One-pot green synthesis of ZIF-8/IgG composite for the precise orientation and protection of antibody and its application in purification and detection of aflatoxins in peanut oil.

This study presents a novel approach toward the one-pot green synthesis of ZIF-8/IgG composite, focusing on its precise orientation and protection of the anti-aflatoxins antibody. The antibody orientation is achieved through the specific binding of IgG to the Fc region of the antibody, while the antibody protection is accomplished by the structural change restriction of ZIF-8 framework to the antibody. Consequently, the antibody exhibits enhanced target capability and significantly improved tolerance to organic solvents. The ZIF-8/IgG/anti-AFT was employed for the purification and detection of AFTs by coupling with UPLC. Under optimized conditions, the recoveries of spiked AFTs in peanut oils are between 86.1% and 106.4%, with relative standard deviations (RSDs) ranging from 0.8% to 8.8%. The linearity range is 0.5-20.0 ng for AFB1 and AFG1, 0.125-5.0 ng for AFB2 and AFG2, the limit of detection is 0.1 ng for AFB1 and AFG1, 0.03 ng for AFB2 and AFG2.

Development of Anti-Idiotypic Nanobody-Phage Based Immuno-Loop-Mediated Isothermal Amplification Assay for Aflatoxins in Peanuts.

Aflatoxin contamination in agricultural products has posed serious health hazards and brought huge economic loss in the food and feed industries. Monitoring aflatoxins in various foods and feeds has become a crucial means to protect public health. This study aimed to report an immuno-loop-mediated isothermal amplification (iLAMP) assay by using an anti-idiotypic nanobody-phage for on-site and rapid detection of aflatoxin in real samples. The iLAMP method was developed on the basis of a competitive immunoassay and LAMP reaction performed in a simple water bath. This method can provide visualized test results: violet color represents positive samples while sky blue represents negative. The visual detection limits of iLAMP for aflatoxin B1, B2, G1, and G2 in peanut samples were 1.6, 1.6, 3.2, and 16 µg/kg, respectively. The developed assay was verified with high performance liquid chromatography (HPLC) for the analysis of aflatoxins in peanuts, which demonstrated that the iLAMP method can be applied to the detection of aflatoxin in real samples. The novel iLAMP assay eliminates the need for aflatoxin conjugates, the antibody labeling process, and special equipment, and offers an alternative to existing methods with advantages of time-saving, cost-effectiveness, and ease-of-use.

A Sensitive, Point-of-Care Detection of Small Molecules Based on a Portable Barometer: Aflatoxins In Agricultural Products.

Sensitive and point-of-care detection of small toxic molecules plays a key role in food safety. Aflatoxin, a typical small toxic molecule, can cause serious healthcare and economic issues, thereby promoting the development of sensitive and point-of-care detection. Although ELISA is one of the official detection methods, it cannot fill the gap between sensitivity and point-of-care application because it requires a large-scale microplate reader. To employ portable readers in food safety, Pt-catalysis has attracted increasing attention due to its portability and reliability. In this study, we developed a sensitive point-of-care aflatoxin detection (POCAD) method via a portable handheld barometer. We synthesized and characterized Au@PtNPs and Au@PtNPs conjugated with a second antibody (Au@PtNPs-IgG). A competitive immunoassay was established based on the homemade monoclonal antibody against aflatoxins. Au@PtNPs-IgG was used to catalyze the production of O2 from H2O2 in a sealed vessel. The pressure of O2 was then recorded by a handheld barometer. The aflatoxin concentration was inversely proportional to the pressure recorded via the barometer reading. After optimization, a limit of detection of 0.03 ng/mL and a linear range from 0.09 to 16.0 ng/mL were achieved. Recovery was recorded as 83.1%-112.0% along with satisfactory results regarding inner- and inter-assay precision (relative standard deviation, RSD < 6.4%). Little cross-reaction was observed. Additionally, the POCAD was validated by high-performance liquid chromatography (HPLC) by using peanut and corn samples. The portable POCAD exhibits strong potential for applications in the on-site detection of small toxic molecules to ensure food safety.

An Automated and High-Throughput Immunoaffinity Magnetic Bead-Based Sample Clean-Up Platform for the Determination of Aflatoxins in Grains and Oils Using UPLC-FLD.

Sample clean-up remains the most time-consuming and error-prone step in the whole analytical procedure for aflatoxins (AFTs) analysis. Herein, an automated and high-throughput sample clean-up platform was developed with a disposable, cost-effective immunoaffinity magnetic bead-based kit. Under optimized conditions, the automated method takes less than 30 min to simultaneously purify 20 samples without requiring any centrifugation or filtering steps. When coupled to ultra-high performance liquid chromatography with fluorescence detection, this new analysis method displays excellent accuracy and precision as well as outstanding efficiency. Furthermore, an interlaboratory study was performed in six laboratories to validate the novel protocol. Mean recovery, repeatability, reproducibility, and Horwitz ratio values were within 91.9%-107.4%, 2.5%-7.4%, 2.7%-10.6%, and 0.26%-0.90, respectively. Results demonstrate that the developed sample clean-up platform is a reliable alternative to most widely adopted clean-up procedures for AFTs in cereals and oils.

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.

The effects of aflatoxin exposure on Hepatitis B-vaccine induced immunity in Kenyan children.

BACKGROUND: Globally, approximately three million children die each year from vaccine preventable infectious diseases mainly in developing countries. Despite the success of the expanded immunization program, not all infants and children around the world develop the same protective immune response to the same vaccine. A vaccine must induce a response over the basal immune response that may be driven by population-specific, environmental or socio-economic factors. Mycotoxins like aflatoxins are immune suppressants that are confirmed to interfere with both cell-mediated and acquired immunity. The mechanism of aflatoxin toxicity is through the binding of the bio-activated AFB1-8, 9-epoxide to cellular macromolecules. METHODS: We studied Hepatitis B surface antibodies [anti-HBs] levels to explore the immune modulation effects of dietary exposure to aflatoxins in children aged between one and fourteen years in Kenya. Hepatitis B vaccine was introduced for routine administration for Kenyan infants in November 2001. To assess the effects of aflatoxin on immunogenicity of childhood vaccines Aflatoxin B1-lysine in blood serum samples were determined using High Performance Liquid Chromatography with Fluorescence detection while anti-HBs were measured using Bio-ELISA anti-HBs kit. RESULTS: The mean ± SD of AFB1-lysine adducts in our study population was 45.38 ± 87.03 pg/mg of albumin while the geometric mean was 20.40 pg/mg. The distribution of AFB1-lysine adducts was skewed to the right. Only 98/205 (47.8%) of the study population tested positive for Hepatitis B surface antibodies. From regression analysis, we noted that for every unit rise in serum aflatoxin level, anti-HBs dropped by 0.91 mIU/ml (-0.9110038; 95% C.I -1.604948, -0.21706). CONCLUSION: Despite high coverage of routine immunization, less than half of the study population had developed immunity to HepB. Exposure to aflatoxin was high and weakly associated with low anti-HBs antibodies. These findings highlight a potentially significant role for environmental factors that may contribute to vaccine effectiveness warranting further research.

Effect of dietary supplementation of mannanoligosaccharides on hepatic gene expressions and humoral and cellular immune responses in aflatoxin-contaminated broiler chicks.

The present study was conducted to investigate the effects of dietary supplementation of mannanoligosaccharides (MOS) on expression of hepatic immunological genes and immune responses in aflatoxin-contaminated broiler chicks. A total of 336 seven-day-old Ross broiler chicks were randomly allotted to 7 experimental treatments with 4 replicates and 12 birds per replicate. Experimental treatments consisted of 2 aflatoxin levels (0.5 and 2 ppm) and 3 supplemental MOS levels (0, 1 and 2 g/kg) as a 2 × 3 factorial arrangement in comparison with a control group (unchallenged group). The chicks were challenged with a mix of aflatoxins during 7-28 d of age. Results showed that aflatoxin challenge resulted in the lower antibody titers against infectious bronchitis (IBV) and bursal (IBD) diseases viruses. In addition, aflatoxin-contaminated birds had a lower (P < 0.0001) lymphocyte percentage and a decline in (P < 0.01) interleukin-2 (IL-2) mRNA abundance. Likewise, heterophil proportion, heterophil to lymphocyte ratio and gene expressions of hepatic interleukin-6 (IL-6) and C reactive protein (CRP) were raised (P < 0.001) by increasing dietary aflatoxin level. Dietary inclusion of MOS increased (P < 0.05) antibody titers against IBV, IBD and Newcastle disease virus. Lymphocyte proportion and hepatic IL-2 gene expression were greater (P < 0.0001) in MOS-supplemented birds. Furthermore, supplemental MOS decreased hepatic IL-6 and CRP abundances. Additionally, inclusion of 2 g/kg MOS resulted in the upregulation (P < 0.01) of hepatic IL-2 gene expression in birds contaminated with 0.5 ppm aflatoxin. The present results indicate that supplemental MOS could improve cellular immunity via the upregulation of hepatic IL-2 gene expression in birds challenged with aflatoxins.

Aflatoxin-specific monoclonal antibody selection for immunoaffinity column development.

Antibodies are the basic components of immunoanalytical systems used for detection of a wide range of analytes. Although there are some ground rules for antibody selection, analyte- and assay-specific criteria are the ones that determine the ultimate success of the immunoassays. In this study, we introduced an effective antibody selection procedure for the development of immunoaffinity columns for aflatoxins. The designed scheme puts emphasis on solvent- and matrix-related characterization steps and was used to comparatively evaluate eight monoclonal antibodies. The selected antibody was tolerant to 40% methanol, 20% acetonitrile, 30% acetone and 40% ethanol and did not interact with corn, red pepper or hazelnut extracts. Immunoaffinity columns developed with the selected antibody were validated by 15 independent aflatoxin analysis laboratories.

Combining E-Nose and Lateral Flow Immunoassays (LFIAs) for Rapid Occurrence/Co-Occurrence Aflatoxin and Fumonisin Detection in Maize.

The aim of this study was to evaluate the potential use of an e-nose in combination with lateral flow immunoassays for rapid aflatoxin and fumonisin occurrence/co-occurrence detection in maize samples. For this purpose, 161 samples of corn have been used. Below the regulatory limits, single-contaminated, and co-contaminated samples were classified according to the detection ranges established for commercial lateral flow immunoassays (LFIAs) for mycotoxin determination. Correspondence between methods was evaluated by discriminant function analysis (DFA) procedures using IBM SPSS Statistics 22. Stepwise variable selection was done to select the e-nose sensors for classifying samples by DFA. The overall leave-out-one cross-validated percentage of samples correctly classified by the eight-variate DFA model for aflatoxin was 81%. The overall leave-out-one cross-validated percentage of samples correctly classified by the seven-variate DFA model for fumonisin was 85%. The overall leave-out-one cross-validated percentage of samples correctly classified by the nine-variate DFA model for the three classes of contamination (below the regulatory limits, single-contaminated, co-contaminated) was 65%. Therefore, even though an exhaustive evaluation will require a larger dataset to perform a validation procedure, an electronic nose (e-nose) seems to be a promising rapid/screening method to detect contamination by aflatoxin, fumonisin, or both in maize kernel stocks.

Feasibility of A Novel On-Site Detection Method for Aflatoxin in Maize Flour from Markets and Selected Households in Kampala, Uganda.

In sub-Saharan Africa, there is a high demand for affordable and accessible methods for on-site detection of aflatoxins for appropriate food safety management. In this study, we validated an electrochemical immunosensor device by the on-site detection of 60 maize flour samples from six markets and 72 samples from households in Kampala. The immunosensor was successfully validated with a linear range from 0.7 ± 0.1 to 11 ± 0.3 µg/kg and limit of detection (LOD) of 0.7 µg/kg. The maize flour samples from the markets had a mean total aflatoxin concentration of 7.6 ± 2.3 µg/kg with approximately 20% of the samples higher than 10 µg/kg, which is the maximum acceptable level in East Africa. Further down the distribution chain, at the household level, approximately 45% of the total number contained total aflatoxin levels higher than the acceptable limit. The on-site detection method correlated well with the established laboratory-based HPLC and ELISA-detection methods for aflatoxin B1 with the correlation coefficients of 0.94 and 0.98, respectively. This study shows the feasibility of a novel on-site detection method and articulates the severity of aflatoxin contamination in Uganda.

Design of Multifunctional Nanostructure for Ultrafast Extraction and Purification of Aflatoxins in Foodstuffs.

Aflatoxins (AFs) are a class of carcinogens, associated with liver cancers, that exist in foodstuffs. There are extremely low maximum limits of AFs in foodstuffs (0.025-20 µg·kg-1). Quick and sensitive detection of such low concentration of AFs in foodstuffs is dominated by the efficiency and selectivity of the AF enrichment process, which is extremely challenging although substantial efforts have been made in recent decades. Here we design and synthesize a multilayer nanoarchitecture composed of a broad-spectrum aflatoxin monoclonal antibody shell, chitosan middle layer, and magnetic bead core (denoted AF-mAb/CTS/Fe3O4). The efficiency of AF-mAb/CTS/Fe3O4 in extracting AFs has been found to be more than 60 times higher than both conventional immunoaffinity chromatography and solid-phase extraction. Furthermore, the nanocomposite displays excellent selectivity and good reusability as well as outstanding efficiency. When coupled to ultraperformance liquid chromatography-tandem quadrupole mass spectrometry, this new nanoarchitecture enables us to probe six AFs at concentrations as low as 0.003 µg·kg-1 in foodstuffs with free matrix effects, which is nearly 10 times smaller than the regulated maximum tolerated does. It is believed that the new nanoarchitecture will provide an efficient and fast pathway to detect AFs in foodstuffs to protect human being from some critical liver cancers.

An On-Site, Ultra-Sensitive, Quantitative Sensing Method for the Determination of Total Aflatoxin in Peanut and Rice Based on Quantum Dot Nanobeads Strip.

An on-site, ultra-sensitive, and quantitative sensing method was developed based on quantum dot nanobeads (QDNBs) and a test strip for the determination of total aflatoxins (AFTs) in rice and peanuts. The monoclonal antibody against AFT (mAbAFT) was homemade and labeled with QDNB. After the pre-coating of the AFT antigen on the test line (T line), the competitive immunoreactions were conducted between AFT and AFT antigen on the T line with QDNBs-mAbAFT. Under optimal conditions, this approach allowed a rapid response towards AFT with a considerable sensitivity of 1.4 pg/mL and 2.9 pg/mL in rice and peanut matrices, respectively. The put-in and put-out durations were within 10 min. The recoveries for AFT in rice and peanut sample matrices were recorded from 86.25% to 118.0%, with relative deviations (RSD) below 12%. The assay was further validated via the comparison between this QDNB strip and the conventional HPLC method using spiked samples. Thus, the design provided a potential alternative for on-site, ultra-sensitive, and quantitative sensing of AFT that could also be expanded to other chemical contaminants for food safety.

A highly specific competitive direct enzyme immunoassay for sterigmatocystin as a tool for rapid immunochemotaxonomic differentiation of mycotoxigenic Aspergillus species.

A simplified method to produce specific polyclonal rabbit antibodies against sterigmatocystin (STC) was established, using a STC-glycolic acid-ether derivative (STC-GE) conjugated to keyhole limpet haemocyanin (immunogen). The competitive direct enzyme immunoassay (EIA) established for STC had a detection limit (20% binding inhibition) of 130 pg ml-1 . The test was highly specific for STC, with minor cross-reactivity with O-methylsterigmatocystin (OMSTC, 0·87%) and negligible reactivity with aflatoxins (<0·02%). STC-EIA was used in combination with a previously developed specific EIA for aflatoxins (<0·1% cross-reactivity with STC and OMSTC), to study the STC/aflatoxin production profiles of reference strains of Aspergillus species. This immunochemotaxonomic procedure was found to be a convenient tool to identify STC- or aflatoxin-producing strains. SIGNIFICANCE AND IMPACT OF THE STUDY: The carcinogenic mycotoxin sterigmatocystin (STC) is produced by several Aspergillus species, either alone or together with aflatoxins. Here, we report a very simple and straightforward procedure to obtain highly sensitive and specific anti-STC antibodies, and their use in the first ever real STC-specific competitive direct enzyme immunoassay (EIA). In combination with a previous EIA for aflatoxins, this study for the first time demonstrates the potential of a STC/aflatoxin EIA pair for what is branded as 'immunochemotaxonomic' identification of mycotoxigenic Aspergillus species. This new analytical tool enhances analytical possibilities for differential analysis of STC and aflatoxins.

Monoclonal IgA Antibodies for Aflatoxin Immunoassays.

Antibody based techniques are widely used for the detection of aflatoxins which are potent toxins with a high rate of occurrence in many crops. We developed a murine monoclonal antibody of immunoglobulin A (IgA) isotype with a strong binding affinity to aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1 (AFG1), aflatoxin G2 (AFG2) and aflatoxin M1 (AFM1). The antibody was effectively used in immunoaffinity column (IAC) and ELISA kit development. The performance of the IACs was compatible with AOAC performance standards for affinity columns (Test Method: AOAC 991.31). The total binding capacity of the IACs containing our antibody was 111 ng, 70 ng, 114 ng and 73 ng for AFB1, AFB2, and AFG1 andAFG2, respectively. Furthermore, the recovery rates of 5 ng of each AF derivative loaded to the IACs were determined as 104.9%, 82.4%, 85.5% and 70.7% for AFB1, AFB2, AFG1 and AFG2, respectively. As for the ELISA kit developed using non-oriented, purified IgA antibody, we observed a detection range of 2-50 µg/L with 40 min total test time. The monoclonal antibody developed in this research is hitherto the first presentation of quadruple antigen binding IgA monoclonal antibodies in mycotoxin analysis and also the first study of their utilization in ELISA and IACs. IgA antibodies are valuable alternatives for immunoassay development, in terms of both sensitivity and ease of preparation, since they do not require any orientation effort.

A toxin-free enzyme-linked immunosorbent assay for the analysis of aflatoxins based on a VHH surrogate standard.

A toxin-free enzyme-linked immunosorbent assay (ELISA) for aflatoxins was developed using an anti-idiotype nanobody VHH 2-5 as surrogate standard. Anti-idiotype nanobody VHH 2-5 was generated by immunizing an alpaca with anti-aflatoxin monoclonal antibody 1C11. This assay was used to detect aflatoxins in agro-products after a simple extraction with 75 % methanol/H2O. Aflatoxin concentration was calculated by a two-step approach: the concentration of VHH 2-5 was first obtained by a four-parameter logistic regression from the detected absorbance value at 450 nm, and then converted to aflatoxin concentration by a linear equation. The assay exhibits a limit of detection (LOD) of 0.015 ng mL(-1), which is better than or comparable with conventional immunoassays. The performance of our VHH surrogate-based ELISA was further validated with a high-performance liquid chromatography (HPLC) method for total aflatoxins determination in 20 naturally contaminated peanut samples, displaying a good correlation (R (2) = 0.988). In conclusion, the proposed assay represents a first example applying an anti-idiotype VHH antibody as a standard surrogate in ELISA. With the advantages of high stability and ease of production, the VHH antibody-based standard surrogate can be extended in the future to immunoassays for other highly toxic compounds. Graphical Abstract ᅟ.

Development of an Enzyme-Linked Immunosorbent Assay Method Specific for the Detection of G-Group Aflatoxins.

To detect and monitor G-group aflatoxins in agricultural products, we generated class-specific monoclonal antibodies that specifically recognized aflatoxins G1 and G2. Of the final three positive and stable hybridomas obtained, clone 2G6 produced a monoclonal antibody that had equal sensitivity to aflatoxins G1 and G2, and did not cross-react with aflatoxins B1, B2, or M1. Its IC50 values for aflatoxins G1 and G2 were 17.18 ng·mL(-1) and 19.75 ng·mL(-1), respectively. Using this new monoclonal antibody, we developed a competitive indirect enzyme-linked immunosorbent assay (CI-ELISA); the method had a limit of detection of 0.06 ng·mL(-1). To validate this CI-ELISA, we spiked uncontaminated peanut samples with various amounts of aflatoxins G1 and G2 and compared recovery rates with those determined by a standard HPLC method. The recovery rates of the CI-ELISA ranging from 94% to 103% were comparable to those of the HPLC (92% to 102%). We also used both methods to determine the amounts of G-group aflatoxins in five peanut samples contaminated by aflatoxin B1-positive, and their relative standard deviations ranged from 8.4% to 17.7% (under 20%), which demonstrates a good correlation between the two methods. We further used this CI-ELISA to assess the ability of 126 fungal strains isolated from peanuts or field soils to produce G-group aflatoxins. Among these, seven stains producing different amounts of G-group aflatoxins were identified. Our results showed that the monoclonal antibody 2 G6-based CI-ELISA was suitable for the detection of G-group aflatoxins present in peanuts and also those produced by fungi.

Flow-based impedimetric immunosensor for aflatoxin analysis in milk products.

Label-free detection technique based on impedance was investigated for aflatoxin M1 (AFM1) and aflatoxin M2 (AFM2) analysis in milk products. The impedance change resulting from antigen-antibody interaction was studied using a two-electrode setup made up of silver (Ag) wire. Processed milk such as drinking yogurt and flavored milk samples were analyzed in a flow-based setup. Two microflow pumps were used to construct the flow system where analytes (AFM1 and AFM2) were injected and impedance was measured using functionalized Ag wire electrodes. The flow system was optimized by adjusting both inlet and outlet flows to maintain the reaction volume optimum for impedance measurements. Using Bode plot, the matrix effect was investigated for detection of AFM1 and AFM2 in various matrices. Good recoveries were obtained even at low-AFM1 concentrations in the range of 1-100 pg/mL. The influence of AFM2 on the detection of AFM1 was also investigated. The proposed method provides good scope for online monitoring of such hazardous toxins in milk products.