Cross-Reactivity of Antibodies with Phenolic Compounds in Pistachios during Quantification of Ochratoxin A by Commercial Enzyme-Linked Immunosorbent Assay Kits

2014 ◽  
Vol 77 (10) ◽  
pp. 1754-1759 ◽  
Author(s):  
HYUN JUNG LEE ◽  
ALEXANDER D. MELDRUM ◽  
NICHOLAS RIVERA ◽  
DOJIN RYU
Keyword(s):  
Phenolic Compounds ◽  
Ochratoxin A ◽  
Immunosorbent Assay ◽  
False Positive ◽  
High Performance ◽  
Cross Reactivity ◽  
Total Phenolic ◽  
Enzyme Linked Immunosorbent Assay ◽  
Wide Range ◽  
The Cross

Ochratoxin A (OTA), a nephrotoxic mycotoxin, naturally occurs in wide range of agricultural commodities. Typical screening of OTA involves various enzyme-linked immunosorbent assay (ELISA) methods. Pistachio (Pistacia vera L.) is a rich source of phenolic compounds that may result in a false positive due to structural similarities to OTA. The present study investigated the cross-reactivity profiles of phenolic compounds using two commercial ELISA test kits. High-performance liquid chromatography was used to confirm the concentration of OTA in the pistachio samples and compared with the results obtained from ELISA. When the degree of interaction and 50% inhibitory concentration of phenolic compounds were determined, the cross-reactivity showed a pattern similar to that observed with the commercial ELSIA kits, although quantitatively different. In addition, the degree of interaction increased with the increasing concentration of phenolic compounds. The ELISA value had stronger correlations with the content of total phenolic compound, gallic acid, and catechin (R2 = 0.757, 0.732, and 0.729, respectively) compared with epicatechin (R2 = 0.590). These results suggest that phenolic compounds in pistachio skins may cross-react with the OTA antibody and lead to a false positive or to an overestimation of OTA concentration in ELISA-based tests.

An Enzyme-Linked Immunosorbent Assay for the Detection of Aspergillus parasiticus and Aspergillus flavus

1997 ◽  
Vol 60 (8) ◽  
pp. 978-984 ◽  
Author(s):  
GUO-JANE TSAI ◽  
SHOU-CHIN YU
Keyword(s):  
Aspergillus Flavus ◽  
Immunosorbent Assay ◽  
False Positive ◽  
Aspergillus Parasiticus ◽  
False Negative ◽  
Zealand White Rabbit ◽  
Cross Reactivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Molecular Weights ◽  
Sds Page

An enzyme-linked immunosorbent assay (ELISA) was established for the specific detection of Aspergillus parasiticus and Aspergillus flavus. A New Zealand white rabbit was immunized intravenously with 100 μg of A. parasiticus CCRC 30117 mycelial protein extracts. The antibodies were separated and purified. The optimal concentration of the antibody and antibody-peroxidase conjugate used in the established ELISA was 10 μg/ml with a detection limit of 1 μg/ml. Among the 126 strains tested (including 21 strains of A. parasiticus, 11 strains of A. flavus, 34 isolates of A. parasiticus/A. flavus from cereals, and 60 strains of non-A. parasiticus/A. flavus fungi), the false-negative and false-positive rates were 1.5 and 3.3%, respectively. Strains of Aspergillus flavofrucatis and Aspergillus sojae produced false-positive reactions. However, their antigens had much lower cross-reactivity with the antibodies raised against A. parasiticus, as shown from I50 values. The molecular weights of the main antigens of A. parasiticus were 94, 82, and 40 kDa. The two heavier antigens had higher sugar contents, as demonstrated by SDS-PAGE and immunoblotting. A good correlation (r = 0.97) was found between mycelium measurement by weighing and by ELISA for A. parasiticus grown in yeast extract sucrose broth (YESB) at 25°C.

scholarly journals Rapid Mycobacterial Liquid Culture-Screening Method for Mycobacterium avium Complex Based on Secreted Antigen-Capture Enzyme-Linked Immunosorbent Assay

2009 ◽  
Vol 16 (5) ◽  
pp. 613-620 ◽  
Author(s):  
Sung Jae Shin ◽  
Kelly Anklam ◽  
Elizabeth J. B. Manning ◽  
Michael T. Collins
Keyword(s):  
Immunosorbent Assay ◽  
Mycobacterium Avium ◽  
False Positive ◽  
High Performance ◽  
Liquid Culture ◽  
False Negative ◽  
Screening Method ◽  
Clinical Samples ◽  
Enzyme Linked Immunosorbent Assay ◽  
Specific Test

ABSTRACT Sensors in automated liquid culture systems for mycobacteria, such as MGIT, BacT/Alert 3D, and Trek ESP II, flag growth of any type of bacteria; a positive signal does not mean that the target mycobacteria are present. All signal-positive cultures thus require additional and often laborious testing. An immunoassay was developed to screen liquid mycobacterial cultures for evidence of Mycobacterium avium complex (MAC). The method, called the MAC-enzyme-linked immunosorbent assay (ELISA), relies on detection of MAC-specific secreted antigens in liquid culture. Secreted MAC antigens were captured by the MAC-ELISA with polyclonal anti- Mycobacterium avium subsp. paratuberculosis chicken immunoglobulin Y (IgY), detected using rabbit anti-MAC IgG, and then revealed using horseradish peroxidase-conjugated goat anti-rabbit IgG. When the MAC-ELISA was evaluated using pure cultures of known mycobacterial (n = 75) and nonmycobacterial (n = 17) organisms, no false-positive or false-negative MAC-ELISA results were found. By receiver operator characteristic (ROC) analysis of 1,275 previously identified clinical isolates, at the assay optimal cutoff the diagnostic sensitivity and specificity of the MAC-ELISA were 92.6% (95% confidence interval [95% CI], 90.3 to 94.5) and 99.9% (95% CI, 99.2 to 100), respectively, with an area under the ROC curve of 0.992. Prospective evaluation of the MAC-ELISA with an additional 652 clinical samples inoculated into MGIT ParaTB medium and signaling positive per the manufacturer's instructions found that the MAC-ELISA was effective in determining those cultures that actually contained MAC species and warranting the resources required to identify the organism by PCR. Of these 652 MGIT-positive cultures, the MAC-ELISA correctly identified 96.8% (of 219 MAC-ELISA-positive cultures) as truly containing MAC mycobacteria, based on PCR or high-performance liquid chromatography (HPLC) as reference tests. Only 6 of 433 MGIT signal-positive cultures (1.4%) were MAC-ELISA false negative, and only 7 of 219 MGIT signal-negative cultures (3.2%) were false positive. The MAC-ELISA is a low-cost, rapid, sensitive, and specific test for MAC in liquid cultures. It could be used in conjunction with or independent of automated culture reading instrumentation. For maximal accuracy and subspecies-specific identification, use of a confirmatory multiplex MAC PCR is recommended.

Immunoassay of Ochratoxin A, Using Antibodies Developed Against a New Ochratoxin-Albumin Conjugate

1992 ◽  
Vol 75 (5) ◽  
pp. 824-828 ◽  
Author(s):  
Anna Breitholtz-Emanuelsson ◽  
Gunnel Dalhammar ◽  
Karl Hult
Keyword(s):  
Ochratoxin A ◽  
Immunosorbent Assay ◽  
Preparation Method ◽  
Detection Limits ◽  
Cross Reactivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Plasma Samples ◽  
Sample Preparation Method ◽  
Ochratoxin Α ◽  
Incubation Buffer

Abstract A derivative of ochratoxin A was linked to bovine serum albumin in such a way that the carboxylic group of ochratoxin A was left unmodified. Lysine was substituted for phenylalanine in ochratoxin A, and the ε-amino group was linked to the protein. The conjugate was injected into 2 rabbits; antibodies against ochratoxin A were developed and used to develop an indirect competitive enzyme-linked immunosorbent assay. The detection limits for ochratoxin A in incubation buffer were 0.07 and 0.02 ng ochratoxin A/mL with the 2 antisera, respectively. Three hundred human plasma samples were purified by a novel sample preparation method and were analyzed by the immunosorbent assay. The detection limits for ochratoxin A in plasma samples were 0.2 and 0.1 ng ochratoxin A/mL with the 2 antisera, respectively. The cross-reactivity of the 2 antiochratoxin A sera was high for the ochratoxin A methyl ester, about 20% for ochratoxin C, and low for (4R)-4-hydroxyochratoxin A, ochratoxin α, ochratoxin B, and 4-hydroxyochratoxin B. No cross-reactivity was seen for phenylalanine and lysine.

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.

Quantification of Ochratoxin A in Swine Kidneys by Enzyme-linked Immunosorbent Assay Using a Simplified Sample Preparation Procedure

1994 ◽  
Vol 57 (11) ◽  
pp. 991-995 ◽  
Author(s):  
J. R. CLARKE ◽  
R. R. MARQUARDT ◽  
A. A. FROHLICH ◽  
R. J. PITURA
Keyword(s):  
High Performance Liquid Chromatography ◽  
Liquid Chromatography ◽  
Sample Preparation ◽  
Ochratoxin A ◽  
Immunosorbent Assay ◽  
High Performance ◽  
Correlation Coefficients ◽  
Enzyme Linked Immunosorbent Assay ◽  
Coefficients Of Variation ◽  
High Degree

An improved procedure for sample preparation and quantitation of ochratoxin A (OA) in swine kidneys was developed. Kidney samples were homogenized in acidified ethyl acetate, centrifuged, sub-sampled, dried, reconstituted with methanol and directly assayed using an indirect competitive enzyme-linked immunosorbent assay (ELISA). The rabbit antisera used in the development of this assay was found to have a high degree of cross-reaction with ochratoxins A and C but not with ochratoxins B, α, 4-OH-OA and two structurally similar molecules L-phenylalanine and citrinin with the values being 100, 80, 3.33, 10, 1.4, 0 and 0.04%, respectively. Extraction recoveries as determined by high performance liquid chromatography in kidneys spiked with 0.97 to 15.62 ppb OA were determined. The recovery values ranged from 91 to 110% with acceptable inter-assay coefficients of variation (CV) being obtained at the 3.9 ppb spiking concentration or higher. The lowest reproducible OA detection limit for the ELISA in the spiked swine kidney samples was 7.81 ppb with inter-assay CV of 8.85%. The ELISA analysis of the spiked samples correlated highly with conventional high-performance liquid chromatography (HPLC) analysis but was dependent on the conditions of the assay. Standards prepared in methanol or extract prepared from a kidney had correlation coefficients ® of 0.91 ± 0.09 and 0.94 ± 0.07, respectively. The assay is sensitive, specific, simple and sufficiently accurate for routine analysis of swine kidneys.

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 Evaluation of Oxytetracycline Metabolites Cross-Reactivity with Oxytetracycline Enzyme-Linked Immunosorbent Assay (ELISA)

Antibiotics ◽  
2020 ◽  
Vol 9 (4) ◽  
pp. 183
Author(s):  
Faraj Hijaz ◽  
Nabil Killiny
Keyword(s):  
Immunosorbent Assay ◽  
Parent Compound ◽  
Cross Reactivity ◽  
Linear Range ◽  
Plant Diseases ◽  
Enzyme Linked Immunosorbent Assay ◽  
Main Metabolite ◽  
The Cross ◽  
Related Compounds

Antibiotics have been successfully used for the control of several plant diseases for many years. Recently, streptomycin and oxytetracycline have been approved for the treatment of Huanglongbing (HLB) in Florida. The enzyme-linked immunosorbent assay (ELISA) is the most commonly used assay for the detection of these antibiotics because it is quick, simple, and can be used to analyze many samples at the same time. However, ELISA can react with the metabolites of the parent compound and its structurally related compounds. In this study, we investigated the cross-reactivity of the oxytetracycline ACCEL ELISA kitTM with three of oxytetracycline metabolites (4-epi-oxytetracycline, α-apo-oxytetracycline, and β-apo-oxytetracycline). The α-apo-oxytetracycline and β-apo-oxytetracycline metabolite did not show any cross-reactivity in the linear range (1.5–50 ng mL−1) of the assay. Whereas 4-epi-oxytetracycline showed high cross-reactivity, and its response was similar to oxytetracycline. Our results indicated that the oxytetracycline ELISA kits estimate the level of oxytetracycline as well as its main metabolite, 4-epi-oxytetracycline.

scholarly journals Seasonal Variation of Health-Promoting Bioactives in Broccoli and Methyl-Jasmonate Pre-Harvest Treatments to Enhance Their Contents

Foods ◽  
2020 ◽  
Vol 9 (10) ◽  
pp. 1371 ◽  
Author(s):  
Vanesa Nuñez-Gómez ◽  
Nieves Baenas ◽  
Inma Navarro-González ◽  
Javier García-Alonso ◽  
Diego A. Moreno ◽  
...  
Keyword(s):  
High Performance Liquid Chromatography ◽  
Liquid Chromatography ◽  
Phenolic Compounds ◽  
Methyl Jasmonate ◽  
Bioactive Compounds ◽  
High Performance ◽  
Total Phenolic ◽  
Diode Array ◽  
Total Carotenoids ◽  
Health Promoting

Broccoli is a source of bioactive compounds that provide an important nutritional value. The content of these compounds can vary depending on agronomic and environmental conditions, as well as on elicitation. In this study, three crop trials were carried out to evaluate the effects of the cultivation season, the application of different dosages of methyl-jasmonate (MeJA) on the overall quality and on the total content of bioactive compounds of ‘Parthenon’ broccoli cultivated under the field conditions of southeastern Spain. Color parameters, chlorophyll content, total phenolic compounds, total flavonoids and antioxidant activity were measured to evaluate the overall quality. Moreover, individual carotenoids, phenolic compounds and glucosinolates were evaluated by high performance liquid chromatography with diode array detection (HPLC-DAD) and high performance liquid chromatography equipped with diode array detector coupled to mass spectrometer using electro spray ionization (HPLC-DAD-ESI/MSn). The content of total carotenoids, phenolic compounds and glucosinolates were higher in autumn compared with spring, showing increases of 2.8-fold, 2-fold and 1.2-fold, respectively. Moreover, a double application of MeJA increased the contents of total carotenoids, phenolic compounds and glucosinolates by 22%, 32% and 39%, respectively, relative to the untreated samples. Considering our results, the controlled and timely application of 250 µM MeJA to the aerial parts of the plants four days before harvest, on two consecutive days, seems to be a valid agronomic strategy to improve the health-promoting capacity of Parthenon broccoli, without compromising its overall quality.

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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