title: Bactrian camel nanobody-based immunoassay for specific and sensitive detection of Cry1Fa toxin.
abstract: The variable domain of the heavy-chain-only antibody (VHH) or nanobody (Nb), derived from camelids, begins to play an important role on the detection of protein markers. In this study, we constructed a phage-displayed library of VHHs against Cry1Fa by immunizing a healthy Bactrian camel with Cry1Fa toxin. After a series of bio-panning and screening by phage display technology, three anti-Cry1Fa nanobodies (Nbs) with great difference in complementarity determining region 3 (CDR3) were obtained and they were highly specific to Cry1Fa as well as showed full of activity when exposed to 70 °C for 3 h. Through modifying Nbs with Horseradish Peroxidase (HRP) and biotin, two Nbs which can recognize the different epitopes of Cry1Fa were determined and they were used to establish a novel sandwich immune ELISA based on biotin-SA interaction for Cry1Fa detection. The immunoassay exhibited a linear range from 1 to 100 ng/mL with a detection limit of 0.88 ng/mL. The recoveries from spiked corn and soybean samples were ranged from 83.33 to 117.17%, with a coefficient of variation (C.V) less than 6.0%. All together, the proposed immunoassay will be a promising way for sensitive and accurate determination of Cry1Fa toxin.
authors.affiliation: The Key Laboratory of Developmental Genes and Human Disease, Ministry of Education, Institute of Life Sciences, Southeast University, Nanjing 210096, PR China.
authors.forename: Pingyan
authors.initials: P
authors.lastname: Wang
authors.affiliation: The Key Laboratory of Developmental Genes and Human Disease, Ministry of Education, Institute of Life Sciences, Southeast University, Nanjing 210096, PR China.
authors.forename: Guanghui
authors.initials: G
authors.lastname: Li
authors.affiliation: The Key Laboratory of Developmental Genes and Human Disease, Ministry of Education, Institute of Life Sciences, Southeast University, Nanjing 210096, PR China.
authors.forename: Junrong
authors.initials: J
authors.lastname: Yan
authors.affiliation: College of 543Biotechnology and Pharmaceutical Engineering, Nanjing University of 544Technology, Nanjing 210009, PR China.
authors.forename: Yonghong
authors.initials: Y
authors.lastname: Hu
authors.affiliation: Institute of Food Safety, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, PR China.
authors.forename: Cunzheng
authors.initials: C
authors.lastname: Zhang
authors.affiliation: Institute of Food Safety, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, PR China.
authors.forename: Xianjin
authors.initials: X
authors.lastname: Liu
authors.affiliation: The Key Laboratory of Developmental Genes and Human Disease, Ministry of Education, Institute of Life Sciences, Southeast University, Nanjing 210096, PR China; Jiangsu Nanobody Engineering and Research Center, Nantong 226010, PR China. Electronic address: ywansystemsbiology@gmail.com.
authors.forename: Yakun
authors.initials: Y
authors.lastname: Wan
mesh_terms: D000818:Animals; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000276:immunology*; D002162:Camelus / Q000276:immunology*; D004731:Endotoxins / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D018604:Epitope Mapping; D004926:Escherichia coli; D005620:Freund's Adjuvant; D006460:Hemolysin Proteins / Q000276:immunology*; D007118:Immunoassay / Q000379:methods*; D019151:Peptide Library; D061905:Single-Domain Antibodies / Q000302:isolation & purification*
chemical_list: D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D004731:Endotoxins; D006460:Hemolysin Proteins; D019151:Peptide Library; D061905:Single-Domain Antibodies; C030956:insecticidal crystal protein, Bacillus Thuringiensis; D005620:Freund's Adjuvant
keywords: Biotin; Cry1Fa; Nanobody; Phage-displayed library; Sandwich immune ELISA; Streptavidin