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 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.

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.

scholarly journals Antibody Responses to CampylobacterInfections Determined by an Enzyme-Linked Immunosorbent Assay: 2-Year Follow-Up Study of 210 Patients

2001 ◽  
Vol 8 (2) ◽  
pp. 314-319 ◽  
Author(s):  
Mette Aagaard Strid ◽  
Jørgen Engberg ◽  
Lena Brandt Larsen ◽  
Kamilla Begtrup ◽  
Kåre Mølbak ◽  
...  
Keyword(s):  
Immunosorbent Assay ◽  
Serum Antibody ◽  
Large Degree ◽  
Elisa Test ◽  
Enzyme Linked Immunosorbent Assay ◽  
Control Group ◽  
Campylobacter Coli ◽  
Negative Results ◽  
Heat Stable Antigen

ABSTRACT An enzyme-linked immunosorbent assay (ELISA) was adapted to measure immunoglobulin G (IgG), IgM, and IgA classes of human serum antibody toCampylobacter jejuni and Campylobacter coli. Heat-stable antigen, a combination of C. jejuni serotype O:1,44 and O:53 in the ratio 1:1, was used as a coating antigen in the ELISA test. A total of 631 sera from 210 patients with verifiedCampylobacter enteritis were examined at various intervals after infection, and a control group of 164 sera were tested to determine the cut-off for negative results. With a 90th percentile of specificity, IgG, IgM, and IgA showed a sensitivity of 71, 60, and 80%, respectively. By combining all three antibody classes, the sensitivity was 92% within 35 days after infection, whereas within 90 days after infection, a combined sensitivity of 90% was found (IgG 68%, IgM 52%, and IgA 76%). At follow-up of the patients, IgG antibodies were elevated 4.5 months after infection but exhibited a large degree of variation in antibody decay profiles. IgA and IgM antibodies were elevated during the acute phase of infection (up to 2 months from onset of infection). The antibody response did not depend on Campylobacter species or C. jejuniserotype, with the important exception of response to C. jejuni O:19, the serotype most frequently associated with Guillain-Barré syndrome. All of the patients infected with this serotype had higher levels of both IgM (P = 0.006) and IgA (P = 0.06) compared with other C. jejuni and C. coli serotypes.

scholarly journals Schmallenberg virus – Is it present in South Africa?

2013 ◽  
Vol 84 (1) ◽  
Author(s):  
Rhoda Leask ◽  
Albertha M. Botha ◽  
Gareth F. Bath
Keyword(s):  
South Africa ◽  
Rift Valley Fever Virus ◽  
Haemagglutination Inhibition ◽  
Elisa Test ◽  
Schmallenberg Virus ◽  
Enzyme Linked Immunosorbent Assay ◽  
Gauteng Province ◽  
Valley Fever ◽  
Mpumalanga Province ◽  
Negative Results

In July 2006, a case of two out of three lambs born to one ewe in a flock of 45 had signs that, in retrospect, were possibly consistent with Schmallenberg virus infection. This occurred in the Onderstepoort area (Gauteng Province) but a definitive diagnosis was not made. Then, in May 2008, a farmer in the Delmas area (Mpumalanga Province) reported that deformed lambs had been born to several ewes in the flock. Six of the approximately 50 mated ewes gave birth to lambs showing varying degrees of arthrogryposis, torticollis, kyphosis, mandibular brachygnathia and hydrocephalus. Of these, only two were born alive but they died within a few hours. Blood was collected from the ewes with deformed lambs, a random sample of ewes that had given birth to normal lambs and a lamb that was normal but had a twin that was deformed. The samples were tested for Wesselsbron and Akabane antibodies using a complement fixation test and a haemagglutination/haemagglutination inhibition test that were available at that time. Bluetongue virus antibodies were also tested for using a commercial Enzyme-linked immunosorbent assay (ELISA) test. All samples showed negative results for all diseases tested. At the time Rift Valley fever virus had not been diagnosed in that region for many years and so it was not included in the testing. It is unlikely that this was the cause as no liver pathology was detected on postmortem examination of the lambs and no adult ewes had died. The farmer reported that another farm just a few kilometres away experienced the same deformities in some of their lambs but this farm was not investigated. During investigation it was thought that the cause was possibly a new strain of Akabane virus, although there was no way to confirm it. However, with the recent discovery of the Schmallenberg virus, it is possible that this virus has been present in South Africa for at least the last four years without being identified.

scholarly journals Validation of the method of quantitative determination of florphenicol in muscle samples by the method of immuno-enzyme analysis

2020 ◽  
pp. 40-45
Author(s):  
M. V. Kostyuk ◽  
◽  
K. S. Myagka ◽  
G. S. Kochetova ◽  
◽  
...  
Keyword(s):  
Immunosorbent Assay ◽  
Screening Method ◽  
Enzyme Analysis ◽  
Enzyme Linked Immunosorbent Assay ◽  
Laboratory Diagnostics ◽  
Screening Methods ◽  
Detection Ability ◽  
Wide Range ◽  
Control Samples

Introduction. Amphenicols are a group of chemical compounds with antibacterial activity, including chloramphenicol (HAF), thiamphenicol (TAF), florfenicol (FF) and their derivatives. Florfenicol (FF) is a synthetic antimicrobial agent with a broad spectrum of action and is one of the most commonly used drugs in poultry, and was developed specifically for veterinary medicine. Given the wide range of activity of florfenicol, the high therapeutic effect combined with low toxicity makes it important for use in animal husbandry. The known method of enzyme-linked immunosorbent assay (ELISA) differs favorably from other screening methods by high sensitivity, specificity, simplicity and speed of performance, availability and stability of reagents, the ability to computer processing of measurement results and automation of test steps, which provides high test efficiency. The aim of the work. To validate the method of enzyme-linked immunosorbent assay to determine the residues of florfenicol in the samples of months of different species of animals (cattle, pigs, chickens, geese, turkeys, rabbits) and fish. Materials and methods. The research was conducted on the basis of the State Research Institute for Laboratory Diagnostics and Veterinary Sanitary Expertise. The material for the study was a solution of florfenicol with a concentration of 1 mg/liter. Results of research and discussion. It was found that the highest value (highest response) for control samples is 0.24 μg/kg and the lowest value for enriched samples (lowest response) - 3.62 μg/kg. According to the obtained results, none of the answers for the enriched samples coincides with the range of answers for the control samples. It follows that the detection ability (CCβ) for this screening method reduces or decreases 5.0 μg/kg The cut-off rate of this test is 3.62 μg/kg. Conclusions and prospects for further research. Validation characteristics have been established for the determination of florfenicol residues in muscle samples, such as: detection ability (CCβ) is 5.0 μg/kg, cut-off level is 3.62 μg/kg. The lowest content of florfenicol that can be determined is 0.2 μg/kg. The percentage of return for enriched samples of both groups is 93 %, which corresponds to the specified production.

Determination of Aflatoxin Concentrations in Peanut Butter by Enzyme-Linked Immunosorbent Assay (ELISA): Study of Three Commercial ELISA Kits

1989 ◽  
Vol 72 (6) ◽  
pp. 965-969
Author(s):  
Alan L Patey ◽  
Matthew Sharman ◽  
Roger Wood ◽  
John Gilbert
Keyword(s):  
United Kingdom ◽  
Liquid Chromatography ◽  
Immunosorbent Assay ◽  
Peanut Butter ◽  
Statistical Treatment ◽  
Enzyme Linked Immunosorbent Assay ◽  
Analytical Laboratories

Abstract Sixteen United Kingdom analytical laboratories participated in an evaluation of 3 commercially available enzyme-linked immunosorbent assay (ELISA) kits for analysis of aflatoxin in peanut butter. Each laboratory was sent 3 sets of 10 randomly numbered samples of peanut butter. Each set consisted of 5 pairs of undisclosed duplicates. Four of the sets of duplicates were naturally contaminated butters with “target” aflatoxin values (estimated by liquid chromatography) between 8 and 81 fig/kg. The fifth pair was a blank peanut butter containing approximately 3 fig/kg of total aflatoxins. A statistical treatment of the results of the study is presented, together with discussion of the relative merits of the different kits.

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.

scholarly journals Thin-Layer Chromatographic Determination of Monensin in Feeds: Screening Method

1998 ◽  
Vol 81 (4) ◽  
pp. 844-847 ◽  
Author(s):  
Wynne W Landgraf ◽  
P Frank Ross
Keyword(s):  
Solid Phase Extraction ◽  
Thin Layer ◽  
Solid Phase ◽  
Screening Method ◽  
Chromatographic Determination ◽  
Phase Extraction ◽  
Color Development ◽  
Layer Chromatography ◽  
Positive Results

Abstract Monensin is extracted from feed with methanol and purified by solvent-partitioning solid-phase extraction. After solvent reduction, monensin is separated by thin-layer chromatography on silica gel and visualized by color development with vanillin. No false-positive results were obtained in validation studies by submitting or peer laboratories when blank samples were analyzed. Three of 20 samples spiked with 5 ppm monensin were reported as containing no monensin. All samples spiked with 10 ppm monensin were reported positive for monensin.

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