New Approach for Development of Sensitive and Environmentally Friendly Immunoassay for Mycotoxin Fumonisin B₁ 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.

Anti-idiotypic VHH phage display-mediated immuno-PCR for ultrasensitive determination of mycotoxin zearalenone in cereals

“…The filamentous phage-displayed library has been proved to be an effective way in generating substitutes for mycotoxin conjugates [24,25,26]. Moreover, phages binding to the interest compound can be isolated, and rapidly reproduced [27].…”

Deoxynivalenol-mimic nanobody isolated from a naïve phage display nanobody library and its application in immunoassay

“…Although heterology format is M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT 4 very useful, the procedures of chemical synthesis of hapten-protein conjugates are over elaborative and complicated. Especially, in regard to the synthesis of toxin-carrier conjugates, which are usually chemosynthesized by "trial and error" approach, involving handling of the toxin which pose a threat to the environment and human health, and the lot-to-lot variation, affecting immunoassay results [18,19].…”

Development and comparative study of chemosynthesized antigen and mimotope-based immunoassays for class-specific analysis of O,O-dimethyl organophosphorus pesticides

“…The optimum antigen was selected by immunoassays717 or molecular modeling1618. These strategies were proved to be feasible in previous studies, but the preparation of candidate haptens was always “trial or error” and required considerable effort in chemical synthesis1619. In addition, the high toxicity of some haptens posed a threat to the environment and human health1219.…”

“…These strategies were proved to be feasible in previous studies, but the preparation of candidate haptens was always “trial or error” and required considerable effort in chemical synthesis1619. In addition, the high toxicity of some haptens posed a threat to the environment and human health1219. Since the peptide mimotope-based immunoassay was successfully developed for low molecular weight compound (deoxynivalenol)20, an increasing number of researchers have begun to select environmentally friendly phage-display peptides (mimotopes), which can mimic the antibody binding site, to replace the chemically synthesized conjugates121920212223242526.…”

“…In addition, the high toxicity of some haptens posed a threat to the environment and human health1219. Since the peptide mimotope-based immunoassay was successfully developed for low molecular weight compound (deoxynivalenol)20, an increasing number of researchers have begun to select environmentally friendly phage-display peptides (mimotopes), which can mimic the antibody binding site, to replace the chemically synthesized conjugates121920212223242526. The immunoassays based on mimotopes, used as peptide-phage12212223, peptide-MBP fusion protein1924 or synthesized peptide forms2526, have shown their advantage in low molecular weight compound detection.…”