Molecular Characterization of Monoclonal Antibodies against Aflatoxins: A Possible Explanation for the Highest Sensitivity

Abstract

We screened and established seven hybridoma cell lines that secrete anti-aflatoxin monoclonal antibodies with different sensitivities. Among these antibodies, 1C11 exhibited the highest sensitivity against all four major kinds of aflatoxins (AFB1, AFB2, AFG1, and AFG2) (IC(50) 0.0012-0.018 ng mL(-1) in the enzyme linked immunosorbent assay (ELISA) system, visual limit of detection of 0.03-0.25 ng mL(-1)). To better understand the interactions between these antibodies and aflatoxins, as well as to guide their potential sensitivity improvement in recombinant antibodies, we used multiple sequence alignment and molecular modeling combined with molecular docking to clarify the molecular mechanism of the highest sensitivity of 1C11 against aflatoxins. Our results show that hydrogen bond and hydrophobic interaction formed by Ser-H49 and Phe-H103 in the antibody with the hapten played the most important roles in determining the binding affinity. Further experiments performed on antibody mutants, designed on the basis of the computational models, supported the prediction of the interaction mode between the antibody and the hapten. Although the factors that influence antibody sensitivity are highly interdependent, our experimental and modeling studies clearly demonstrate how structural differences influence the binding properties of antibodies against the target hapten with different sensitivities.

Highly sensitive electrochemical impedance spectroscopy immunosensor for the detection of AFB1 in olive oil

“…Ionic liquids are ion conductive, and display a wide electrochemical potential window (Opallo & bundles, which are physically cross-linked due to cation-p and pp interactions between the imidazolium cations and CNTs (Fujita, Murata, Masuda, Nakamura, & Ohno, 2012). The three-dimensional network will provide numerous conductive microcavities for the antibodies immobilization and small molecule hapten transfer (Li et al, 2012;Moghaddam, Løbersli, Gebhardt, Braunagel, & Marvik, 2001). The ionic liquid provides excellent biocompatible microenvironment to maintain activity of the antibody, and can be used as an excellent conductive binder to immobilize antibodies and MWCNTs onto the surface of electrode (Opallo & Lesniewski, 2011).…”

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

“…(Since the recombinant antibody fragments are more convenient in molecular modeling [33], the anti-DON single-chain variable fragment (ScFv) was used in this study and its sequence data was available in supplement materials Figure …”

Application of quantitative structure‐activity relationship analysis on an antibody and alternariol‐like compounds interaction study

“…More and more studies have shown that the specificity of the antibody is of great significance for improving immunoassays . An antibody with high specificity (low cross‐reactivity [CR]) is suitable to be used in the immunoassay detecting a single compound, while an antibody with broad specificity (high CR) is suitable to be used in the immunoassay detecting multiple compounds . However, because of the lack of knowledge about the antibody recognition and the ambiguous structure‐activity relationship, artificial control of the specificity of the antibody remains a huge challenge …”