title: Uniform orientation of biotinylated nanobody as an affinity binder for detection of Bacillus thuringiensis (Bt) Cry1Ac toxin.
abstract: Nanobodies are the smallest natural fragments with useful properties such as high affinity, distinct paratope and high stability, which make them an ideal tool for detecting target antigens. In this study, we generated and characterized nanobodies against the Cry1Ac toxin and applied them in a biotin-streptavidin based double antibodies (nanobodies) sandwich-ELISA (DAS-ELISA) assay. After immunizing a camel with soluble Cry1Ac toxin, a phage displayed library was constructed to generate Nbs against the Cry1Ac toxin. Through successive rounds of affinity bio-panning, four nanobodies with greatest diversity in CDR3 sequences were obtained. After affinity determination and conjugating to HRP, two nanobodies with high affinity which can recognize different epitopes of the same antigen (Cry1Ac) were selected as capture antibody (Nb61) and detection antibody (Nb44). The capture antibody (Nb61) was biotinylated in vivo for directional immobilization on wells coated with streptavidin matrix. Both results of specificity analysis and thermal stability determination add support for reliability of the following DAS-ELISA with a minimum detection limit of 0.005 μg·mL-1 and a working range 0.010-1.0 μg·mL-1. The linear curve displayed an acceptable correlation coefficient of 0.9976. These results indicated promising applications of nanobodies for detection of Cry1Ac toxin with biotin-streptavidin based DAS-ELISA system.
authors.affiliation: The Key Laboratory of Developmental Genes and Human Disease, Ministry of Education, Institute of Life Sciences, Sipailou NO. 2, Southeast University, Nanjing 210096, China. damin.li@163.com.
authors.forename: Min
authors.initials: M
authors.lastname: Li
authors.affiliation: The Key Laboratory of Developmental Genes and Human Disease, Ministry of Education, Institute of Life Sciences, Sipailou NO. 2, Southeast University, Nanjing 210096, China. zhumin555111@163.com.
authors.forename: Min
authors.initials: M
authors.lastname: Zhu
authors.affiliation: Institute of Food Safety, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China. zhcz2003@hotmail.com.
authors.forename: Cunzheng
authors.initials: C
authors.lastname: Zhang
authors.affiliation: Institute of Food Safety, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China. jaasliu@jaas.ac.cn.
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, Sipailou NO. 2, Southeast University, Nanjing 210096, China. ywansystemsbiology@gmail.com.
authors.forename: Yakun
authors.initials: Y
authors.lastname: Wan
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000918:Antibody Specificity; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000302:isolation & purification*; D001710:Biotin / Q000737:chemistry; D019921:Biotinylation; D002162:Camelus / Q000276:immunology; D004731:Endotoxins / Q000302:isolation & purification*; D004797:Enzyme-Linked Immunosorbent Assay; D006460:Hemolysin Proteins / Q000302:isolation & purification*; D057230:Limit of Detection; D008969:Molecular Sequence Data; D019151:Peptide Library; D015203:Reproducibility of Results; D061905:Single-Domain Antibodies / Q000737:chemistry*; D019809:Streptavidin / Q000737:chemistry
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; D001710:Biotin; D019809:Streptavidin