Nanobody-Based Enzyme Immunoassay for Aflatoxin in Agro-Products with High Tolerance to Cosolvent Methanol

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• 作者：Ting He ; Yanru Wang ; Peiwu Li ; Qi Zhang ; Jiawen Lei ; Zhaowei Zhang ; Xiaoxia Ding ; Haiyan Zhou ; Wen Zhang
• 刊名：Analytical Chemistry
• 出版年：2014
• 出版时间：September 2, 2014
• 年：2014
• 卷：86
• 期：17
• 页码：8873-8880
• 全文大小：519K
• ISSN：1520-6882

文摘

A phage-displayed library of variable domain of heavy chain of the heavy chain antibody (VHH) or nanobody (Nb) was constructed after immunizing an alpaca with aflatoxin B1 (AFB₁) conjugated with bovine serum albumin (AFB₁鈥揃SA). Two AFB₁-specific nanobodies were selected. The obtained nanobodies were compared to an aflatoxin-specific monoclonal antibody B5 with respect to stability under organic solvents and high temperature. The two nanobodies could bind antigen specifically after exposure to temperatures as high as 95 掳C. Besides, the nanobodies showed better or similar tolerance to organic solvents. A competitive ELISA with nanobody Nb26 was developed for the analysis of AFB₁, exhibiting an IC₅₀ value of 0.754 ng/mL (2.4 渭M), linear range from 0.117 to 5.676 ng/mL. Due to the high tolerance to methanol, sample extracts were analyzed by nanobody-based ELISA without dilution. The recovery from spiked peanut, rice, corn and feedstuff ranged from 80 to 115%. In conclusion, the isolated nanobodies are excellent candidates for immunoassay application in aflatoxin determination.