Highly Specific Aptamer-Antibody Birecognized Sandwich Module for Ultrasensitive Detection of a Low Molecular Weight Compound.

Herein, the aptamer-antibody sandwich module was first introduced to accurately recognize a low molecular weight compound (mycotoxin). Impressively, compared with the large steric hindrance of a traditional dual-antibody module, the aptamer-antibody sandwich with low Gibbs free energy and a low dissociation constant has high recognition efficiency; thus, it could reduce false positives and false negatives caused by a dual-antibody module. As a proof of concept, a sensitive electrochemiluminescence (ECL) biosensor was constructed for detecting mycotoxin zearalenone (ZEN) based on an aptamer-antibody sandwich as a biological recognition element and porous ZnO nanosheets (Zn NSs) supported Cu nanoclusters (Cu NCs) as the signal transduction element, in which the antibody was modified on the vertex of a tetrahedral DNA nanostructure (TDN) with a rigid structure to increase the kinetics of target recognition for promoting the detection sensitivity. Moreover, the Cu NCs/Zn NSs exhibited an excellent ECL response that was attributed to the aggregation-induced ECL enhancement through electrostatic interactions. The sensing platform achieved trace detection of ZEN with a low detection limit of 0.31 fg/mL, far beyond that of the enzyme-linked immunosorbent assay (ELISA, the current rapid detection method) and high-performance liquid chromatography (HPLC, the national standard detection method). The strategy has great application potential in food analysis, environmental monitoring, and clinical diagnosis.

Mycotoxin Zearalenone Attenuates Innate Immune Responses and Suppresses NLRP3 Inflammasome Activation in LPS-Activated Macrophages.

Zearalenone (ZEA) is a mycotoxin that has several adverse effects on most mammalian species. However, the effects of ZEA on macrophage-mediated innate immunity during infection have not been examined. In the present study, bacterial lipopolysaccharides (LPS) were used to induce the activation of macrophages and evaluate the effects of ZEA on the inflammatory responses and inflammation-associated signaling pathways. The experimental results indicated that ZEA suppressed LPS-activated inflammatory responses by macrophages including attenuating the production of proinflammatory mediators (nitric oxide (NO) and prostaglandin E2 (PGE2)), decreased the secretion of proinflammatory cytokines (tumor necrosis factor (TNF)-α, interleukin (IL)-1ß and IL-6), inhibited the activation of c-Jun amino-terminal kinase (JNK), p38 and nuclear factor-κB (NF-κB) signaling pathways, and repressed the nucleotide-binding and oligomerization domain (NOD)-, leucine-rich repeat (LRR)- and pyrin domain-containing protein 3 (NLRP3) inflammasome activation. These results indicated that mycotoxin ZEA attenuates macrophage-mediated innate immunity upon LPS stimulation, suggesting that the intake of mycotoxin ZEA-contaminated food might result in decreasing innate immunity, which has a higher risk of adverse effects during infection.

Preparation and Characterization of Monoclonal Antibodies with High Affinity and Broad Class Specificity against Zearalenone and Its Major Metabolites.

This study aimed to detect and monitor total Zearalenone (ZEN) and its five homologs (ZENs) in cereals and feed. The monoclonal antibodies (mAbs) with a high affinity and broad class specificity against ZENs were prepared, and the conditions of a heterologous indirect competitive ELISA (icELISA) were preliminarily optimized based on the ZEN mAbs. The immunogen ZEN-BSA was synthesized using the oxime active ester method (OAE) and identified using infrared (IR) and ultraviolet (UV). The coating antigen ZEN-OVA was obtained via the 1,4-butanediol diglycidyl ether method (BDE). Balb/c mice were immunized using a high ZEN-BSA dose with long intervals and at multiple sites. A heterologous indirect non-competitive ELISA (inELISA) and an icELISA were used to screen the suitable cell fusion mice and positive hybridoma cell lines. The ZEN mAbs were prepared by inducing ascites in vivo. The standard curve was established, and the sensitivity and specificity of the ZEN mAbs were determined under the optimized icELISA conditions. ZEN-BSA was successfully synthesized at a conjugation ratio of 17.2:1 (ZEN: BSA). Three hybridoma cell lines, 2D7, 3C2, and 4A10, were filtered, and their mAbs corresponded to an IgG1 isotype with a κ light chain. The mAbs titers were between (2.56 to 5.12) × 102 in supernatants and (1.28 to 5.12) × 105 in the ascites. Besides, the 50% inhibitive concentration (IC50) values were from 18.65 to 31.92 µg/L in the supernatants and 18.12 to 31.46 µg/L in the ascites. The affinity constant (Ka) of all of the mAbs was between 4.15 × 109 and 6.54 × 109 L/mol. The IC50 values of mAb 2D7 for ZEN, α-ZEL, ß-ZEL, α-ZAL, ß-ZAL and ZAN were 17.23, 16.71, 18.27, 16.39, 20.36 and 15.01 µg/L, and their cross-reactivities (CRs, %) were 100%, 103.11%, 94.31%, 105.13%, 84.63%, and 114.79%, respectively, under the optimized icELISA conditions. The limit of detection (LOD) for ZEN was 0.64 µg/L, and its linear working range was between 1.03 and 288.55 µg/L. The mAbs preparation and the optimization of icELISA conditions promote the potential development of a rapid test ELISA kit, providing an alternative method for detecting ZEN and its homologs in cereals and feed.

Development of a multi-channel magnetic bead micro-probe assay for high-throughput detection of zearalenone in edible and medicinal Coix seed.

A multi-channel magnetic bead micro-probes assay (MBPA) based on indirect competitive principle was developed for high-throughput detection of zearalenone (ZEA) in edible and medicinal Coix seed. This strategy introduced magnetic beads as the carriers, the specific primary antibodies as the capture probes for targets and the secondary antibodies functionalized goat anti-mouse immunoglobulin G labeled fluorescein isothiocyanate as the fluorescence signal probes. Through the competitive reaction of ZEA in Coix seed samples and that covalently coupled on the surface of MBs with their specific antibodies, as well as fast magnetic separation and sensitive fluorescence detection, the developed MBPA strategy allowed low limit of detection (2.03 ng/mL) with broad dynamic range (2.03-440.67 ng/mL), as well as excellent accuracy with the average recovery rate of 96.39% and relative standard deviation (RSD) of 5.48% for ZEA. 36 samples could realize simultaneous analysis in one operation within less than 20 min only needing 50 µL of solution and 30 s of sampling, avoiding large consumption of time and organic solvents. Multiple centrifugation and cleanup steps were omitted because of magnetic separation, avoiding the loss of targets. Diverse capture and fluorescent probes can be randomly bound onto the surface of MBs, making the MBPA strategy a promising tool for on-site high-throughput monitoring of various trace hazard factors in food safety, and environmental monitoring.

An Optical Planar Waveguide-Based Immunosensors for Determination of Fusarium Mycotoxin Zearalenone.

A planar waveguide (PW) immunosensor working as a polarisation interferometer was developed for the detection of mycotoxin zearalenone (ZON). The main element of the sensor is an optical waveguide consisting of a thin silicon nitride layer between two thicker silicon dioxide layers. A combination of a narrow waveguiding core made by photolithography with an advanced optical set-up providing a coupling of circular polarised light into the PW via its slanted edge allowed the realization of a novel sensing principle by detection of the phase shift between the p- and s-components of polarised light propagating through the PW. As the p-component is sensitive to refractive index changes at the waveguide interface, molecular events between the sensor surface and the contacting sample solution can be detected. To detect ZON concentrations in the sample solution, ZON-specific antibodies were immobilised on the waveguide via an electrostatically deposited polyelectrolyte layer, and protein A was adsorbed on it. Refractive index changes on the surface due to the binding of ZON molecules to the anchored antibodies were detected in a concentration-dependent manner up to 1000 ng/mL of ZON, allowing a limit of detection of 0.01 ng/mL. Structurally unrelated mycotoxins such as aflatoxin B1 or ochratoxin A did not exert observable cross-reactivity.

Dual fluorescent immunochromatographic assay for simultaneous quantitative detection of citrinin and zearalenone in corn samples.

The presence of multiple mycotoxins in the agricultural products poses a serious threat to the health of humans and animals. Citrinin (CIT) causes slow growth in animals and damages the kidney function. Zearalenone (ZEN) causes chronic poisoning, abnormal functioning and even death in animals. Herein, a dual fluorescent immunochromatographic assay (DF-ICA) based on europium nanoparticles (EuNPs) was developed for the simultaneous detection of CIT and ZEN in the corn samples. After optimization, the limits of detection (LODs), IC50 and average recoveries for the simultaneous determination of CIT and ZEN were 0.06 and 0.11 ng/mL, 0.35 and 0.76 ng/mL, from 86.3% to 111.6% and from 86.6% to 114.4%, respectively. Moreover, the DF-ICA was validated by high performance liquid chromatography (HPLC) analyses, and a satisfactory consistency was obtained. In brief, this work demonstrates the feasibility of DF-ICA for simultaneous monitoring of CIT and ZEN in the corn samples.

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.

Analysis of multiple mycotoxins-contaminated wheat by a smart analysis platform.

This study describes a smart analysis platform capable of quantitative measurements using a multiplex lateral flow strip. Using the multi-mycotoxin strip, five fungal toxins were simultaneously and quantitatively detected in naturally contaminated wheat. First, a matrix-based standard curve was established for the detection of aflatoxin B1 (AFB1), fumonisin B1 (FB1), T-2, deoxynivalenol (DON), and zearalenone (ZEN). Established on an open android system, the platform is able to read 6 lines on the strip simultaneously. The platform is equipped with a Quick Response code scanning model, which reads the established standard curves, and then rapidly quantify mycotoxins in naturally contaminated wheat. All the data and sample information are stored on a central server through the platform which is linked to the cloud. The limits of detection (LOD) for AFB1, FB1, T-2, DON, and ZEN in wheat were 4, 20, 10, 200, and 40 µg/kg and the visual cut off values was 20, 1000, 200, 4000, and 400 µg/kg, separately. To validate the platform and the multi-mycotoxin detection method, 10 wheat samples were analyzed and the results were in a good agreement with those obtained by LC-MS/MS. The platform will be a powerful tool for crop monitoring services.

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.

Preparation monoclonal ß-type anti-idiotype antibody of zearalenone and development of green ELISA quantitative detecting technique.

Immunoassay has been widely used in the screening of mycotoxins, which may be hazardous to the operator or the environment. This study was to develop a green way to measure zearalenone (ZEN) with a monoclonal ß-type anti-idiotype antibody (Ab2ß) against ZEN in place of ZEN standard. Six monoclonal ß-type anti-idiotype antibodies were prepared. The 50% inhibitory concentration (IC50) value to ZEN of the six antibodies was between 34.45 ± 1.12-182.12 ± 15.40 nM. A green ELISA was then developed and validated. The quantitative conversion formula between ZEN and the monoclonal Ab2ß against ZEN was y = 0.092x0.722, R2 = 0.990. The working range was 2.63-100.64 ng ml-1. The recovery rate in spiked feed samples was from 82.15% to 102.79%, and the within-assay and between-assay coefficient variation (CV) level were less than 10.00%. A good correlation was obtained by high-performance liquid chromatography method (HPLC) to validate the developed method.

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.

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.

Anti-idiotypic nanobody-phage display-mediated real-time immuno-PCR for sensitive, simultaneous and quantitative detection of total aflatoxins and zearalenone in grains.

An anti-idiotypic nanobody-phage display-mediated immuno-polymerase chain reaction (PD-IPCR) method was developed for simultaneous quantitative detection of total aflatoxins and zearalenone in cereals. Two phages, displaying the variable domain of the heavy chain anti-idiotypic nanobody that binds aflatoxin- or zearalenone-specific monoclonal antibody (1C11 or 2D3), were used as competitors for corresponding analytes. Specific DNA sequences encoding anti-idiotypic nanobodies were used to design the primers for PCR amplification. The results indicated that detection limits for total aflatoxins and zearalenone in a sample were 0.03 and 0.09 ng mL-1, respectively. Recoveries of spiked aflatoxins and zearalenone were 80-118% and 76.7-111%, respectively. Validation results were in good agreement with the gold-standard high-performance liquid chromatography method. This report is the first to describe PD-IPCR for simultaneous quantitative detection of total aflatoxins and zearalenone in cereals.

Generation of human and rabbit recombinant antibodies for the detection of Zearalenone by phage display antibody technology.

This article reports the identification, engineering and characterisation of recombinant single chain variable fragment (scFv) antibody against Zearalenone (ZEN), an oestrogenic mycotoxin, using phage display antibody technology. To increase the chance of obtaining clones that can bind to free toxin, the conjugated proteins of the target antigen, i.e. bovine serum albumin ZEN-BSA and ovalbumin ZEN-OVA, were switched during the biopanning. One phage-displayed scFv clone specific to free ZEN, designated yZEN2A8, could be isolated. The gene encoding the yZEN2A8 scFv was sub-cloned into the pET-21d (+) and pKP300 delta III vectors to generate the recombinant scFv and scFv-AP antibody formats, respectively. After ELISA optimisation by checkerboard titration, the sensitivities of the recombinant yZEN2A8 scFv antibody and scFv-AP fusion were improved approx. 2 and 60 folds, respectively. Competitive ELISA indicated that the median inhibition concentration (IC50) of recombinant yZEN2A8 scFv antibody and scFv-AP fusion after ELISA optimisation were 90 and 14 ng mL-1, with a limit of detection (LOD) of 20 and 2 ng mL-1, respectively. No cross-reactivity to other common mycotoxins was observed. Homology modelling illustrated specific binding of the recombinant antibody to ZEN and demonstrated the role of complementary determining regions (CDRs) of both the variable heavy and light chains in antibody-antigen interactions. Efficient application of scFv-AP for the detection of ZEN contamination in corns and wheat samples were investigated for the first time. The antibody in the form of scFv-AP can be used as a prototype for the development of a convenient reagent for the detection of ZEN contamination in various format, including biosensor-based.

Three kinds of lateral flow immunochromatographic assays based on the use of nanoparticle labels for fluorometric determination of zearalenone.

Colloidal gold, quantum dots and polystyrene microspheres were used as labels in three kinds of lateral flow immunochromatographic assays (ICAs) for the detection of zearalenone (ZEN) in cereal samples. The assays allow ZEN to be quantified within 20 min. The LODs are 10 µg·L-1 of ZEN for the colloidal gold-based ICA, and 1 µg·L-1 for both the quantum dot and polystyrene microsphere based ICAs. The respective data are 60 µg·kg-1, 6 µg·kg-1 and 6 µg·kg-1, respectively, for spiked samples and cereals. Only minor cross-sensitivity occurred between ZEN and fusarium toxins, and no cross-sensitivity if found for aflatoxin B1, T-2 mycotoxin, ochratoxin A, deoxynivalenol, and fumonisin B1. LODs of the three assays are lower than the maximum limits of ZEN set by most standardization agencies. Graphical abstract Schematic presentation of three lateral flow immunochromatographic assays (ICAs) based on the use of (a) colloidal gold (CG), (b) fluorescent quantum dots (QD), and

An On-Site Simultaneous Semi-quantification of Aflatoxin B1, Zearalenone, and T-2 Toxin in Maize- and Cereal-based Feed via Multicolor Immunochromatographic Assay.

Multiple-mycotoxin contamination has been frequently found in the agro-food monitoring due to the coexistence of fungi. However, many determination methods focused on a single mycotoxin, highlighting the demand for on-site determination of multiple mycotoxins in a single run. We develop a multicolor-based immunochromatographic strip (ICS) for simultaneous determination of aflatoxin B1 (AFB1), zearalenone (ZEN) and T-2 toxin in maize- and cereal-based animal feeds. The nanoparticles with different colors are conjugated with three monoclonal antibodies, which serve as the immunoassay probes. The decrease in color intensity is observed by the naked eyes, providing simultaneous quantification of three mycotoxins. The visible limits of detection for AFB1, ZEN and T-2 are estimated to be 0.5, 2, and 30 ng/mL, respectively. The cut-off values are 1, 10, and 50 ng/mL, respectively. Considerable specificity and stability are found using real samples. The results are in excellent agreement with those from high-performance liquid chromatography/tandem mass spectrometry. The multi-color ICS boasts sensitive and rapid visual differentiation and simultaneous semi-quantification of aflatoxin B1, zearalenone and T-2 toxin in maize- and cereal-based feed samples within 20 min.

Preparation of a broad-spectrum anti-zearalenone and its primary analogues antibody and its application in an indirect competitive enzyme-linked immunosorbent assay.

A broad-spectrum monoclonal antibody (mAb)-based indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) has been developed for rapidly screening zearalenone (ZEN) and its primary analogues in various samples using an easy sample preparation procedure. Primarily, a group-specific mAb, 6C2, was produced, which had IC50 values for ZEN, α-zearalenol, ß-zearalenol, α-zearalanol, ß-zearalanol and zearalanone of 114.0, 127.4, 290.4, 114.9, 205.6 and 257.1 ng L-1, respectively. The limit of detection and limit of quantitation of this method for ZEN and its five primary analogues in various matrix samples ranged from 114.2 to 812.3 ng L-1 and 237.1 to 1653.9 ng L-1, respectively. The recoveries of the above samples spiked with ZEN and its five primary analogues were in the range of 62.9-113.6%. The CVs were less than 13.2%. A good correlation (R2 = 0.995) between the ic-ELISA results and the HPLC-MS/MS results for swine feeds supported the reliability of the developed ic-ELISA.

Development of a nanoarray capable of the rapid and simultaneous detection of zearalenone, T2-toxin and fumonisin.

Fusarium mycotoxins such as trichothecenes, zearalenone and fumonisins occur on a worldwide basis in cereal grains, animal feeds and forages. Practical solutions for multiple mycotoxin determination in samples are required by industry and regulators for cost effective screening purposes. The feasibility of developing a novel multiplex nanoarray for the simultaneous and semi-quantitative detection of three regulated mycotoxins: zearalenone (ZEA), T2-toxin (T2) and fumonisin B1 (FUM) was examined. Additionally, the assay was also able to detect HT2 toxin and fumonisin B2 and B3 due to the cross reactivity profiles of the antibodies used. Individual mycotoxin conjugates specific to the three mycotoxins were nano-spotted onto wells of a microtitre plate. Optimisation of assay parameters and antibodies was undertaken with both individual and multiplex calibration curves generated. A competitive assay format was employed enabling a calibration curve for concentration analysis and duplicate results for up to 40 samples in 70min for the three target mycotoxins. The characteristics and performance of the nanoarray were evaluated including sensitivity and specificity for each target. Additionally, intra and inter spotting precision, cross reactivity, matrix effects and sample analysis in maize and wheat (n=8) was performed. Sensitivity, determined as the concentration causing 50% inhibition, was 70.1, 2.8 and 90.9ppb in PBS, 172.4, 3.2 and 129.3ppb in methanol, 197.4, 0.7 and 216.7ppb in wheat and 43.6, 0.5 and 25.9ppb in maize for ZEA, T2 and FUM respectively. Intra spotting precision was 6%, 11% and 10% for PBS and 5%, 11% and 12% for methanol for ZEA, T2 and FUM respectively. Inter spotting precision was 4%, 14% and 6% for PBS and 3%, 9% and 16% for methanol for ZEA, T2 and FUM respectively. The feasibility of the nanoarray as an easy to use sensitive screening tool in the 96 well format has been demonstrated for the multiplex detection of three regulated mycotoxins. Improvements in automated image and data analysis software for novice end users are required to improve the overall rapidity of analysis.

Novel fluorescent ELISA for the sensitive detection of zearalenone based on H2O2-sensitive quantum dots for signal transduction.

A direct competitive fluorescent enzyme-linked immunosorbent assay (ELISA) was developed for the detection of zearalenone (ZEN) using ZEN labeled catalase (CAT) as a competing antigen with H2O2-sensitive CdTe quantum dots (QDs) for signal transduction. The novel fluorescent ELISA showed very high sensitivity for ZEN detection because it combined the high catalytic activity of CAT to H2O2 and H2O2-sensitive property of QDs. Under optimal conditions, the developed method showed a good dynamic linear detection for ZEN in the range of 2.4pg/mL to 1.25ng/mL with a detection limit of 4.1pg/mL. The median inhibition concentration (IC50) of ZEN was 75pg/mL, which was approximately 17-fold lower than that of horseradish peroxidase-based conventional ELISA. Moreover, our developed method also showed a high reproducibility and an excellent selectivity. In brief, the novel fluorescent ELISA shows great potential for the sensitive and economic detection of mycotoxins and other analytes in food analysis, clinical diagnosis and environmental monitoring.

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

Immunoassay is frequently used to analyze mycotoxin contamination. However, the introduction of mycotoxins or their conjugates in conventional immunoassay threatens the safety of individuals and the environment. The variable domain of heavy-chain antibodies (VHHs) can be used as alternative compounds to produce anti-idiotypic antibodies, which work as non-toxic surrogate reagents in immunoassay. In this work, anti-zearalenone (ZEN) monoclonal antibody (mAb) was used as the target for biopanning anti-idiotypic VHH from a naïve alpaca VHH phage display library. After four panning cycles, one anti-idiotypic VHH phage clone (Z1) was isolated and the Z1 based phage ELISA for ZEN showed a half inhibitory concentration (IC50) of 0.25±0.02ng/mL, a linear range of 0.11-0.55ng/mL, and a limit of detection (LOD) of 0.08ng/mL. Furthermore, the phage particles of Z1 were also applied to immuno-polymerase chain reaction (PD-IPCR), which supplied both the detection antigens and deoxyribonucleic acid (DNA) templates. Compared with that of phage ELISA, the LOD of Z1 based PD-IPCR was 12-fold improved, with a detection limit of 6.5pg/mL and a linear range of 0.01-100ng/mL. The proposed method was then validated with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Results showed the reliability of PD-IPCR for the determination of ZEN in cereal samples. The use of anti-idiotypic VHH phage as non-toxic surrogate and signal-amplification function of PCR make it a promising method for actual ZEN analysis in cereals.