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Aflatoxins and zearalenone (ZEN) are highly common mycotoxins in maize and maize-based products. This study aimed to report a time-resolved fluorescence immunochromatographic assay (TRFICA) developed using two idiotypic nanobodies for rapid, quantitative, and simultaneous detection of aflatoxin B1 (AFB1) and ZEN in maize and its products. A novel Eu/Tb(III) nanosphere with enhanced fluorescence was prepared as a label and conjugated to anti-idiotypic nanobody (AIdnb) and monoclonal antibody (mAb). On the basis of nanosphere--antibody conjugation, two patterns of competitive time-resolved strip methods (AIdnb--TRFICA and mAb--TRFICA) were established and compared. The half inhibition concentration of AIdnb--TRFICA was 0.46 and 0.86 ng⋅mL-1 for AFB1 and ZEN, which was 18.3- and 20.3-fold more sensitive than that of mAb--TRFICA for AFB1 and ZEN, respectively. Under optimal conditions, AIdnb--TRFICA for dual mycotoxin was established and provided a quantitative relationship ranging from 0.13 to 4.54 ng⋅mL-1 for AFB1 and 0.20 to 2.77 ng⋅mL-1 for ZEN, with a detection limit of 0.05 and 0.07 ng⋅mL-1 in the buffer solution, respectively. AIdnb--TRFICA showed good recoveries (72.6%-106.6%) in samples and was applied to detect dual mycotoxin in maize samples with satisfying results. To the best of our knowledge, it is the first report about a time-resolved strip method based on AIdnbs for dual mycotoxin. [ABSTRACT FROM AUTHOR]

Aflatoxins are a group of extremely toxic small molecules that have been involved in human hepatic and extrahepatic carcinogenesis as causative agents. Herein, we developed a real-time immuno polymerase chain reaction (1PCR) assay for the accurately quantitative detection of aflatoxins in agri-products base on a M13 phage containing aflatoxin anti-idiotypic nanobody and its encoding DNA which was used to design the specific primers. The limit of detection (LOD) of the assay is 0.02 ng/mL, which exhibits a 4-fold improvement over traditional phage ELISA The developed method was successfally validated with the samples of com, nee, peanut, and feedstuff, which are major aflatoidn-contaminated agri-products. And the recoveries were from 77.05 to 122.16%. hor farther validation, the developed assay was also compared with a reference HPLC method for the analysis of aflatoxins in com and peanuts, and concordant results (R2 = 0.991) were obtained In this context, this study provides a novel opportunity to analyze aflatoxins in agri-products. [ABSTRACT FROM AUTHOR]

Anti-idiotypic antibodies recognize the antigenic determinants of an antibody, thus they can be used as surrogate antigens. Single-domain antibodies from camlid heavy-chain antibodies with the benefit features of small size, thermostability, and ease in expression, are leading candidates to produce anti-idiotypic antibodies. In this work, we constructed an antibody phage library from the mRNA of an alpaca immunized with an antiaflatoxin monoclonal antibody (mAb) 1C11. Three anti-idiotypic VHH antibodies were isolated and applied to immunoassay toward aflatoxin as a coating antigen. The best immunoassay developed with one of these VHH antibodies shows an IC50 of 0.16 ng/mL toward aflatoxin B1 and cross-reactivity toward aflatoxin B2, G1, and G2 of 90.4%, 54.4%, and 37.7%, respectively. The VHH-based immunoassay was successfully applied to the analysis of peanuts, corn, and rice, which are the predominant commodities regularly contaminated by aflatoxins. A good correlation (r² = 0.89) was found between the data obtained from the conventional ELISA and the ELISA based on a VHH coating antigen for the analysis of aflatoxins in peanuts and feedstuff. The use of biotechnology in developing the surrogate, the absence of standard aflatoxin and organic solvents in the synthesis procedures, and the reproducibility of the VHH antibody makes it an ideal strategy for replacing conventional synthesized antigens. [ABSTRACT FROM AUTHOR]

We screened and established seven hybridoma cell lines that secrete anti-aflatoxin monodonal antibodies with different sensitivities. Among these antibodies, 1C11 exhibited the highest sensitivity against all four major kinds of allatoxins (AFB1, AFB2, AFG1, and AFG2) (IC50 0.0012-0.018 ng mL-1 in the enzyme linked immwiosorbent sassy (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 daril;, the molecular mechanism of the highest sensitivity of ICII 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 dearly demonstrate how structural differences influence the binding properties of antibodies against the target hapten with different sensitivities. [ABSTRACT FROM AUTHOR]