Journal
ANALYTICAL CHEMISTRY
Volume 84, Issue 12, Pages 5229-5235Publisher
AMER CHEMICAL SOC
DOI: 10.1021/ac202747u
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Funding
- National Natural Science Foundation of China [31171702]
- Project of National Science & Technology Pillar Plan [2012BAB19B09, 2012BAK08B03, 2011BAD02D02, 2010BAD01B07]
- Special Fund for Agro-scientific Research in the Public Interest [201203094, 201203037]
- earmarked fund for China Agriculture research system [CARS-13]
- Key Project of the Ministry of Agriculture [2011-G5, 2010-G1]
- Special Foundation of President of the Chinese Agricultural Academy of Sciences [2012ZL042, 1610172010003]
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We screened and established seven hybridoma cell lines that secrete anti-aflatoxin monoclonal antibodies with different sensitivities. Among these antibodies, IC11 exhibited the highest sensitivity against all four major kinds of aflatoxins (AFB1, AFB2, AFG1, and AFG2) (IC50 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.
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