title: Nanobody-based enzyme immunoassay for aflatoxin in agro-products with high tolerance to cosolvent methanol.
abstract: 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-BSA). 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 °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 μM), 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.
authors: Oil Crops Research Institute of the Chinese Academy of Agricultural Sciences , Wuhan 430062, People's Republic of China.
authors: Ting
authors: T
authors: He
authors: Yanru
authors: Y
authors: Wang
authors: Peiwu
authors: P
authors: Li
authors: Qi
authors: Q
authors: Zhang
authors: Jiawen
authors: J
authors: Lei
authors: Zhaowei
authors: Z
authors: Zhang
authors: Xiaoxia
authors: X
authors: Ding
authors: Haiyan
authors: H
authors: Zhou
authors: Wen
authors: W
authors: Zhang
mesh_terms: D016604:Aflatoxin B1 / Q000276:immunology*; D000348:Aflatoxins / Q000032:analysis*; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001667:Binding, Competitive; D002161:Camelids, New World / Q000378:metabolism; D002417:Cattle; D061505:Cell Surface Display Techniques; D002623:Chemistry Techniques, Analytical / Q000379:methods*; D004797:Enzyme-Linked Immunosorbent Assay; D008297:Male; D000432:Methanol / Q000737:chemistry*; D008969:Molecular Sequence Data; D055550:Protein Stability; D016415:Sequence Alignment; D012709:Serum Albumin / Q000276:immunology*; D061905:Single-Domain Antibodies / Q000276:immunology*; D012997:Solvents / Q000737:chemistry; D013696:Temperature
chemical_list: D000348:Aflatoxins; D000911:Antibodies, Monoclonal; D012709:Serum Albumin; D061905:Single-Domain Antibodies; D012997:Solvents; C052669:aflatoxin B1-bovine serum albumin; D016604:Aflatoxin B1; D000432:Methanol