Enzyme-Linked Immunosorbent Assay for Screening Aflatoxin B1 in Cottonseed Products and Mixed Feed: Collaborative Study

1989 ◽  
Vol 72 (2) ◽  
pp. 326-332 ◽  
Author(s):  
Douglas L Park ◽  
Brinton M Miller ◽  
L Patrick Hart ◽  
George Yang ◽  
James McVey ◽  
...  
Keyword(s):  
Cottonseed Meal ◽  
Aflatoxin B1 ◽  
Collaborative Study ◽  
Screening Method ◽  
The United States ◽  
Poultry Feed ◽  
Enzyme Linked Immunosorbent Assay ◽  
Contamination Level ◽  
Mixed Feed ◽  
Incomplete Removal

Abstract A joint AOAC/IUPAC (International Union of Pure and Applied Chemistry) interlaboratory study of an enzyme-linked immunosorbent screening assay (ELISA) for aflatoxins was conducted in laboratories in Canada, France, Japan, South Africa, Switzerland, The Netherlands, Tunisia, and the United States. Twenty-eight samples of raw and roasted peanuts, corn, whole cottonseed, cottonseed meal, ammoniated cottonseed meal, and poultry feed containing various quantities of natural aflatoxins and supplemented when appropriate with aflatoxin B1 were distributed to participating laboratories for testing. The assay is based on conjugation of pure aflatoxin B1 to an enzyme and the competition between this conjugate and (free) aflatoxins in the product for aflatoxin-specific antibodies coated onto microtiter well walls. After a wash step to remove all unbound aflatoxins, a substrate, added to each well, is catalyzed from a colorless to a green solution by any bound enzyme-conjugated aflatoxin Bf present. The intensity of the color decreases as the amount of free aflatoxin B1 in the product increases. Overall correlation was good between ELISA and thin-layer chromatographic (TLC) results for cottonseed products and mixed feed. Variable results were reported for corn and peanut product samples. Although some positive samples (> 15 ng/g) of cottonseed products and mixed feed were reported to contain < 15 ng/g by visual determination, a review of data for absorbance measurements showed that the contamination level was close to the ≥ 15 ng/g standard and would not have been reported as negative under routine screening. Variation in ELISA results may have been due to several factors such as: lack of homogeneity of the aflatoxin contamination in the samples (prestudy TLC analysis samples were collected randomly from a pool of subsamples), interferences that resulted from incomplete removal of hexane during the filtration step, and antibody strips at or past their expiration date. The ELISA method has been adopted official first action as a screening method to determine the presence or absence of aflatoxin B1 at a concentration of ≥ 15 ng/g in cottonseed products and mixed feed.

Determination of Total Aflatoxin Levels in Peanut Butter by Enzyme-Linked Immunosorbent Assay: Collaborative Study

1992 ◽  
Vol 75 (4) ◽  
pp. 693-697 ◽  
Author(s):  
Alan L Patey ◽  
Matthew Sharman ◽  
John Gilbert
Keyword(s):  
Immunosorbent Assay ◽  
Aflatoxin B1 ◽  
Peanut Butter ◽  
Collaborative Study ◽  
The United States ◽  
Enzyme Linked Immunosorbent Assay ◽  
Total Aflatoxin ◽  
Aflatoxin Level ◽  
Relative Standard

Abstract Laboratories in Australia, Japan, Spain, the United Kingdom, and the United States participated in a collaborative study to evaluate a commercial enzyme- linked immunosorbent assay for the determination of total aflatoxin. Collaborators were sent 10 randomly numbered samples (5 blind duplicates) of roasted peanut butter. Two pairs were "blank" peanut butters to which aflatoxin B1, B2, G1, and G2 standards had been added. The other 3 pairs of peanut butters were 1 low aflatoxin level sample and 2 naturally contaminated samples. The assay is based on indirect competition. Test samples containing (free) aflatoxin, added to aflatoxin-coated microwells, compete for specific monoclonal rat anti-aflatoxin. As the concentration of aflatoxin in the test samples increases, the amount of rat antiaflatoxin binding to the aflatoxin attached to the well decreases. After a wash step to remove unbound material, the amount of rat anti-aflatoxin bound to the well is determined by its reaction with peroxidase conjugated rabbit anti-rat globulin. Bound peroxidase activity is then determined by the addition of a substrate, whose color development is inversely proportional to the aflatoxin concentration and is measured by absorbance. Coefficients of variation (CV) for total aflatoxin concentrations, for mean levels of 9,30, and 89µg/kg, were between 28 and 37% for the low level and 2 naturally contaminated samples, which contained mainly aflatoxin B1. CVs for the spiked samples were lower (24-25%) for mean levels of 11 and 20 µg/kg; recoveries were 84 and 89%, respectively. Ranges for relative standard deviations for repeatabilty and reproducibility were 9-30% and 25-37%, respectively. The method has been adopted first action by AOAC International.

Enzyme-Linked Immunosorbent Assay for Detection of Zearalenone in Corn, Wheat, and Pig Feed: Collaborative Study

1994 ◽  
Vol 77 (6) ◽  
pp. 1500-1508 ◽  
Author(s):  
Glenn A Bennett ◽  
Terry C Nelsen ◽  
Brjnton M Miller
Keyword(s):  
Immunosorbent Assay ◽  
Collaborative Study ◽  
Screening Method ◽  
The United States ◽  
False Positives ◽  
Enzyme Linked Immunosorbent Assay ◽  
Elisa Method ◽  
False Negatives ◽  
Relative Standard ◽  
Pig Feed

Abstract A direct competitive enzyme-linked immunosorbent assay (ELISA) screening method for zearalenone in corn, wheat, and feed at 500 ng/g was evaluated by 23 collaborators (22 laboratories) in an international collaborative study. Eighteen samples of spiked or naturally contaminated corn, wheat, and pig feed were prepared by the sponsoring laboratory and sent for testing with complete test kits to participating collaborators in Canada, Italy, Sweden, The Netherlands, and the United States. Test samples were extracted with methanolwater solution (70 + 30) by shaking on a wrist-action shaker for 3 min. A portion of the extract was mixed with an equal volume of zearalenone-enzyme conjugate, and the mixture was incubated with zearalenone-specific monoclonal antibodies coated onto microtiter wells. All test samples were assayed in duplicate. One of 52 (2%) blanks was reported positive. Thirty-nine of the 52 (75%) samples that were spiked at 500 ng/g were reported as positive. Forty-nine of the 51 (96%) samples with concentrations at or above 1000 ng/g were reported as positive. The overall incidence of false negatives was 6.0% and the incidence of false positives was 22.7% by the ELISA method. Only one (3.4%) false negative was reported for samples containing ≥800 ng/g. In the spectrophotometric method, 8 collaborators determined approximate levels of zearalenone in test samples from standard curves constructed from spiked extracts (0–3000 ng/g of each commodity tested). This method gave and overall incidence of false negatives of 5.7% and false positives of 17.8%. Average relative standard deviations, RSDr (repeatability) and RSDR (reproducibility), were 11.6 and 25.1% for spiked samples and 11.7 and 33.1% for naturally contaminated samples, respectively. Standard curves were constructed with each set of samples assayed. Comparison of absorbance values from these standard curves indicate the performance of reagents and antibody used in the assay. The ELISA method has been adopted first action by AOAC INTERNATIONAL as a screening method for zearalenone at ≥800 ng/g in corn, wheat, and pig feed.

Enzyme-Linked Immunosorbent Assay of Aflatoxins Bi, B2, and Gi in Corn, Cottonseed, Peanuts, Peanut Butter, and Poultry Feed: Collaborative Study

1989 ◽  
Vol 72 (6) ◽  
pp. 957-962 ◽  
Author(s):  
Mary W Trucksess ◽  
Michael E Stack ◽  
Stanley Nesheim ◽  
Douglas L Park ◽  
Albert E Pohland
Keyword(s):  
Immunosorbent Assay ◽  
Polyclonal Antibodies ◽  
Peanut Butter ◽  
Collaborative Study ◽  
Screening Method ◽  
Steam Bath ◽  
Test Sample ◽  
Positive Test ◽  
Poultry Feed ◽  
Enzyme Linked Immunosorbent Assay

Abstract A direct competitive enzyme-linked immunosorbent assay (ELISA) screening method for aflatoxins at 20 ng/g was studied by 12 collaborators. Test samples of peanut butter were extracted by blending with methanol-water-hexane (55 + 45 + 100) and heating the test extracts on a steam bath; test samples of the other commodities were extracted by blending with methanol-water (80 + 20). All test extracts were filtered and the filtrates were diluted with buffer to a final methanol concentration of <30%. Each diluted filtrate was applied to a cup containing a filter with immobilized polyclonal antibodies specific to aflatoxins B1, B2, and G1. Aflatoxin B1-peroxidase conjugate was added, the cup was washed with water, and a mixture of hydrogen peroxide and tetramethylbenzidine was added. The test sample was judged to contain >20 ng aflatoxins/g when, after exactly 1 min, no color was observed on the filter; when a blue or gray color developed, the test sample was judged to contain <20 ng aflatoxins/g. All collaborators correctly identified naturally contaminated corn and raw peanut positive test samples. No false positives were found for controls containing <2 ng aflatoxins/g. The correct responses for positive test samples spiked at levels of 10, 20, and >30 ng aflatoxins/g (the ratio of B1, B2, and G1 was 10:1:3) were 52, 86, and 96%, respectively. The method, which is rapid and simple, has been adopted official first action for screening for aflatoxins at >20 ng/g in cottonseed and peanut butter and for aflatoxins at >30 ng/g in corn and raw peanuts. The procedure will require further study for poultry feed. Positive test samples may require reanalysis by an official, quantitative method.

scholarly journals A Case for Regular Aflatoxin Monitoring in Peanut Butter in Sub-Saharan Africa: Lessons from a 3-Year Survey in Zambia

2016 ◽  
Vol 79 (5) ◽  
pp. 795-800 ◽  
Author(s):  
SAMUEL M. C. NJOROGE ◽  
LIMBIKANI MATUMBA ◽  
KENNEDY KANENGA ◽  
MOSES SIAMBI ◽  
FARID WALIYAR ◽  
...  
Keyword(s):  
South Africa ◽  
Aflatoxin B1 ◽  
Peanut Butter ◽  
Aflatoxin Contamination ◽  
Comprehensive Analysis ◽  
Sub Saharan Africa ◽  
Enzyme Linked Immunosorbent Assay ◽  
Contamination Level ◽  
Batch Number ◽  
Sub Saharan

ABSTRACT A 3-year comprehensive analysis of aflatoxin contamination in peanut butter was conducted in Zambia, sub-Saharan Africa. The study analyzed 954 containers of 24 local and imported peanut butter brands collected from shops in Chipata, Mambwe, Petauke, Katete, and Nyimba districts and also in Lusaka from 2012 to 2014. For analysis, a sample included six containers of a single brand, from the same processing batch number and the same shop. Each container was quantitatively analyzed for aflatoxin B1 (AFB1) in six replicates by using competitive enzyme-linked immunosorbent assay; thus, aflatoxin contamination level of a given sample was derived from an average of 36 test values. Results showed that 73% of the brands tested in 2012 were contaminated with AFB1 levels >20 μg/kg and ranged up to 130 μg/kg. In 2013, 80% of the brands were contaminated with AFB1 levels >20 μg/kg and ranged up to 10,740 μg/kg. Compared with brand data from 2012 and 2013, fewer brands in 2014, i.e., 53%, had aflatoxin B1 levels >20 μg/kg and ranged up to 1,000 μg/kg. Of the eight brands tested repeatedly across the 3-year period, none consistently averaged ≤20 μg/kg. Our survey clearly demonstrates the regular occurrence of high levels of AF B1 in peanut butter in Zambia. Considering that some of the brands tested originated from neighboring countries such as Malawi, Zimbabwe, and South Africa, the current findings provide a sub-Saharan regional perspective regarding the safety of peanut butter.

scholarly journals LOCATE Enzyme-Linked Immunosorbent Assay for Detection of Salmonella in Food: Collaborative Study

1998 ◽  
Vol 81 (2) ◽  
pp. 419-437 ◽  
Author(s):  
Vidhya Gangar ◽  
Michael S Curiale ◽  
Armando D'onorio ◽  
Carol Donnelly ◽  
Paul Dunnigan ◽  
...  
Keyword(s):  
Collaborative Study ◽  
Screening Method ◽  
Culture Method ◽  
Microtiter Plate ◽  
Black Pepper ◽  
Soy Flour ◽  
Food Type ◽  
Enzyme Linked Immunosorbent Assay ◽  
Data Sets ◽  
Whole Egg

abstract A collaborative study was performed in 27 laboratories to validate the enzyme-linked immunosorbent procedure LOCATE for rapid detection of Salmonella in foods. Results were read visually and with a microtiter plate reader. The LOCATE method was compared with the Bacteriological Analytical Manual (BAM)/AOAC INTERNATIONAL culture method for detecting Salmonella in 6 foods: milk chocolate, nonfat dry milk, dried whole egg, soy flour, ground black pepper, and ground raw turkey. Two foods—dried whole egg and black pepper—required repeat rounds because insufficient data sets were produced initially (AOAC INTERNATIONAL stipulates a minimum of 15 sets per food type). Each laboratory tested one or more of the 6 foods. A total of 1 439 samples were analyzed, and no significant differences (P <0.05) were observed between LOCATE with either visual or reader detection and BAM/AOAC INTERNATIONAL results. The LOCATE screening method with visual or reader detection is recommended for Official First Action Approval

Comparison of Two Immunochemical Methods with Thin-Layer Chromatographic Methods for Determination of Aflatoxins

1990 ◽  
Vol 73 (3) ◽  
pp. 425-428
Author(s):  
Mary W Trucksess ◽  
Kathryn Young ◽  
Kevin F Donahue1 ◽  
Deborah K Morris ◽  
Ernie Lewis
Keyword(s):  
Thin Layer ◽  
Correct Response ◽  
Peanut Butter ◽  
Screening Method ◽  
Elisa Test ◽  
Poultry Feed ◽  
Enzyme Linked Immunosorbent Assay ◽  
Affinity Column ◽  
Negative Results

Abstract Three different methods were compared for the determination of total flatoxins in corn and peanuts naturally contaminated with aflatoxins and In corn, peanuts, cottonseed, peanut butter, and poultry feed spiked with aflatoxins Bi, B2, and d . The 3 methods were an enzyme-linked Immunosorbent assay (ELISA) screening test; a monoclonal antibody-affinity column-solld-phase separation method; and the AOAC official thin-layer chromatography (TLC) methods for all except poultry feed, for which Shannon's TLC method for mixed feed was used. The ELISA test is designed to provide only positive results for total aflatoxins at >20 ng/g or negative results at <20 ng/g. The affinity column separation Is coupled with either brominatlon solution fluorometry to estimate total aflatoxins or liquid chromatography (LC) to quantltate Individual aflatoxins. Fluorodensitometry was used to determine aflatoxins In commodities analyzed by the TLC methods. The LC and TLC results were In good agreement for all the analyses. The results for the affinity column using brominatlon solution fluorometry were similar except those for cottonseed, which were about 60% higher. The ELISA screening method correctly Identified naturally contaminated corn and peanut positive samples. No false positives were found for controls. The correct response for spiked corn, raw peanuts, peanut butter, and cottonseed at >20 ng aflatoxlns/g was about 90%. The correct response for spiked poultry feed at >20 ng aflatoxlns/ g was about 50%.

Enzyme-Linked Immunosorbent Assay of Total Aflatoxins B1, B2, and G1 in Corn: Follow-up Collaborative Study

1994 ◽  
Vol 77 (3) ◽  
pp. 655-658 ◽  
Author(s):  
Mary W Trucksess ◽  
Michael E Stack
Keyword(s):  
Immunosorbent Assay ◽  
Polyclonal Antibodies ◽  
Collaborative Study ◽  
Screening Method ◽  
Test Sample ◽  
Enzyme Linked Immunosorbent Assay ◽  
Test Device ◽  
Total Aflatoxin ◽  
Peroxidase Conjugate

Abstract A direct competitive enzyme-linked immunosorbent assay screening method for af latoxins at 20 ng/g in corn was studied by 15 collaborating laboratories. Test samples of corn were extracted by blending with methanol-water (8 + 2). The extracts were filtered and the filtrates were diluted with buffer to a final methanol concentration of <30%. Each diluted filtrate was applied to a test device containing a filter with immobilized polyclonal antibodies specific to aflatoxins B1, B2, and G1. Aflatoxin Bi-peroxidase conjugate was added, the test device was washed with water, and a mixture of hydrogen peroxide and tetramethylbenzidine was added. A test sample was judged to contain ≥20 ng af latoxins/g when, after exactly 1 min, no color was observed on the filter; if a blue or gray color developed, the test sample was judged to contain <20 ng aflatoxins/g. All laboratories correctly identified naturally contaminated corn test samples. Only one false positive was found for controls containing no aflatoxins. The correct responses for positive test samples spiked at levels of 10,20, and 30 ng aflatoxins/g (the ratio of B1:B2:G1 was 15:1:3) were 67,97, and 100%, respectively. This method was adopted first action by AOAC INTERNATIONAL as a change in method for 990.34 for screening for aflatoxins B1, B2, and G1 in corn at total aflatoxin concentrations of ≥20 ng/g.

Collaborative Study of a Screening Method for the Detection of Aflatoxins in Mixed Feeds, Other Agricultural Products, and Foods

1976 ◽  
Vol 59 (1) ◽  
pp. 110-117
Author(s):  
Thomas R Romer ◽  
Alfred D Campbell
Keyword(s):  
Cottonseed Meal ◽  
Peanut Butter ◽  
Collaborative Study ◽  
Screening Method ◽  
Peanut Meal ◽  
Fluorescent Band ◽  
Yellow Corn ◽  
Pistachio Nuts ◽  
Cleanup Procedure ◽  
Mixed Feeds

Abstract A screening method for aflatoxins was collaboratively tested on 11 different agricultural and food products: white and yellow corn, peanuts, peanut butter, pistachio nuts, peanut meal, cottonseed meal, chicken, pig, and turkey starter rations, and dairy cattle feed. The method involves a rapid extraction and cleanup procedure followed by the detection of total aflatoxins (B1 + B2 + G1 + G2) as a fluorescent band on the Florisil layer of a Velasco-type minicolumn. The results of 32 collaborators from 10 different countries are presented. Samples containing 0, 5, 10, 15, 20, and 25 μg aflatoxins/kg were analyzed. Eighty-four per cent of the negative samples and 89% of the samples containing 10–25 μg total aflatoxins/kg were correctly identified. This method has been adopted as official first action for the detection of aflatoxins in corn, peanuts, peanut butter, peanut meal, cottonseed meal, mixed feeds, and pistachio nuts.

Enzyme-Linked Immunosorbent Assay of Mycotoxins Using Nylon Bead and Terasaki Plate Solid Phases

1984 ◽  
Vol 47 (4) ◽  
pp. 305-308 ◽  
Author(s):  
J. J. PESTKA ◽  
F. S. CHU
Keyword(s):  
Immunosorbent Assay ◽  
Aflatoxin B1 ◽  
Linear Response ◽  
Screening Method ◽  
Detection Limits ◽  
Microtiter Plate ◽  
Aflatoxin M1 ◽  
Enzyme Linked Immunosorbent Assay ◽  
Solid Phases ◽  
New Procedures

Nylon beads and Terasaki plates were tested as solid phases for the enzyme-linked immunosorbent assay (ELISA) of the mycotoxins aflatoxin B1 (AFB1), aflatoxin M1 (AFM1) and T-2 toxin. Both methods had detection limits comparable to that of mycotoxin microtiter plate ELISAs. Using the nylon bead ELISA, ELISA competition curves for AFB1, AFM1 and T-2 toxin exhibited linear response between 1.0 to 100, 0.1 to 100, and 0.1 to 10.0 ng/ml, respectively. Response ranges for Terasaki plate ELISAs of AFB1, AFM1 and T-2 toxin were 1.0 to 50, 0.05 to 0.50, and 0.5 to 1.0 ng/ml, respectively. The new procedures did not require specialized instrumentation and may be used as an economical screening method for mycotoxins in the field and to diagnose certain mycotoxicoses.

scholarly journals Determination of Total Fumonisins in Corn by Competitive Direct Enzyme-Linked Immunosorbent Assay: Collaborative Study

2002 ◽  
Vol 85 (2) ◽  
pp. 404-410 ◽  
Author(s):  
Chuck B Bird ◽  
Bruce Malone ◽  
Larry G Rice ◽  
P Frank Ross ◽  
Robert Eppley ◽  
...  
Keyword(s):  
Immunosorbent Assay ◽  
Fusarium Species ◽  
Collaborative Study ◽  
The United States ◽  
Enzyme Linked Immunosorbent Assay ◽  
Test Portion ◽  
Relative Standard ◽  
Standard Deviations ◽  
Different Levels

Abstract Fumonisins—mycotoxins produced by some Fusarium species—have been shown to be the causative agent of diseases in horses and other domesticated animals as well as possible carcinogens in humans. A collaborative study was conducted to evaluate the effectiveness of a competitive direct enzyme-linked immunosorbent assay (CD-ELISA) for the determination of total fumonisins (B1, B2, and B3) in corn. The test portion was extracted with methanol–water (7 + 3), filtered, diluted, and tested on the CD-ELISA. Naturally and artificially contaminated corn test portions were sent to 13 collaborators in the United States. Naturally contaminated field test portions were prepared at 3 different levels. Artificially contaminated test portions were spiked at 1.0, 3.0, and 5.0 mg/kg total fumonisins (B1, B2, and B3). Average recoveries of total fumonisins were 120, 100, and 90%, respectively. The relative standard deviations for repeatability ranged from 13.3 to 23.3% and the relative standard deviations for reproducibility ranged from 15.8 to 30.3% across all levels tested. HORRAT values, calculated for each individual sample, ranged from 1.24 to 1.94. This method demonstrated acceptable intra- and interlaboratory precision at the levels tested.

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