Competitive-Type Pressure-Dependent Immunosensor for Highly Sensitive Detection of Diacetoxyscirpenol in Wheat via Monoclonal Antibody.

Diacetoxyscirpenol (DAS) is a type A trichothecene mycotoxin with low molecular weight, and with respect to its toxicity and the occurrence in food and feed, it is known as a potential risk for public and animal health. In the present study, first, a sensitive and specific monoclonal antibody (5E7) was developed. Then, the antibody was applied to develop a competitive-type pressure-dependent immunosensor (CTPDI). The Au@PtNP was synthesized and labeled with goat antimouse antibody (Au@PtNPs-IgG). Finally, the concentration of DAS was negatively correlated with the pressure signal. In the presence of optimal conditions, matrix-matched calibration curves were plotted for wheat samples, in which an optimal IC50 value (half maximal inhibitory concentration) of 3.08 ng/g was achieved. The CTPDI was further applied to detect natural and blind wheat samples, and validation was carried out by liquid chromatography-tandem mass spectrometry. The results showed that CTPDI was highly appropriate and accurate for detection of DAS in wheat.

Fluorometric lateral flow immunoassay for simultaneous determination of three mycotoxins (aflatoxin B1, zearalenone and deoxynivalenol) using quantum dot microbeads.

A fluorometric lateral flow immunoassay (LFA) is described for the simultaneous determination of the mycotoxins aflatoxin B1 (AFB1), zearalenone (ZEN) and deoxynivalenol (DON). The method is based on the use of CdSe/SiO2 quantum dot microbeads (QBs) with a mean diameter of 106 nm. These have strong red luminescence (with excitation/emission peaks at 365/622 nm) which results in enhanced sensitivity. The QBs binding with monoclonal antibodies (mAbs) as the signal probes can react specifically with AFB1, ZEN and DON, respectively. There is an inverse correlation between the fluorescence signal intensity of test line and the analyte content, which can realize the quantitative analysis of analytes within 15 min. The limits of detection in solution are 10, 80 and 500 pg mL-1 for AFB1, ZEN and DON, respectively. Besides, the average recoveries from spiked feed range from 85.5 to 119.0%, and the relative standard deviations are less than 16.4% for both intra- and inter-day assays. The method was used to analyze naturally contaminated feedstuff, and this resulted in a good agreement with data obtained by LC-MS/MS. Graphical abstractSchematic representation of a fluorometric method for the simultaneous determination of three mycotoxins. Quantum dot microbeads (QBs) binding with monoclonal antibodies (mAbs) are signal probes. There is an inverse correlation between the fluorescence intensity of test line and the analyte concentration.

[Polyclonal antibody and immunomagnetic beads preparation technology for type A trichothecenes].

OBJECTIVE: Preparing immunomagnetic beads for enrichment and purification of mask type A trichothecenes. METHODS: Through immunizing New Zealand white rabbits with family A toxin artificial antigen 3-Ac-NEOS-HS-BSA, the polyclonal antibodies of type A trichothecenes( type A family) were isolated, as well as its antibody titre were determinated and purified. The coupling condition of high titer antibody and immunomagnetic beads were optimized and applied. RESULTS: After 5 times of immunization with type A trichothecene family toxin, the rabbit serum inhibited the highest antibody titer( 1 : 64 000). The optimal coupling condition of antibody and immunomagnetic beads were: temperature = 24 ℃, rotation capture in pH 7. 4, 0. 02 mol/L PBS buffer solution with 10% methanol in 24 h. CONCLUSION: The establishment of preparation method of type A single family toxin polyclonal antibody and immunomagnetic beads can make a standard for enrichment and purification of type A matrix complex single family toxins.

Biochip Spray: Simplified Coupling of Surface Plasmon Resonance Biosensing and Mass Spectrometry.

A simplified coupling of surface plasmon resonance (SPR) immuno-biosensing with ambient ionization mass spectrometry (MS) was developed. It combines two orthogonal analysis techniques: the biosensing capability of SPR and the chemical identification power of high resolution MS. As a proof-of-principle, deoxynivalenol (DON), an important mycotoxin, was captured using an SPR gold chip containing an antifouling layer and monoclonal antibodies against the toxin and, after washing, the chip could be taken out and analyzed by direct spray MS of the biosensor chip to confirm the identity of DON. Furthermore, cross-reacting conjugates of DON present in a naturally contaminated beer could be successfully identified, thus showing the potential of rapid identification of (un)expected cross-reacting molecules.

Insight into the Working Mechanism of Quenchbody: Transition of the Dye around Antibody Variable Region That Fluoresces upon Antigen Binding.

Recently, we reported a novel immunoassay reagent Quenchbody (Q-body): a single chain antibody variable region (scFv) fragment labeled with fluorescent dye, whose fluorescence intensity increases when it binds to the antigen. Here we analyze its working mechanism by immuno- and fluorescence polarization (FP) assays. In an enzyme-linked immunosorbent assay, we found that in the presence of antigen osteocalcin peptide (BGP-C7), more TAMRA-labeled Q-bodies bound to anti-TAMRA antibody than in its absence. Moreover, we found that anti-BGP Q-body with the shortest linker that exhibits the largest antigen-dependency in fluorescence showed the highest binding signal. Similar results were obtained with anti-bisphenol A (BPA) Q-bodies, with inversed correlation with their linker lengths. In the FP assay, when the ATTO 520 labeled Q-body was added with antigen, the Brownian motion of the dye became more active, which resulted in reduced fluorescence anisotropy r. In other words, in the presence of antigen, 1/r showing that the dye mobility is larger than in the absence of its antigen. In addition, anti-BGP Q-body with the largest antigen-dependency in fluorescence showed the highest mobility. Overall, these results clearly suggest that the antigen-dependent fluorescence quenching and recovery of Q-body is caused by the movement of the dye within and around scFv, which moves out of scFv upon binding with its antigen.

Modification of a deoxynivalenol-antigen-mimicking nanobody to improve immunoassay sensitivity by site-saturation mutagenesis.

A nanobody (N-28) which can act as a deoxynivalenol (DON) antigen has been generated, and its residues Thr102-Ser106 were identified to bind with anti-DON monoclonal antibody by alanine-scanning mutagenesis. Site-saturation mutagenesis was used to analyze the plasticity of five residues and to improve the sensitivity of the N-28-based immunoassay. After mutagenesis, three mutants were selected by phage immunoassay and were sequenced. The half-maximal inhibitory concentrations of the immunoassay based on mutants N-28-T102Y, N-28-V103L, and N-28-Y105F were 24.49 ± 1.0, 51.83 ± 2.5, and 35.65 ± 1.6 ng/mL, respectively, showing the assay was, respectively, 3.2, 1.5, and 2.2 times more sensitive than the wild-type-based assay. The best mutant, N-28-T102Y, was used to develop a competitive phage ELISA to detect DON in cereals with high specificity and accuracy. In addition, the structural properties of N-28-T102Y and N-28 were investigated, revealing that the affinity of N-28-T102Y decreased because of increased steric hindrance with the large side chain. The lower-binding-affinity antigen mimetic may contribute to the improvement of the sensitivity of competitive immunoassays. These results demonstrate that nanobodies would be a favorable tool for engineering. Moreover, our results have laid a solid foundation for site-saturation mutagenesis of antigen-mimicking nanobodies to improve immunoassay sensitivity for small molecules.

Application on gold nanoparticles-dotted 4-nitrophenylazo graphene in a label-free impedimetric deoxynivalenol immunosensor.

In this paper, we report a new concept to construct a label-free electrochemical inhibition-based immunosensor for the detection of the mycotoxin deoxynivalenol (DON) in cereal samples. The electrochemical impedance spectroscopy of tris(bipyridine) ruthenium (II) chloride was used as a marker enhanced with gold nanoparticles-dotted 4-nitrophenylazo functionalized graphene (AuNp/G/PhNO2) nanocatalyst mediated in Nafion on a glassy carbon electrode. Under the optimized conditions, the formation of immunocomplexes inhibited electron flow and increased the charge transfer resistance of the sensing interface linearly. The change in impedance was proportional to DON concentrations in the range of 6-30 ng/mL with a sensitivity and detection limit of 32.14 ΩL/ng and 0.3 µg/mL, respectively, which compares favorably with the ELISA result. The proposed sensor had a stability of 80.3%, good precision and selectivity in DON standard solution containing different interfering agents, indicating promising application prospect for this strategy in designing impedimetric, electrochemiluminescent, voltammetric or amperometric sensors.

Monoclonal-Antibody-Based Immunoassays for the Mycotoxins NX-2 and NX-3 in Wheat.

The fungal infestation of crops can cause major economic losses. Toxins produced by the causative fungi (mycotoxins) represent a potential safety hazard to people and livestock consuming them. One such mycotoxin is deoxynivalenol (DON, also known as vomitoxin), a trichothecene associated with Fusarium Head Blight of wheat. DON is commonly found in cereal crops worldwide. A group of trichothecene mycotoxins closely related to DON, the NX toxins, have been reported to occur in the northeastern United States and southern Canada. While many commercial immunoassays are available to detect DON, there are no rapid screening assays for the NX toxins. We describe the development and isolation of three monoclonal antibodies (mAbs) specific towards two NX toxins: NX-2 and NX-3. The mAbs did not recognize DON or several other closely related trichothecenes. One of the mAbs was selected for development of an enzyme-linked immunosorbent assay (ELISA) for NX-2 and NX-3 in wheat. The dynamic ranges for the assay were 7.7 to 127 µg/kg for NX-2 and 59 µg/kg to 1540 µg/kg for NX-3 in wheat. Recoveries from spiked wheat averaged 84.4% for NX-2 and 99.3% for NX-3, with RSDs of 10.4% and 11.3%, respectively (n = 24). The results suggest that this assay can be used to screen for NX toxins in wheat at levels relevant to human food and animal feed safety.

A novel biosensor regulated by the rotator of F0F1-ATPase to detect deoxynivalenol rapidly.

A novel biosensor (immuno-rotary biosensor) was developed by conjugating deoxynivalenol (DON) monoclonal antibodies with the "rotator" ε-subunit of F(0)F(1)-ATPase within chromatophores with an ε-subunit monoclonal antibody-biotin-avidin-biotin linker to capture DON residues. The conjugation conditions were then optimized. The capture of DON was based on the antibody-antigen reaction and it is indicated by the change in ATP synthetic activity of F(0)F(1)-ATPase, which is measured via chemiluminescence using the luciferin-luciferase system with a computerized microplate luminometer analyzer. 10(-7)mg/ml of DON can be detected. The whole detection process requires only about 20min. This method has promising applications in the detection of small molecular compounds because of its rapidity, simplicity, and sensitivity.

An Automatic Immunoaffinity Pretreatment of Deoxynivalenol Coupled with UPLC-UV Analysis.

An immunoaffinity magnetic beads (IMBs) based automatic pretreatment method was developed for the quantitative analysis of deoxynivalenol (DON) by ultra-performance liquid chromatography and ultraviolet detector (UPLC-UV). First, N-hydroxysuccinimide-terminated magnetic beads (NHS-MBs) with good magnetic responsivity and dispersibility were synthesized and characterized by optical microscopy, scanning electron microscopy (SEM), and laser diffraction-based particle size analyzer. Then, the amino groups of anti-DON monoclonal antibody (mAb) and the NHS groups of NHS-MBs were linked by covalent bonds to prepare IMB, without any activation reagent. The essential factors affecting the binding and elution of DON were meticulously tuned. Under optimal conditions, DON could be extracted from a real sample and eluted from IMB by water, enabling environmentally friendly and green analysis. Hence, there was no need for dilution or evaporation prior to UPLC-UV analysis. DON in 20 samples could be purified and concentrated within 30 min by the mycotoxin automated purification instrument (MAPI), allowing for automated, green, high-throughput and simple clean-up. Recoveries at four distinct spiking levels in corn and wheat ranged from 92.0% to 109.5% with good relative standard deviations (RSD, 2.1-7.0%). Comparing the test results of IAC and IMB in commercial samples demonstrated the reliability and superiority of IMB for quantitatively analyzing massive samples.

Trichothecene mycotoxins activate inflammatory response in human macrophages.

Damp building-related illnesses have caused concern for years in many countries. Although the problem is extensive, the knowledge of the immunological reactions behind damp building-related illnesses is still quite limited. Trichothecene mycotoxins form one major group of toxins, which possibly contribute to the illnesses. Stachybotrys chartarum is a well-known, but also controversial damp building mold and many strains of this mold are capable of producing trichothecenes. In this report, we have examined the effect of S. chartarum and trichothecene mycotoxins on the proinflammatory cytokine response in human macrophages. As a result, satratoxin-positive S. chartarum activated inflammasome-associated caspase-1, which is needed for proteolytic processing of IL-1beta and IL-18. Furthermore, purified trichothecene mycotoxins, roridin A, verrucarin A, and T-2 toxin activated caspase-1, and these mycotoxins also strongly enhanced LPS-dependent secretion of IL-1beta and IL-18. The satratoxin-positive strain of S. chartarum and the trichothecenes also triggered the activation of caspase-3, which is an effector caspase of apoptosis. Satratoxin-negative S. chartarum was not able to activate either caspase-1 or caspase-3. In conclusion, our results indicate that human macrophages sense trichothecene mycotoxins as a danger signal, which activates caspase-1, and further enables the secretion of IL-1beta and IL-18 from the LPS-primed cells.

Dual near-infrared fluorescence-based lateral flow immunosensor for the detection of zearalenone and deoxynivalenol in maize.

A novel dual near-infrared fluorescence-based lateral flow immunosensor was developed to determine zearalenone and deoxynivalenol in maize. Two near-infrared dyes with distinct fluorescence characteristics were utilized to separately label the anti-zearalenone and anti-deoxynivalenol antibodies as detection reagents. The capture antigens zearalenone-BSA and deoxynivalenol-BSA were mixed and immobilized on the same test line of nitrocellulose membrane. This assay format facilitates simultaneous detection of the two mycotoxins on a single test line. After optimizing experimental parameters, the limits of detection for zearalenone and deoxynivalenol were as low as 0.55 µg/kg and 3.8 µg/kg in maize, respectively. The spiking experiment yielded recovery ratios ranging from 81.7% to 107.3% with coefficients of variation less than 14% demonstrating high assay accuracy and precision. Moreover, the actual sample analysis produced consistent results between this method and instrumental method. Therefore, the developed immunosensor can serve as an accurate and efficient approach for monitoring mycotoxins in agricultural products.

Human Milk Oligosaccharide 3'-GL Improves Influenza-Specific Vaccination Responsiveness and Immunity after Deoxynivalenol Exposure in Preclinical Models.

Deoxynivalenol (DON), a highly prevalent mycotoxin food contaminant, is known to have immunotoxic effects. In the current study, the potential of dietary interventions with specific mixtures of trans-galactosyl-oligosaccharides (TOS) to alleviate these effects were assessed in a murine influenza vaccination model. Vaccine-specific immune responses were measured in C57Bl/6JOlaHsd mice fed diets containing DON, TOS or a combination, starting 2 weeks before the first vaccination. The direct effects of TOS and its main oligosaccharide, 3'-galactosyl-lactose (3'-GL), on DON-induced damage were studied in Caco-2 cells, as an in vitro model of the intestinal epithelial barrier. Exposure to DON significantly reduced vaccine-specific immune responses and the percentages of Tbet+ Th1 cells and B cells in the spleen. DON significantly altered epithelial structure and integrity in the ileum and reduced the SCFA levels in the cecum. Adding TOS into DON-containing diets significantly improved vaccine-specific immune responses, restored the immune cell balance in the spleen and increased SCFA concentrations in the cecum. Incubating Caco-2 cells with TOS and 3'-GL in vitro further confirmed their protective effects against DON-induced barrier disruption, supporting immune modulation. Overall, dietary intervention with TOS can attenuate the adverse effects of DON on Th1-mediated immune responses and gut homeostasis. These beneficial properties might be linked to the high levels of 3'-GL in TOS.

Intracellular expression of a single domain antibody reduces cytotoxicity of 15-acetyldeoxynivalenol in yeast.

15-Acetyldeoxynivalenol (15-AcDON) is a low molecular weight sesquiterpenoid trichothecene mycotoxin associated with Fusarium ear rot of maize and Fusarium head blight of small grain cereals. The accumulation of mycotoxins such as deoxynivalenol (DON) and 15-AcDON within harvested grain is subject to stringent regulation as both toxins pose dietary health risks to humans and animals. These toxins inhibit peptidyltransferase activity, which in turn limits eukaryotic protein synthesis. To assess the ability of intracellular antibodies (intrabodies) to modulate mycotoxin-specific cytotoxocity, a gene encoding a camelid single domain antibody fragment (V(H)H) with specificity and affinity for 15-AcDON was expressed in the methylotropic yeast Pichia pastoris. Cytotoxicity and V(H)H immunomodulation were assessed by continuous measurement of cellular growth. At equivalent doses, 15-AcDON was significantly more toxic to wild-type P. pastoris than was DON. In turn, DON was orders of magnitude more toxic than 3-acetyldeoxynivalenol. Intracellular expression of a mycotoxin-specific V(H)H within P. pastoris conveyed significant (p = 0.01) resistance to 15-AcDON cytotoxicity at doses ranging from 20 to 100 mug.ml(-1). We also documented a biochemical transformation of DON to 15-AcDON to account for the attenuation of DON cytotoxicity at 100 and 200 mug.ml(-1). The proof of concept established within this eukaryotic system suggests that in planta V(H)H expression may lead to enhanced tolerance to mycotoxins and thereby limit Fusarium infection of commercial agricultural crops.

Production and characterization of monoclonal antibody and its recombinant single chain variable fragment specific for a food-born mycotoxin, fumonisin B1.

Fumonisin B(1) (FMB(1)) is a food-born mycotoxin produced by Fusarium moniliforme. Monoclonal antibody against FMB(1) (anti-FMB(1) mAb) was produced in the hybridoma DV9, which was established from a BALB/c mouse immunized with bovine serum albumin conjugated FMB(1) (FMB(1)-BSA). A competitive direct enzyme-linked immunosorbent assay (ELISA) showed that anti-FMB(1) mAb has about 10 ppb of minimum FMB(1) detection concentration and 220 ppb of 50% inhibition concentration (IC(50)). Much lower cross-reactivity of anti-FMB(1) mAb on ochratoxin A, aflatoxin B(1) and deoxynivalenol provided that anti-FMB(1) mAb was specific for FMB(1). The gene coding single chain variable fragment against FMB(1) (anti-FMB(1) scFv) was cloned from the hybridoma DV9 and was expressed in recombinant Escherichia coli. Insoluble anti-FMB(1) scFv required optimization of its refolding condition, and hence functional scFv was obtained. By using indirect ELISA, about 12-fold lower binding activity of anti-FMB(1) scFv on FMB(1)-BSA was obtained in comparison with that of the parental mAb.

Three-dimensional ordered macroporous magnetic photonic crystal microspheres for enrichment and detection of mycotoxins (I): Droplet-based microfluidic self-assembly synthesis.

Ordered porous materials are attracting enormous attention due to their uniform pore structures, particularly the magnetic photonic crystal microspheres (PCMs) which not only possess unique photonic crystal structure but also can achieve separation easily based on magnet. Here, a two-phase microfluidic self-assembly synthetic system was established simply and employed for the preparation of three dimensional PCMs (3DPCMs) by using the emulsion droplet approach. One phase (dispersed phase) was an aqueous emulsion containing Fe3O4, silica (SiO2) and polystyrene (PS) nanoparticles; another phase (continuous phase) was pure silicone oil. The droplets were formed by introducing the dispersed phase into the continuous phase through a tee valve. By heating the droplets, the water would evaporate and the nanoparticles would finally assemble into solid microspheres, which could be changed into macroporous 3DPCMs after removal of the PS nanoparticles by calcination. The contents and particle sizes of Fe3O4, SiO2 and PS nanoparticles in the dispersed phase were investigated in detail and optimized to prepare macroporous magnetic 3DPCMs with high quality. The morphologies, surface crystal structure, magnetic property, particle size distribution, specific surface area and pore size of the macroporous magnetic 3DPCMs were characterized. The expected 3DPCM displayed regular and uniform photonic crystal structure, narrow particle size distribution and strong magnetic property. The macroporous magnetic 3DPCMs grafted with vomitoxin (DON)-antibodies could be applied for selective enrichment of DON in real samples.

Silanized Luminescent Quantum Dots for the Simultaneous Multicolor Lateral Flow Immunoassay of Two Mycotoxins.

A critical point for the successful development of a fluorescent quantum dot (QD)-based immunoassay is maintaining the high fluorescence quantum yield of QDs during hydrophilization and bioconjugation. In this paper, we carefully designed CdSe/CdS and CdSe/CdS/ZnS core-shell heterostructures and extended them with silica coating of different surface composition allowing preservation of fluorescence quantum yield as high as 70% in aqueous media. The silanized QDs containing epoxy and carboxy surface groups were bioconjugated with monoclonal antibodies. The synthesized fluorescent conjugates were used in a multicolor lateral flow immunoassay for simultaneous determination of two mycotoxins. Zearalenone and deoxynivalenol were chosen as a proof of concept. Cutoff levels for the zearalenone and deoxynivalenol detection were adjusted to be at 40 and 400 µg kg-1, respectively, complying with the European Commission regulation. Validation of the developed test was performed by analysis of 34 naturally contaminated maize and wheat samples; as a confirmatory method, LC-MS/MS was used.

Immune response among patients exposed to molds.

Macrocyclic trichothecenes, mycotoxins produced by Stachybotrys chartarum, have been implicated in adverse reactions in individuals exposed to mold-contaminated environments. Cellular and humoral immune responses and the presence of trichothecenes were evaluated in patients with mold-related health complaints. Patients underwent history, physical examination, skin prick/puncture tests with mold extracts, immunological evaluations and their sera were analyzed for trichothecenes. T-cell proliferation, macrocyclic trichothecenes, and mold specific IgG and IgA levels were not significantly different than controls; however 70% of the patients had positive skin tests to molds. Thus, IgE mediated or other non-immune mechanisms could be the cause of their symptoms.

Ultrasensitive and eco-friendly immunoassays based monoclonal antibody for detection of deoxynivalenol in cereal and feed samples.

Ultrasensitive immunoassays, including an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) and a lateral-flow immunochromatographic assay (ICA), were developed based on a monoclonal antibody for the analysis of deoxynivalenol in food and feed samples. With 0.01 M PBS, 20% ethanol-PBS, and 60% ethanol-PBS extraction, which are environmentally safe, the 50% inhibitory concentration (IC50) and limit of detection (LOD) values were 1.83-4.68 µg/kg and 0.241-0.664 µg/kg, respectively, with recovery rates of 87.7%-137% and coefficient variation values of 3.99-9.88% (intra-assay) and 4.17-9.81% (inter-assay) for the ic-ELISA relative to the results obtained by liquid chromatography-tandem mass spectrometry (LC-MS). For the ICA strip, the visual LODs were 10-150 µg/kg, the cut-off values were 50-750 µg/kg, and the calculated LODs were 1.97-46.8 µg/kg, with different sample extraction solutions, and the recovery rates were 66.7%-127%. These methods are sensitive, simple and safe, providing an auxiliary analytical tool for screening the massive samples in markets.

Simultaneous detection of aflatoxin B1, ochratoxin A, zearalenone and deoxynivalenol in corn and wheat using surface plasmon resonance.

Mycotoxins are toxic metabolites produced by fungi or molds, which may cause serious harm to human health through polluted cereal foods. In order to measure the typical mycotoxin contaminations in wheat and corn, a surface plasmon resonance (SPR) method was established using SPR sensor chip that was fabricated based on self-assembled monolayer. The minimum detection limit of aflatoxin B1, ochratoxin A, zearalenone and deoxynivalenol were identified as 0.59 ng/mL, 1.27 ng/mL, 7.07 ng/mL and 3.26 ng/mL, respectively. The cross-reactivity for all four mycotoxins were demonstrated to be low. Moreover, the test data were compared with HPLC-MS/MS confirmatory analysis results and good agreement was found between them. In conclusion, the SPR method for simultaneously detecting four mycotoxins has been developed with high sensitivity, good linearity and specificity, which can meet the detection requirements of cereal foods.