 2 , 5 . Charericterization of a Phage-Displayed Nanobody Imitating Aflatoxin Antigen[J].Scientia Agricultura Sinica, 2014, 47(4): 685-692.

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