Application of ELISA to Retail Survey of Aflatoxin B1 in Peanut Butter

A simple procedure was devised for the routine screening of aflatoxin B1 (AFB1) in peanut butter using enzyme-linked immunosorbent assay (ELISA). Peanut butter samples (5 g) were artificially contaminated with AFB1 and extracted by blending with 25 ml of 55% methanol and 10 ml of hexane. The extract was filtered and aqueous filtrate analyzed by a direct competitive ELISA. Recovery of AFB1 added to peanut butter samples ranged from 85 to 112%, with an average inter-well coefficient of variation of 18.4%. The inter-assay coefficient of variation was 22.7%. Using this procedure, only 3 of 63 commercial samples of peanut butter had detectable levels (>5.0 μg/kg) of AFB1.

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

Journal of AOAC International ◽

10.1093/jaoac/75.4.693 ◽

1992 ◽

Vol 75 (4) ◽

pp. 693-697 ◽

Cited By ~ 3

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.

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The Development of a Quantitative ELISA for Aflatoxin B1 in Ground Peanuts Using Antibodies Isolated From the Yolk of a Laying Hen

Journal of Food Protection ◽

10.4315/0362-028x-57.11.1022 ◽

1994 ◽

Vol 57 (11) ◽

pp. 1022-1024 ◽

Cited By ~ 4

Author(s):

SUZHEN LI ◽

RONALD R. MARQUARDT ◽

ANDREW A. FROHLICH ◽

HAO XIAO ◽

JAMES R. CLARKE

Keyword(s):

Immunosorbent Assay ◽

Aflatoxin B1 ◽

Laying Hens ◽

Egg Yolk ◽

Laying Hen ◽

Competitive Elisa ◽

Enzyme Linked Immunosorbent Assay ◽

Coating Antigen ◽

Extraction Protocol

Antibodies directed against Aflatoxin B1 (AFB1) were produced in laying hens, isolated from egg yolk and applied in an enzyme-linked immunosorbent assay (ELISA) for AFB1 in ground peanuts. The ELISA sensitivity was improved by reduction of the amount of the coating antigen absorbed onto the microplate well surfaces. The main aflatoxins B1, B2, G1, G2, M1, aflatoxicol, sterigmatocystin were found to cross-react in the competitive ELISA, 100, 25, 23, 4, 0, 0, 0%, respectively. Aflatoxin B1 could be reproducibly detected and quantified in spiked ground peanuts at levels greater than 5 ppb using a hexane and methanol solvent based peanut extraction protocol.

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Enzyme linked immunosorbent assay (ELISA) for the determination of protein-A

Bioscience Reports ◽

10.1007/bf01114968 ◽

1986 ◽

Vol 6 (11) ◽

pp. 933-936 ◽

Cited By ~ 8

Author(s):

P. J. Considine ◽

P. Duggan ◽

A. Eadie

Keyword(s):

Immunosorbent Assay ◽

Protein A ◽

Competitive Elisa ◽

Routine Screening ◽

Enzyme Linked Immunosorbent Assay ◽

Culture Supernatants

A competitive ELISA for the determination of protein-A concentration in culture supernatants is described. The sensitivity of the assay is 50 ng/ml and it may be used to determine concentrations of protein-A up to 1000 ng/ml. The assay is specific, rapid and suitable for routine screening of protein-A producing microorganisms.

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Indirect Enzyme-Linked Immunosorbent Assay for Detection of Aflatoxin B1 in Corn and Peanut Butter

Journal of Food Protection ◽

10.4315/0362-028x-47.4.263 ◽

1984 ◽

Vol 47 (4) ◽

pp. 263-266 ◽

Cited By ~ 24

Author(s):

TITAN S.L. FAN ◽

FUNS. CHU

Keyword(s):

Immunosorbent Assay ◽

Aflatoxin B1 ◽

Solid Phase ◽

Peanut Butter ◽

Enzyme Linked Immunosorbent Assay ◽

Corn Meal ◽

Additional Advantage ◽

Microtiter Plates ◽

Sample Extract ◽

Direct Elisa

An indirect enzyme-linked immunosorbent assay (ELISA) for aflatoxin B1 (AFB1) was developed. The method involves coating AFB1-polylysine conjugate on microtiter plates as immobilized antigen, followed by incubation with free toxin standard or sample extract and anti-aflatoxin antibody from rabbits. The amount of antibody bound to the solid phase was determined by subsequent incubation with a secondary antibody conjugated with an enzyme, i.e., goat anti-rabbit IgG-horseradish peroxidase conjugate, and reaction with the chromogenic substrate. Aflatoxins were extracted from peanut butter and corn meal samples according to the BF and CB method of the Association of Official Analytical Chemists, respectively. Extracts without column cleanup treatment were dissolved in assay buffer for subsequent ELISA. Using this technique, 79.5 to 98.6% and 68 to 97% of AFB1 added in the range of 5 to 40 (μg/kg to the corn meal and peanut butter samples were recovered, respectively. The indirect ELISA achieved the same sensitivity and specificity for AFB1 as that obtained from the direct ELISA, with an additional advantage that much less antibody (100 times less) was required for the assay.

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Quantitation of Aflatoxin B1-N7-Guanine Adduct in Urine by Enzyme-Linked Immunosorbent Assay Coupled with Immunoaffinity Chromatography

Journal of AOAC International ◽

10.1093/jaoac/80.5.1013 ◽

1997 ◽

Vol 80 (5) ◽

pp. 1013-1022 ◽

Cited By ~ 5

Author(s):

Tfflrumurthy Vidyasagar ◽

Vadapalli Vyjayanthi ◽

Nayak Sujatiia ◽

Beedu Sashidhar Rao ◽

Ramesh Venkataramana Bhat

Keyword(s):

Immunosorbent Assay ◽

Aflatoxin B1 ◽

Polyclonal Antibodies ◽

Immunoaffinity Chromatography ◽

Competitive Elisa ◽

Aflatoxin M1 ◽

Enzyme Linked Immunosorbent Assay ◽

Immunoaffinity Column ◽

Indirect Competitive Elisa

Abstract IndiaA specific and sensitive method to quantitate aflatoxin B1-N7-guanine adduct in urine samples by immunoaffinity chromatography coupled with indirect competitive enzyme-linked immunosorbent assay (ELISA) is reported. A novel in vitro method to synthesize an antigen (bovine serum albumin-guanine-aflatoxin B1) and its use to produce polyclonal antibodies specific to the hapten aflatoxin B-N7-guanine are discussed. An indirect competitive ELISA developed to quantitate aflatoxin adduct showed a 50% inhibition at 15.6 pmol aflatoxin B1-N7-guanine (y=66.73+ (-19.8)x, r=-0.997). Interference by aflatoxin B1 was less than 5%, and no interference by guanine, aflatoxin Gι and aflatoxin M1 was observed. An immunoaffinity column was also developed, by using these polyclonal antibodies, for single-step purification of aflatoxin B1-N7-gua-nine. The immunoaffinity column and the indirect competitive ELISA were evaluated and validated by quantitation of aflatoxin B1-N7-guanine in urine from rats dosed with aflatoxin B1 (1 mg/kg body weight). Spiking studies with standard aflatoxin B1-N7-guanine adduct at 2 and 4 μg/mL phosphate- buffered saline gave 96 and 100% recoveries, respectively, for immunoaffinity cleanup column. The method also was tested successfully for quantitating aflatoxin B1-N7-guanine adduct in spiked human urine. Recoveries of the adduct were 79-90%.

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

Journal of Food Protection ◽

10.4315/0362-028x.jfp-15-542 ◽

2016 ◽

Vol 79 (5) ◽

pp. 795-800 ◽

Cited By ~ 17

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.

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