Enzymatic and Immunological Approaches for the Quantitation and Confirmation of Ochratoxin A in Swine Kidneys

1997 ◽  
Vol 60 (2) ◽  
pp. 172-176 ◽  
Author(s):  
ANDREW A. FROHLICH ◽  
RONALD R. MARQUARDT ◽  
JAMES R. CLARKE
Keyword(s):  
Ochratoxin A ◽  
High Performance ◽  
Hplc Analysis ◽  
Linear Regression Analysis ◽  
Enzyme Linked Immunosorbent Assay ◽  
Enzymatic Method ◽  
Naturally Occurring ◽  
Related Compounds ◽  
Enzymatic Procedure ◽  
Swine Kidney

Several techniques for the quantitation and confirmation of ochratoxin A (OA) in swine kidneys were examined. Naturally and artificially contaminated swine kidneys were analyzed for OA by conventional high-performance liquid chromatography (HPLC) analysis. Samples were additionally tested by enzyme-linked immunosorbent assay (ELISA) or treated with carboxypeptidase A followed by HPLC analysis (enzymatic method). Correlations (r values) between the conventional HPLC procedure and the ELISA, using artificially contaminated samples, were 0.88 and 0.81 (P < 0.05) respectively, while the corresponding values between the conventional HPLC procedure and the enzymatic method were 0.89 and 0.98 (P < 0.05). The ELISA gave a more direct estimation of OA contamination than the enzymatic procedure. The enzymatic method also had a reproducible tendency to underestimate or overestimate the amounts of OA in kidney. This was found to be dependent on the source of contamination, as artificially and naturally contaminated kidney samples resulted in linear regression analysis slopes of 0.38 and 2.8, whereas the slopes for the ELISA method were 1.13 and 0.92, respectively. The results with the naturally contaminated kidneys suggest that other naturally occurring forms of OA also occurred in swine kidney. Regardless of this effect, the enzymatic method accurately confirms the presence of OA and related compounds in kidney. The techniques are simple and will complement conventional HPLC analysis for the detection, quantitation, and confirmation of OA in swine kidneys.

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.

Polyclonal Antibody-Based ELISA and HPLC Methods for the Determination of Fumonisins in Corn: A Comparative Study

1996 ◽  
Vol 59 (8) ◽  
pp. 893-897 ◽  
Author(s):  
ERIC W. SYDENHAM ◽  
SONJA STOCKENSTRÖM ◽  
PIETER G. THIEL ◽  
JOHN P. RHEEDER ◽  
M. BRUNO DOKO ◽  
...  
Keyword(s):  
Comparative Study ◽  
Polyclonal Antibody ◽  
High Performance ◽  
Linear Regression Analysis ◽  
Correlation Coefficients ◽  
Hplc Method ◽  
Enzyme Linked Immunosorbent Assay ◽  
Regression Slopes ◽  
The Individual ◽  
The Comparative Study

The performance of an experimental polyclonal antibody (PAb)-based competitive direct enzyme-linked immunosorbent assay (CD-ELISA) developed for the analysis of fumonisins in corn was assessed by comparison with an established high-performance liquid chromatography (HPLC) method. The comparative study was conducted using a series of 20 corn samples naturally contaminated with combined fumonisin levels ranging from <0.05 to >5 μg/g (ppm). Linear regression analysis between the results generated by HPLC and CD-ELISA provided correlation coefficients (r) and regression slopes (b) of r = 0.960, b = 1.493 (P < 0.001); r = 0.865, b = 3.903 (P < 0.001); and r = 0.832, b = 0.107 (P < 0.001) for the individual fumonisins B1 (FB1), B2 (FB2) and B3 (FB3), respectively, while corresponding values of r = 0.967, b = 1.059 (P < 0.001) were obtained for the combined FB1, FB2, and FB3 concentrations. In 3 of 18 fumonisin-positive corn samples, combined fumonisin levels determined by CD-ELISA were between 85 and 100% higher than those determined in the same extracts by HPLC, while in 13 other samples, CD-ELISA results were between 1.8 and 53% higher than those determined by HPLC. Conversely, in 2 of 18 samples, CD-ELISA results were lower than those determined by HPLC. The differences recorded between HPLC and the experimental PAb-based CD-ELISA were far less than those previously recorded for other mono- and polyclonal antibody-based CD-ELISA systems. The results indicate that the experimental PAb-based CD-ELISA may be effectively applied for the initial screening for fumonisins in corn.

scholarly journals Multiple Mycotoxins in Kenyan Rice

Toxins ◽  
2021 ◽  
Vol 13 (3) ◽  
pp. 203
Author(s):  
Samuel K. Mutiga ◽  
J. Musembi Mutuku ◽  
Vincent Koskei ◽  
James Kamau Gitau ◽  
Fredrick Ng’ang’a ◽  
...  
Keyword(s):  
Ochratoxin A ◽  
High Performance ◽  
Low Frequency ◽  
Rice Cultivar ◽  
Enzyme Linked Immunosorbent Assay ◽  
Milled Rice ◽  
Regulatory Limits ◽  
Liquid Chromatography Tandem Mass ◽  
Rice Samples ◽  
Time Of Harvest

Multiple mycotoxins were tested in milled rice samples (n = 200) from traders at different milling points within the Mwea Irrigation Scheme in Kenya. Traders provided the names of the cultivar, village where paddy was cultivated, sampling locality, miller, and month of paddy harvest between 2018 and 2019. Aflatoxin, citrinin, fumonisin, ochratoxin A, diacetoxyscirpenol, T2, HT2, and sterigmatocystin were analyzed using ultra-high-performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS). Deoxynivalenol was tested using enzyme-linked immunosorbent assay (ELISA). Mycotoxins occurred in ranges and frequencies in the following order: sterigmatocystin (0–7 ppb; 74.5%), aflatoxin (0–993 ppb; 55.5%), citrinin (0–9 ppb; 55.5%), ochratoxin A (0–110 ppb; 30%), fumonisin (0–76 ppb; 26%), diacetoxyscirpenol (0–24 ppb; 20.5%), and combined HT2 + T2 (0–62 ppb; 14.5%), and deoxynivalenol was detected in only one sample at 510 ppb. Overall, low amounts of toxins were observed in rice with a low frequency of samples above the regulatory limits for aflatoxin, 13.5%; ochratoxin A, 6%; and HT2 + T2, 0.5%. The maximum co-contamination was for 3.5% samples with six toxins in different combinations. The rice cultivar, paddy environment, time of harvest, and millers influenced the occurrence of different mycotoxins. There is a need to establish integrated approaches for the mitigation of mycotoxin accumulation in the Kenyan rice.

Short HPLC gradient method for 20-Hydroxyecdysone (20E) quantification in malaria vectors v1

2021 ◽  
Author(s):  
Oswald Yedjinnavenan Djihinto ◽  
Luc S. Djogbenou ◽  
Luisa Nardini ◽  
Armorel Van Eyk ◽  
Lizette L. Koekemoer
Keyword(s):  
Gradient Method ◽  
High Performance ◽  
Chromatographic Method ◽  
Control Strategies ◽  
Malaria Vectors ◽  
Enzyme Linked Immunosorbent Assay ◽  
Biological Processes ◽  
Sample Material ◽  
Enzymatic Method ◽  
Regulatory Pathways

Ecdysteroids are arthropod steroid hormones that are primarily involved in insect moulting. In arthropod vectors, especially in mosquitoes, ecdysteroids of interest include mainly ecdysone (E) and 20-hydroxyecdysone (20E). These two compounds are involved in several important biological processes. Targeting these compounds and their regulatory pathways could lead to the characterisation of novel genetic tools towards implementing new malaria control strategies. To date, there are two main methods for quantifying E and 20E. These methods include an enzymatic method (Enzyme-Linked Immunosorbent Assay (ELISA)) and a chromatographic method (High-Performance Liquid Chromatography (HPLC)). However, for ecdysteroids quantification, the HPLC methods available in literature go from 30 minutes to one hour. Here, we developed a short HPLC gradient method for 20-hydroxyecdysone quantification in the malaria vector Anopheles gambiae. This method was developed specifically when sample material is limited as well as to save time and cost.

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 Development of an Anti-Idiotypic VHH Antibody and Toxin-Free Enzyme Immunoassay for Ochratoxin A in Cereals

Toxins ◽  
2019 ◽  
Vol 11 (5) ◽  
pp. 280 ◽  
Author(s):  
Caixia Zhang ◽  
Qi Zhang ◽  
Xiaoqian Tang ◽  
Wen Zhang ◽  
Peiwu Li
Keyword(s):  
Enzyme Immunoassay ◽  
Ochratoxin A ◽  
High Performance ◽  
Elisa Test ◽  
Hplc Method ◽  
Free Enzyme ◽  
Agricultural Products ◽  
Enzyme Linked Immunosorbent Assay ◽  
Highly Sensitive ◽  
Ic50 Value

Enzyme-linked immunosorbent assay (ELISA) test kits have been widely used for the determination of mycotoxins in agricultural products and foods, however, this test uses toxin standards with high toxicity and carcinogenicity that seriously threaten human health. In this work, the anti-idiotypic nanobody VHH 2-24 was first developed and then, using it as a surrogate standard, a toxin-free enzyme immunoassay for ochratoxin A (OTA) was established. The IC50 value of the VHH 2-24 surrogate standard-based ELISA was 0.097 µg/mL, with a linear range of 0.027–0.653 µg/mL. The average recoveries were tested by spike-and-recovery experiments, and ranged from 81.8% to 105.0%. The accuracy of the developed ELISA for detecting OTA was further verified by using the high performance liquid chromatography (HPLC) method, and an excellent correlation was observed. In summary, the toxin-free ELISA established in this study proves the latent use of the anti-idiotypic VHH as a surrogate calibrator for other mycotoxins and highly toxic small molecule analysis to improve assay properties for highly sensitive analyte determination in agricultural products.

Ochratoxin A: Comparison of Extraction Methods from Grapes and Quantitative Determination by Different Competitive Enzyme-Linked Immunosorbent Assay Kits

2008 ◽  
Vol 71 (12) ◽  
pp. 2488-2496 ◽  
Author(s):  
E. ANGELINI ◽  
I. BAZZO ◽  
M. SAVINO ◽  
M. BORGO
Keyword(s):  
Ochratoxin A ◽  
High Performance ◽  
Reference Method ◽  
Cost Effective ◽  
Extraction Methods ◽  
Fluorescent Detection ◽  
Enzyme Linked Immunosorbent Assay ◽  
Lower Sensitivity ◽  
Chromatography Method ◽  
Detection Techniques

The European Community has recently established a maximum limit for ochratoxin A (OTA) concentration in grapevine products, but many practical difficulties remain concerning the establishment of optimum cost-effective methods of quantification. The performance of four extraction procedures and three commercial competitive enzyme-linked immunosorbent assays (cELISAs) for grapes were compared. Results differed for the extractions and the cELISA kits. The advantage of using immunoaffinity columns (IACs) in the extraction was the excellent detection limit, which was between 0.06 and 0.0075 ng ml−1 depending on the cELISA kit used. Despite lower sensitivity (between 1.2 and 0.15 ng ml−1 depending on the cELISA kit), an extraction method in liquid phase, which was simple and inexpensive, was confirmed as suitable for quantifying OTA at levels estimated to be dangerous for human health. Two of the three cELISA kits produced satisfactory results. When these two cELISAs were coupled with IAC extraction, the lower quantification limits were 0.010 and 0.0075 ng ml−1, respectively, and the dynamic ranges were 50 and 27, respectively. The most reliable procedures were then compared with the reference method, high-performance liquid chromatography plus fluorescent detection coupled with an IAC. The results were very similar, although the cELISAs generally provided slightly higher values than did the chromatography method. The IAC method coupled with the cELISA was four times more sensitive than was the IAC method coupled with the chromatography method. The cELISA detection techniques were excellent alternatives to the already established chromatographic protocols, especially for mass screening and for determining concentrations of OTA as low as 0.010 ng ml−1.

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.

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