scholarly journals Eco-Friendly Fluorescent ELISA Based on Bifunctional Phage for Ultrasensitive Detection of Ochratoxin A in Corn

Foods ◽  
2021 ◽  
Vol 10 (10) ◽  
pp. 2429
Author(s):  
Weipeng Tong ◽  
Hao Fang ◽  
Hanpeng Xiong ◽  
Daixian Wei ◽  
Yuankui Leng ◽  
...  
Keyword(s):  
Ochratoxin A ◽  
Limit Of Detection ◽  
High Reliability ◽  
Ultra Performance Liquid Chromatography ◽  
Enzyme Linked Immunosorbent Assay ◽  
Enhanced Sensitivity ◽  
Great Specificity ◽  
Screening Platform ◽  
Mimic Peptide ◽  
Low Sensitivity

Conventional enzyme-linked immunosorbent assay (ELISA) is commonly used for Ochratoxin A (OTA) screening, but it is limited by low sensitivity and harmful competing antigens of enzyme-OTA conjugates. Herein, a bifunctional M13 bacteriophage with OTA mimotopes fused on the p3 protein and biotin modified on major p8 proteins was introduced as an eco-friendly competing antigen and enzyme container for enhanced sensitivity. Mercaptopropionic acid-modified quantum dots (MPA-QDs), which are extremely sensitive to hydrogen peroxide, were chosen as fluorescent signal transducers that could manifest glucose oxidase-induced fluorescence quenching in the presence of glucose. On these bases, a highly sensitive and eco-friendly fluorescent immunoassay for OTA sensing was developed. Under optimized conditions, the proposed method demonstrates a good linear detection of OTA from 4.8 to 625 pg/mL and a limit of detection (LOD) of 5.39 pg/mL. The LOD is approximately 26-fold lower than that of a conventional horse radish peroxidase (HRP) based ELISA and six-fold lower than that of a GOx-OTA conjugate-based fluorescent ELISA. The proposed method also shows great specificity and accepted accuracy for analyzing OTA in real corn samples. The detection results are highly consistent with those obtained using the ultra-performance liquid chromatography-fluorescence detection method, indicating the high reliability of the proposed method for OTA detection. In conclusion, the proposed method is an excellent OTA screening platform over a conventional ELISA and can be easily extended for sensing other analytes by altering specific mimic peptide sequences in phages.

scholarly journals Dramatically Enhancing the Sensitivity of Immunoassay for Ochratoxin A Detection by Cascade-Amplifying Enzyme Loading

Toxins ◽  
2021 ◽  
Vol 13 (11) ◽  
pp. 781
Author(s):  
Zhuolin Song ◽  
Lin Feng ◽  
Yuankui Leng ◽  
Mingzhu Huang ◽  
Hao Fang ◽  
...  
Keyword(s):  
Ochratoxin A ◽  
Limit Of Detection ◽  
High Sensitivity ◽  
Cross Reaction ◽  
Molar Ratio ◽  
Quantitative Detection ◽  
Enzyme Linked Immunosorbent Assay ◽  
Enzyme Loading ◽  
M13 Bacteriophage ◽  
Mimic Peptide

Enzyme-linked immunosorbent assay (ELISA) is widely used in the routine screening of mycotoxin contamination in various agricultural and food products. Herein, a cascade-amplifying system was introduced to dramatically promote the sensitivity of an immunoassay for ochratoxin A (OTA) detection. Specifically, a biotinylated M13 bacteriophage was introduced as a biofunctional competing antigen, in which a seven-peptide OTA mimotope fused on the p3 protein of M13 was used to specifically recognize an anti-OTA monoclonal antibody, and the biotin molecules modified on capsid p8 proteins were used in loading numerous streptavidin-labeled polymeric horseradish peroxidases (HRPs). Owing to the abundance of biotinylated p8 proteins in M13 and the high molar ratio between HRP and streptavidin in streptavidin-polyHRP, the loading amount of HRP enzymes on the M13 bacteriophage were greatly boosted. Hence, the proposed method exhibited high sensitivity, with a limit of detection of 2.0 pg/mL for OTA detection, which was 250-fold lower than that of conventional ELISA. In addition, the proposed method showed a slight cross-reaction of 2.3% to OTB, a negligible cross-reaction for other common mycotoxins, and an acceptable accuracy for OTA quantitative detection in real corn samples. The practicability of the method was further confirmed with a traditional HRP-based ELISA method. In conclusion, the biotinylated bacteriophage and polyHRP structure showed potential as a cascade-amplifying enzyme loading system for ultra-trace OTA detemination, and its application can be extended to the detection of other analytes by altering specific mimic peptide sequences.

scholarly journals Aflatoxigenic and ochratoxigenic moulds and their toxins in tree nuts on Serbian market

2020 ◽  
pp. 9-18
Author(s):  
Irena Rakic ◽  
Gordana Dimic ◽  
Marija Skrinjar ◽  
Suncica Kocic-Tanackov
Keyword(s):  
Aspergillus Niger ◽  
Ochratoxin A ◽  
Immunosorbent Assay ◽  
Alternaria Alternata ◽  
Rhizopus Oryzae ◽  
Limit Of Detection ◽  
Average Concentration ◽  
Enzyme Linked Immunosorbent Assay ◽  
Mean Values ◽  
Tree Nuts

In this study, moulds and mycotoxins presence in different tree nuts were investigated. The results showed that all of the 25 samples were contaminated with moulds. Mean values of total mould count varied from 1-4.9 cfu per grain. The most frequent species in hazelnut samples were Rhizopus oryzae (32.2%) and Aspergillus niger (28.9%). In walnuts A. niger (75.6%), in cashews also A. niger (42.4%) while in pistachio samples Alternaria alternata (20.7%), and Cladosporium cladosporioides (20.7%) were the most dominant. Rhizopus oligosporus was the only identified species in all almond samples (100%). Using Enzyme Linked Immunosorbent Assay (ELISA), the presence of total aflatoxins and ochratoxin A was examinated. In all analyzed samples, levels of ochratoxin A were below the limit of detection. Total aflatoxins were detected only in walnut samples with average concentration of 7.1 ?g/kg.

scholarly journals Change of Amino Acid Residues in Idiotypic Nanobodies Enhanced the Sensitivity of Competitive Enzyme Immunoassay for Mycotoxin Ochratoxin A in Cereals

Toxins ◽  
2020 ◽  
Vol 12 (4) ◽  
pp. 273
Author(s):  
Caixia Zhang ◽  
Weiqi Zhang ◽  
Xiaoqian Tang ◽  
Qi Zhang ◽  
Wen Zhang ◽  
...  
Keyword(s):  
Amino Acid ◽  
Ochratoxin A ◽  
Limit Of Detection ◽  
Basic Knowledge ◽  
Affinity Constant ◽  
Enzyme Linked Immunosorbent Assay ◽  
Amino Acid Residues ◽  
Assay Sensitivity ◽  
Coating Antigen ◽  
Relative Standard

Anti-idiotypic nanobodies, usually expressed by gene engineering protocol, has been shown as a nontoxic coating antigen for toxic compound immunoassays. We here focused on how to increase immunoassay sensitivity by changing the nanobody’s primary sequence. In the experiments, two anti-idiotype nanobodies against monoclonal antibody 1H2, which is specific to ochratoxin A, were obtained and named as nontoxic coating antigen 1 (NCA1) and nontoxic coating antigen 2 (NCA2). Three differences between the nanobodies were discovered. First, there are six amino acid residues (AAR) of changes in the complementarity determining region (CDR), which compose the antigen-binding site. One of them locates in CDR1 (I–L), two of them in CDR2 (G–D, E–K), and three of them in CDR3 (Y–H, Y–W). Second, the affinity constant of NCA1 was tested as 1.20 × 108 L mol−1, which is about 4 times lower than that of NCA2 (5.36 × 108 L mol−1). Third, the sensitivity (50% inhibition concentration) of NCA1 for OTA was shown as 0.052 ng mL−1, which was 3.5 times lower than that of nontoxic coating antigen 2 (0.015 ng mL−1). The results indicate that the AAR changes in CDR of the anti-idiotypic nanobodies, from nonpolar to polar, increasing the affinity constant may enhance the immunoassay sensitivity. In addition, by using the nontoxic coating antigen 2 to substitute the routine synthetic toxic antigen, we established an eco-friendly and green enzyme-linked immunosorbent assay (ELISA) method for rapid detection of ochratoxin A in cereals. The half-maximal inhibitory concentration (IC50) of optimized ELISA was 0.017 ng mL−1 with a limit of detection (LOD) of 0.003 ng mL−1. The optimized immunoassay showed that the average recoveries of spiked corn, rice, and wheat were between 80% and 114.8%, with the relative standard deviation (RSD) ranging from 3.1–12.3%. Therefore, we provided not only basic knowledge on how to improve the structure of anti-idiotype nanobody for increasing assay sensitivity, but also an available eco-friendly ELISA for ochratoxin A in cereals.

Quantitation and Stability of Fumonisins B1 and B2 in Milk

1994 ◽  
Vol 77 (5) ◽  
pp. 1162-1167 ◽  
Author(s):  
Chris M Maragos ◽  
John L Richard
Keyword(s):  
Liquid Chromatography ◽  
Limit Of Detection ◽  
Analytical Techniques ◽  
Enzyme Linked Immunosorbent Assay ◽  
Color Development ◽  
Strong Anion Exchange ◽  
Low Sensitivity ◽  
No Sample Preparation ◽  
Fumonisins B1 And B2 ◽  
Lower Heating

Abstract The well-documented presence of fumonisin myco-toxins B1 and B2 (FB1 and FB2) in corn raises the possibility that these toxins are carried over into the milk of animals fed with contaminated feed. The presence of FB1 and FB2 in milk has not been assessed because of the lack of sensitive analytical techniques for this matrix. Two methods, liquid chromatography (LC) and enzyme-linked immunosorbent assay (ELISA), were adapted for the analysis of milk. The ELISA, produced commercially for screening corn, required no sample preparation and was reproducible but was of low sensitivity [concentration that inhibits color development by 50% (IC50), 1200-1600 ng FB1/mL]. The more sensitive LC method involves serial extraction of milk with methanol-acetone and strong anion exchange followed by derivatization with naphthalene-2,3-dicarboxaldehyde. Recoveries of 50 ng FB1 and FB2/mL from unpasteurized and un-homogenized milk were 84 and 83%, respectively (limit of detection, 5 ng/mL). Recoveries of FB1 from whole homogenized milk (76%) were slightly lower. Heating milk for 30 min at 62°C, to mimic pasteurization, did not significantly reduce FB1 or FB2 recovery, nor did storing milk for 11 days at 4°C. The LC method was applied to 165 samples of milk, only 1 of which was positive. This finding suggests that exposure of humans to FB1 and FB2 from milk is low.

scholarly journals Nanobody-Alkaline Phosphatase Fusion Protein-Based Enzyme-Linked Immunosorbent Assay for One-Step Detection of Ochratoxin A in Rice

Sensors ◽  
2018 ◽  
Vol 18 (11) ◽  
pp. 4044 ◽  
Author(s):  
Zhichang Sun ◽  
Xuerou Wang ◽  
Qi Chen ◽  
Yonghuan Yun ◽  
Zongwen Tang ◽  
...  
Keyword(s):  
Alkaline Phosphatase ◽  
Fusion Protein ◽  
Ochratoxin A ◽  
Immunosorbent Assay ◽  
Limit Of Detection ◽  
Binding Capacity ◽  
Cross Reactivity ◽  
Solvent Tolerance ◽  
Enzyme Linked Immunosorbent Assay ◽  
One Step

Ochratoxin A (OTA) has become one a focus of public concern because of its multiple toxic effects and widespread contamination. To monitor OTA in rice, a sensitive, selective, and one-step enzyme-linked immunosorbent assay (ELISA) using a nanobody-alkaline phosphatase fusion protein (Nb28-AP) was developed. The Nb28-AP was produced by auto-induction expression and retained an intact antigen-binding capacity and enzymatic activity. It exhibited high thermal stability and organic solvent tolerance. Under the optimal conditions, the developed assay for OTA could be finished in 20 min with a half maximal inhibitory concentration of 0.57 ng mL−1 and a limit of detection of 0.059 ng mL−1, which was 1.1 times and 2.7 times lower than that of the unfused Nb28-based ELISA. The Nb28-AP exhibited a low cross-reactivity (CR) with ochratoxin B (0.92%) and ochratoxin C (6.2%), and an ignorable CR (<0.10%) with other mycotoxins. The developed Nb-AP-based one-step ELISA was validated and compared with a liquid chromatography-tandem mass spectrometry method. The results show the reliability of Nb-AP-based one-step ELISA for the detection of OTA in rice.

scholarly journals Co-occurrence of Aflatoxins, Ochratoxin A, Fumonisins, and Zearalenone in Cereals and Feed, Determined by Competitive Direct Enzyme-Linked Immunosorbent Assay and Thin-Layer Chromatography

2009 ◽  
Vol 60 (4) ◽  
pp. 427-434 ◽  
Author(s):  
Maja Klarić ◽  
Zdenka Cvetnić ◽  
Stjepan Pepeljnjak ◽  
Ivan Kosalec
Keyword(s):  
Thin Layer ◽  
Thin Layer Chromatography ◽  
Ochratoxin A ◽  
Immunosorbent Assay ◽  
Limit Of Detection ◽  
Permissible Limit ◽  
Enzyme Linked Immunosorbent Assay ◽  
Wide Range ◽  
Food And Feed ◽  
Layer Chromatography

Co-occurrence of Aflatoxins, Ochratoxin A, Fumonisins, and Zearalenone in Cereals and Feed, Determined by Competitive Direct Enzyme-Linked Immunosorbent Assay and Thin-Layer ChromatographyAspergillus, Penicillium, andFusariumspecies frequently contaminate crops. For this reason mycotoxins such as aflatoxins (AFs), ochratoxin A (OTA), fumonisins (FBs), and zearalenone (ZEA) are found in food and feed in a wide range of concentrations, depending on environmental and storage conditions. Consumption of mycotoxin-contaminated food and feed has been associated with acute and chronic poisoning and carcinoma. The aim of this study was to determine the incidence and co-occurrence of AFs (B1+B2+G1+G2), OTA, FBs (B1+B2+B3), and ZEA in 37 samples of cereals and feed randomly collected in 2007 from households of an endemic nephropathy (EN) area in Croatia. The mycotoxins were determined using the competitive direct ELISA test (CD-ELISA) in combination with thin-layer chromatography (TLC). The most frequent mycotoxin was ZEA (92%, mean 318.3 μg kg-1), followed by FBs (27%, 3690 μg kg-1), AFs (24.3%, 4.6 μg kg-1), and OTA (16.2%, 9.8 μg kg-1). Levels of AFs, ZEA, and FBs detected by CD-ELISA significantly correlated with the TLC results. However, only one OTA-positive sample was confirmed by TLC due to its high limit of detection. The levels of these mycotoxins were below the permissible limit for animal feed. Twenty-nine percent of cereals were contaminated with FBs, OTA, or ZEA in mass fractions above the permissible limit for humans. Co-occurrence of two toxins varied between 4.2% and 54% and of three between 4.2% and 7.6%. Prolonged co-exposure to AFs, OTA, FBs, and ZEA might increase the risk of various chronic diseases.

scholarly journals Rapid and Sensitive Detection of miRNA Based on AC Electrokinetic Capacitive Sensing for Point-of-Care Applications

Sensors ◽  
2021 ◽  
Vol 21 (12) ◽  
pp. 3985
Author(s):  
Nan Wan ◽  
Yu Jiang ◽  
Jiamei Huang ◽  
Rania Oueslati ◽  
Shigetoshi Eda ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Point Of Care ◽  
Low Cost ◽  
Clinical Diagnostics ◽  
Detection Methods ◽  
Capacitive Sensing ◽  
Application Potential ◽  
Mirna Detection ◽  
Mirna 25 ◽  
Low Sensitivity

A sensitive and efficient method for microRNAs (miRNAs) detection is strongly desired by clinicians and, in recent years, the search for such a method has drawn much attention. There has been significant interest in using miRNA as biomarkers for multiple diseases and conditions in clinical diagnostics. Presently, most miRNA detection methods suffer from drawbacks, e.g., low sensitivity, long assay time, expensive equipment, trained personnel, or unsuitability for point-of-care. New methodologies are needed to overcome these limitations to allow rapid, sensitive, low-cost, easy-to-use, and portable methods for miRNA detection at the point of care. In this work, to overcome these shortcomings, we integrated capacitive sensing and alternating current electrokinetic effects to detect specific miRNA-16b molecules, as a model, with the limit of detection reaching 1.0 femto molar (fM) levels. The specificity of the sensor was verified by testing miRNA-25, which has the same length as miRNA-16b. The sensor we developed demonstrated significant improvements in sensitivity, response time and cost over other miRNA detection methods, and has application potential at point-of-care.

scholarly journals Pitfalls in the Immunochemical Determination of β‑Lactam Antibiotics in Water

Antibiotics ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 298
Author(s):  
Alexander Ecke ◽  
Rudolf J. Schneider
Keyword(s):  
Limit Of Detection ◽  
Cost Effective ◽  
Enzyme Linked Immunosorbent Assay ◽  
Degree Of Hydrolysis ◽  
Site Analysis ◽  
Antibody Recognition ◽  
Immunochemical Determination ◽  
Key Factor ◽  
Lactam Ring

Contamination of waters with pharmaceuticals is an alarming problem as it may support the evolution of antimicrobial resistance. Therefore, fast and cost-effective analytical methods for potential on-site analysis are desired in order to control the water quality and assure the safety of its use as a source of drinking water. Antibody-based methods, such as the enzyme-linked immunosorbent assay (ELISA), can be helpful in this regard but can also have certain pitfalls in store, depending on the analyte. As shown here for the class of β-lactam antibiotics, hydrolysis of the β‑lactam ring is a key factor in the immunochemical analysis as it influences antibody recognition. With the antibody used in this study, the limit of detection (LOD) in the immunoassay could be significantly reduced by hydrolysis for the five tested penicillins, with the lowest LOD for carbenicillin (0.2 nmol/L) and the greatest impact on penicillins G and V (reduction by 85%). In addition to enhanced quantification, our strategy also provides access to information about the degree of hydrolysis in water samples as shown for the most abundant penicillin amoxicillin.

scholarly journals Evaluation of Recombinant Proteins of Burkholderia mallei for Serodiagnosis of Glanders

2012 ◽  
Vol 19 (8) ◽  
pp. 1193-1198 ◽  
Author(s):  
Vijai Pal ◽  
Subodh Kumar ◽  
Praveen Malik ◽  
Ganga Prasad Rai
Keyword(s):  
Sensitivity And Specificity ◽  
Recombinant Proteins ◽  
False Negative ◽  
Enzyme Linked Immunosorbent Assay ◽  
Burkholderia Mallei ◽  
Content Type ◽  
Whole Cells ◽  
Negative Results ◽  
Elisa Format ◽  
Low Sensitivity

ABSTRACTGlanders is a contagious disease caused by the Gram-negative bacillusBurkholderia mallei. The number of equine glanders outbreaks has increased steadily during the last decade. The disease must be reported to the Office International des Epizooties, Paris, France. Glanders serodiagnosis is hampered by the considerable number of false positives and negatives of the internationally prescribed tests. The major problem leading to the low sensitivity and specificity of the complement fixation test (CFT) and enzyme-linked immunosorbent assay (ELISA) has been linked to the test antigens currently used, i.e., crude preparations of whole cells. False-positive results obtained from other diagnostic tests utilizing crude antigens lead to financial losses to animal owners, and false-negative results can turn a risk into a possible threat. In this study, we report on the identification of diagnostic targets using bioinformatics tools for serodiagnosis of glanders. The identified gene sequences were cloned and expressed as recombinant proteins. The purified recombinant proteins ofB. malleiwere used in an indirect ELISA format for serodiagnosis of glanders. Two recombinant proteins, 0375H and 0375TH, exhibited 100% sensitivity and specificity for glanders diagnosis. The proteins also did not cross-react with sera from patients with the closely related disease melioidosis. The results of this investigation highlight the potential of recombinant 0375H and 0375TH proteins in specific and sensitive diagnosis of glanders.

scholarly journals A comparison between enzyme immunoassay and HPLC for ochratoxin A detection in green, roasted and instant coffee

2007 ◽  
Vol 50 (2) ◽  
pp. 349-359 ◽  
Author(s):  
Simone Fujii ◽  
Elisabete Yurie Sataque Ono ◽  
Ricardo Marcelo Reche Ribeiro ◽  
Fernanda Garcia Algarte Assunção ◽  
Cássia Reika Takabayashi ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Ochratoxin A ◽  
High Performance ◽  
Screening Tool ◽  
Correlation Coefficients ◽  
Enzyme Linked Immunosorbent Assay ◽  
Elisa Assay ◽  
Quality And Safety ◽  
Green Coffee ◽  
Instant Coffee

An indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) for ochratoxin A (OTA) detection in green, roasted and instant coffees was developed using anti-OTA monoclonal antibody. Immunological reagents prepared were OTA-BSA (4.76 mg/mL), anti-OTA.7 MAb (2x10³-fold dilution) and HRP-anti IgG (10³-fold dilution). The detection limit was 3.73 ng OTA/g and correlation coefficients (r) between this immunoassay and high performance liquid chromatography were 0.98 for green coffee, 0.98 for roasted and 0.86 for instant. OTA levels detected by ic-ELISA were higher than by HPLC, with ELISA/HPLC ratio of 0.66 - 1.46 (green coffee), 0.96 - 1.11 (roasted) and 0.93 - 1.82 (instant). ELISA recoveries for OTA added to coffee (5 - 70 ng/g) were 81.53 % for green coffee, 46.73 % for roasted and 64.35 % for instant, while recoveries by HPLC were 80.54 %, 45.91 % and 55.15 %, respectively. Matrices interferences were minimized by samples dilution before carrying out the ELISA assay. The results indicate that MAb-based ic-ELISA could be a simple, sensitive and specific screening tool for OTA detection, contributing to quality and safety of coffee products.

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