title: New approach for development of sensitive and environmentally friendly immunoassay for mycotoxin fumonisin B(1) based on using peptide-MBP fusion protein as substitute for coating antigen.
abstract: Here, on the basis of mimotope of small analytes, we demonstrated a new approach for development of sensitive and environmentally friendly immunoassay for toxic small analytes based on the peptide-MBP fusion protein. In this work, using mycotoxin fumonisin B1 (FB1) as a model hapten, phage displayed peptide (mimotope) that binds to the anti-FB1 antibody were selected by biopanning from a 12-mer peptide library. The DNA coding for the sequence of peptide was cloned into Escherichia coli ER2738 as a fusion protein with a maltose binding protein (MBP). The prepared peptide-MBP fusion protein are "clonable" homogeneous and FB1-free products and can be used as a coating antigen in the immunoassay. The half inhibition concentration of the quantitative immunoassay setup with fusion protein (F1-MBP and F15-MBP) was 2.15 ± 0.13 ng/mL and 1.26 ± 0.08 ng/mL, respectively. The fusion protein (F1-MBP) was also used to develop a qualitative Elispot assay with a cutoff level of 2.5 ng/mL, which was 10-fold more sensitive than that measured for chemically synthesized FB1-BSA conjugates based Elispot immunoassay. The peptide-MBP fusion protein not only can be prepared reproducibly as homogeneous and FB1-free products in a large-scale but also can contribute to the development of a highly sensitive immunoassay for analyzing FB1. Furthermore, the novel concept might provide potential applications to a general method for the immunoassay of various toxic small molecules.
authors: State Key Laboratory of Food Science and Technology, Sino-German Joint Research Institute, Nanchang University , No. 235 Nanjing East Road, Nanchang 330047, China.
authors: Yang
authors: Y
authors: Xu
authors: Bo
authors: B
authors: Chen
authors: Qing-hua
authors: QH
authors: He
authors: Yu-Lou
authors: YL
authors: Qiu
authors: Xing
authors: X
authors: Liu
authors: Zhen-yun
authors: ZY
authors: He
authors: Zheng-ping
authors: ZP
authors: Xiong
mesh_terms: D000595:Amino Acid Sequence; D000821:Animal Feed / Q000032:analysis / Q000382:microbiology; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D037341:Fumonisins / Q000032:analysis*; D007118:Immunoassay / Q000379:methods*; D057230:Limit of Detection; D058619:Maltose-Binding Proteins / Q000737:chemistry* / Q000235:genetics; D009183:Mycotoxins / Q000032:analysis*; D012275:Oryza / Q000737:chemistry / Q000382:microbiology; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics; D003313:Zea mays / Q000737:chemistry / Q000382:microbiology
chemical_list: D037341:Fumonisins; D058619:Maltose-Binding Proteins; D009183:Mycotoxins; D019151:Peptide Library; D010455:Peptides; D011993:Recombinant Fusion Proteins; C056933:fumonisin B1