Xiaoxia Ding

Multi-component immunochromatographic assay for simultaneous detection of aflatoxin B1, ochratoxin A and zearalenone in agro-food

Mycotoxins are highly toxic contaminants and have induced health threat to human and animals. Aflatoxin B1 (AFB1), ochratoxin A (OTA) and zearalenone (ZEA) commonly occur in food and feed. A multi-component immunochromatographic assay (ICA) was developed for rapid and simultaneous determination of these three mycotoxins in agro-food. The strategy was performed based on the competitive immunoreactions between antibody-colloidal gold nanoparticle conjugate probes and mycotoxins or mycotixin antigens. Each monoclonal antibody specially recognize its corresponding mycotoxin and antigen, and there was no cross reactivity in the assay. Three mycotixin antigens were immobilized as three test lines in the nitrocellulose membrane reaction zone, which enable the simultaneous detection in one single test. The visible ICA results were obtained in 20 min. The visual detection limits of this strip test for the AFB1, OTA and ZEA were 0.25 ng/mL, 0.5 ng/mL and 1 ng/mL, respectively. The assay was evaluated using spiked and naturally contaminated peanuts, maize and rice samples. The results were in accordance with those obtained using enzyme-linked immunosorbent assay. In summary, this developed ICA could provide an effective and rapid approach for onsite detection of multi-mycotoxin in agro-food samples without any expensive instrument.

Production of ultrasensitive generic monoclonal antibodies against major aflatoxins using a modified two-step screening procedure.

Monoclonal antibodies (McAbs) cross-reactive with four major aflatoxins were achieved using a modified two-step screening procedure. The first step was twice modified indirect enzyme-linked immunosorbent assay (ELISA) and resulted in positive hybridomas and hapten-specific antibodies. The modified indirect competitive ELISA (ciELISA) was the second step, in which the competition incubation time was decreased to 30min, aflatoxin B(1), B(2), G(1) and G(2) were all used as competitors, the concentrations of four aflatoxins were gradiently decreased in each screening. 2-3 subclonings were performed after every modified fusion and resulted in eight hybridomas that secreted antibodies with good cross-reactivity and high affinity to four aflatoxins. Five McAbs were chosen for further analysis. Of the five, two antibodies had similar reaction efficiency with aflatoxin B(1), B(2) and G(1) but showed a weak cross-reaction to G(2). Another two had almost identical reaction capability with four aflatoxins. One clone 1C11 exhibited the highest sensitivity for all four aflatoxins. The concentrations of aflatoxin B(1), B(2), G(1) and G(2) at 50% inhibition for 1C11 were 1.2, 1.3, 2.2 and 18.0pgmL(-1) respectively. This is the most sensitive for all four major aflatoxins described so far. The results indicated that the modified two-step screening procedure had superiority and these antibodies could be used for simultaneous analysis of total aflatoxins.

A high selective immunochromatographic assay for rapid detection of aflatoxin B1

To solve the problem of low selectivity of current immunochromatographic assay (ICA) for aflatoxin B(1) (AFB(1)) alone detection, a novel selective ICA was developed here. With very high selectivity, a new AFB(1) monoclonal antibody (MAb) 3G1 was prepared by immunizing Balb/c mice with aflatoxin B(2a)-BSA (AFB(2a)-BSA) rather than AFB(1)-BSA used in other reports and 3G1 possessed the highest selectivity than those used in published ICAs. The ICA with visual detection limit (VDL) of 1 ng mL(-1) showed no cross-reactivity with other aflatoxins. Comparing with previous reports, the ICA here provided the most powerful guarantee for avoiding false positive results leaded by coexistence of other aflatoxins in samples. For validation, naturally contaminated samples including peanut, puer-tea, vegetable oil and feedstuff were respectively assayed by ICA and a standard high performance liquid chromatography (HPLC), and good agreement of results was obtained between two methods. Therefore, the developed ICA could well meet the selective detection of AFB(1) in agro-products.

Nanobody-based enzyme immunoassay for aflatoxin in agro-products with high tolerance to cosolvent methanol.

A phage-displayed library of variable domain of heavy chain of the heavy chain antibody (VHH) or nanobody (Nb) was constructed after immunizing an alpaca with aflatoxin B1 (AFB1) conjugated with bovine serum albumin (AFB1-BSA). Two AFB1-specific nanobodies were selected. The obtained nanobodies were compared to an aflatoxin-specific monoclonal antibody B5 with respect to stability under organic solvents and high temperature. The two nanobodies could bind antigen specifically after exposure to temperatures as high as 95 °C. Besides, the nanobodies showed better or similar tolerance to organic solvents. A competitive ELISA with nanobody Nb26 was developed for the analysis of AFB1, exhibiting an IC50 value of 0.754 ng/mL (2.4 μM), linear range from 0.117 to 5.676 ng/mL. Due to the high tolerance to methanol, sample extracts were analyzed by nanobody-based ELISA without dilution. The recovery from spiked peanut, rice, corn and feedstuff ranged from 80 to 115%. In conclusion, the isolated nanobodies are excellent candidates for immunoassay application in aflatoxin determination.

Monoclonal antibody-quantum dots CdTe conjugate-based fluoroimmunoassay for the determination of aflatoxin B1 in peanuts

A fluoroimmunoassay towards aflatoxin B1 (AFB1) was presented using quantum dots as the fluorescent label. The CdTe QDs were successfully linked to the monoclonal antibody against AFB1. Based on the conjugated complexes, a novel direct competitive fluorescence-linked immunosorbent assay (cFLISA) was developed for AFB1 detection. The 50% inhibition value (IC50) of the cFLISA was 0.149ng/mL in peanuts matrix. The method performance included the limit of detection (LOD) of 0.016ng/mL and considerable recoveries of 85-117% at three fortification levels (0.075, 0.15, and 0.3ng/g) from spiked AFB1 blank peanuts samples, along with coefficients of variation (CVs) below 10%. The cFLISA provided an alternative of rapid and sensitive detection for AFB1 and, moreover provided great potential for multiplexed mycotoxins determination simultaneously.

Effect of ozone on aflatoxins detoxification and nutritional quality of peanuts

Aflatoxins are a group of secondary metabolites produced by Aspergillus flavus and Aspergillus parasiticus with carcinogenicity, teratogenicity, and mutagenicity. Aflatoxins may be found in a wide range of agri-products, especially in grains, oilseeds, corns, and peanuts. In this study, the conditions for detoxifying peanuts by ozonation were optimised. Aflatoxins in peanuts at moisture content of 5% (w/w) were sensitive to ozone and easily degraded when reacted with 6.0mg/l of ozone for 30min at room temperature. The detoxification rates of the total aflatoxins and aflatoxin B1 (AFB1) were 65.8% and 65.9%, respectively. The quality of peanut samples was also evaluated in this research. No significant differences (P>0.05) were found in the polyphenols, resveratrol, acid value (AV), and peroxide value (PV) between treated and untreated samples. The results suggested that ozonation was a promising method for aflatoxin detoxification in peanuts.

Classification and Adulteration Detection of Vegetable Oils Based on Fatty Acid Profiles

The detection of adulteration of high priced oils is a particular concern in food quality and safety. Therefore, it is necessary to develop authenticity detection method for protecting the health of customers. In this study, fatty acid profiles of five edible oils were established by gas chromatography coupled with mass spectrometry (GC/MS) in selected ion monitoring mode. Using mass spectral characteristics of selected ions and equivalent chain length (ECL), 28 fatty acids were identified and employed to classify five kinds of edible oils by using unsupervised (principal component analysis and hierarchical clustering analysis), supervised (random forests) multivariate statistical methods. The results indicated that fatty acid profiles of these edible oils could classify five kinds of edible vegetable oils into five groups and are therefore employed to authenticity assessment. Moreover, adulterated oils were simulated by Monte Carlo method to establish simultaneous adulteration detection model for five kinds of edible oils by random forests. As a result, this model could identify five kinds of edible oils and sensitively detect adulteration of edible oil with other vegetable oils about the level of 10%.

Molecular characterization of monoclonal antibodies against aflatoxins: a possible explanation for the highest sensitivity.

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.

Graphene oxide: an adsorbent for the extraction and quantification of aflatoxins in peanuts by high-performance liquid chromatography.

In this paper, graphene oxide (GO) was synthesized and specifically selected by centrifugation to extract four aflatoxins (B1, B2, G1, and G2) as an effective adsorbent. Then, the amount of aflatoxins was quantitatively measured by high-performance liquid chromatography (HPLC). The GO was characterized by X-ray diffraction (XRD), atomic force microscopy (AFM), and ultraviolet (UV) spectrophotometer. Several parameters that could affect the extraction efficiency, including the GO amount, methanol concentration in the extraction solvent, spiked amount, extraction time, and elution cycle, were also investigated and optimized in this work. Under optimal conditions, good linear relationships were achieved with the correlation coefficient (r) ranging from 0.99217 to 0.99995. The detection limit of this method for the four aflatoxins ranged from 0.08 to 0.65ng/g. Finally, the proposed method has been successfully applied to determine aflatoxins in peanut samples. The results show that the recoveries of the four aflatoxins range from 85.1% to 100.8% with the relative standard deviations between 2.1% and 7.9%.

Current development of microfluidic immunosensing approaches for mycotoxin detection via capillary electromigration and lateral flow technology

Mycotoxin contamination in the food chain has caused serious health issues in humans and animals. Thus, a rapid on‐site and lab‐independent detection method for mycotoxins, such as aflatoxins (AFTs), is desirable. Microfluidic chip based immunosensor technology is one of the most promising methods for fast mycotoxin assays. In this review, we cover the major microfluidic immunosensors used for mycotoxin analysis, via flow‐through (capillary electromigration) and lateral flow technology. Sample preparation from different matrices of agricultural products and foodstuffs is summarized. The choice of materials, fabrication strategies, and detection methods for microfluidic immunosensors are further discussed in detail. The sensors application in mycotoxin determination is also outlined. Finally, future challenges and opportunities are discussed.