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

1984 ◽  
Vol 47 (4) ◽  
pp. 263-266 ◽  
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.

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

1986 ◽  
Vol 49 (10) ◽  
pp. 792-795 ◽  
Author(s):  
BHANU P. RAM ◽  
L. PATRICK HART ◽  
RICHARD J. COLE ◽  
JAMES J. PESTKA
Keyword(s):  
Immunosorbent Assay ◽  
Aflatoxin B1 ◽  
Coefficient Of Variation ◽  
Simple Procedure ◽  
Peanut Butter ◽  
Competitive Elisa ◽  
Routine Screening ◽  
Enzyme Linked Immunosorbent Assay

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.

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 T-2 Toxin Metabolites in Urine

1989 ◽  
Vol 72 (2) ◽  
pp. 345-348
Author(s):  
Rachel C Lee ◽  
Ru-Dong Wei ◽  
Fun S Chu
Keyword(s):  
Immunosorbent Assay ◽  
Cross Reactivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Linear Portion ◽  
Standard Curve ◽  
Sample Extract ◽  
Direct Elisa ◽  
Minimum Detection Level ◽  
Elisa Plate

Abstract A direct competitive enzyme-linked immunosorbent assay (ELISA) for determination of total T-2 toxin metabolites in urine was developed. The assay involves coating anti-3-acetyl-neosolaniol-hemisuccinate- bovine serum albumin conjugate (anti-3-Ac-NEOS-HS-BSA) antibody to the ELISA plate and using 3-Ac-NEOS-HS-peroxidase as the enzyme marker. Competitive ELISA revealed that the antibody had good cross-reactivity with acetyldiacetoxyscirpenol (Ac-DAS), T-2 tetraol tetraacetate, 3'-OH-Ac-T-2, 3-Ac-NEOS, and 3,4,15- triacetyl-12,13-epoxytrichothec-9-en-8-one (Ac-T-2-8-one), but less cross-reactivity with Ac-T-2 toxin and T-2 toxin. All metabolites of T-2 toxin in urine were converted to T-2 tetraol tetraacetate (T-2- 4ol-4Ac) by acetylation of the sample extract before ELISA. To test the ELISA accuracy, a radioimmunoassay (RIA) was performed simultaneously. The linear portion of the standard curve of this direct ELISA for T-2-4ol-4Ac was 0.2-2.0 ng/mL, which was 10 times more sensitive than RIA. The minimum detection level for T-2-4ol- 4Ac was 0.02 ng/mL (0.4 pg/assay) in the absence of urine sample. The overall analytical recoveries for T-2 toxin, HT-2, T-2-4ol, 3'- OH-HT-2, NEOS, and a mixture of these 5 toxins added to the urine samples in the ELISA at concentrations of 0.05 and 0.2 ng/mL were 87 and 94%, respectively.

Enzyme-Linked Immunosorbent Assay of Aflatoxin Bi in Naturally Contaminated Corn and Cottonseed

1986 ◽  
Vol 69 (5) ◽  
pp. 904-907 ◽  
Author(s):  
Bhanu P Ram ◽  
L Patrick Hart ◽  
Odette L Shotwell ◽  
James J Pestka
Keyword(s):  
Correlation Coefficient ◽  
Immunosorbent Assay ◽  
Solid Phase ◽  
Microtiter Plate ◽  
Enzyme Linked Immunosorbent Assay ◽  
Extraction Solvent ◽  
Sample Extract ◽  
Coefficients Of Variation ◽  
Layer Chromatography ◽  
Duplicate Sample

Abstract Naturally contaminated corn and cottonseed samples were screened for aflatoxin B1 (AFB1) by a direct competitive enzyme-linked immunosorbent assay (ELISA). Samples were blended 5 min in an extraction solvent of methanol-water-dimethylformamide (70 + 29 + 1) and filtered. Filtrates were assayed by direct competition between AFBi in the corn and cottonseed samples and AFB1-peroxidase conjugate for binding to specific antibody adsorbed to a solid phase microtiter plate. Standard curves prepared using the extract of AFB1-free corn and cottonseed samples, and extraction solvent only, showed negligible interference by the sample extract in the performance of ELISA. The AFBi content in corn and dehulled cottonseed samples as determined by ELISA ranged from 7 to 422 μg/kg and 7 to 3258 μg/kg, respectively. When ELISA estimates of AFB1 in corn were compared with values obtained by thin layer chromatography (CB method), the correlation coefficient (n = 10) was 0.95. Average interassay and subsample coefficients of variation for ELISA in corn were 21.4 and 22.0%, respectively. When ELISA estimates of AFB1 in cottonseed were compared with values obtained by liquid chromatography (Pons method), the correlation coefficient (n = 15) was 0.96. Using this ELISA, 36 duplicate sample extracts can be screened for AFB1 in less than 2 h.

Enzyme-linked immunosorbent assay for serum amyloid A apolipoprotein with use of specific antibodies against synthetic peptides.

1988 ◽  
Vol 34 (9) ◽  
pp. 1767-1771 ◽  
Author(s):  
R Saïle ◽  
C Delpierre ◽  
P Puchois ◽  
G Hocke ◽  
C Cachera ◽  
...  
Keyword(s):  
Immunosorbent Assay ◽  
Synthetic Peptides ◽  
Solid Phase ◽  
Polyclonal Antibodies ◽  
Enzyme Linked Immunosorbent Assay ◽  
Specific Antibodies ◽  
Amyloid A ◽  
Microtiter Plates ◽  
Sensitivity Specificity ◽  
As Solid Phase

Abstract We describe a noncompetitive enzyme-linked immunosorbent assay for amyloid A apolipoprotein in human serum (apo SAA) in which specific antibodies against synthetic peptides are used. Microtiter plates were used as solid phase and coated with affinity-purified antibodies raised against SAA1-(95-104) peptide. After incubation with delipidated plasmas, the bound apo SAA was revealed by labeled antibodies raised against SAA1-(58-69) peptide. The assay offers several advantages over existing techniques: sensitivity, specificity, simplicity, and non-use of radioisotopes. Results correlate well with those by a nephelometric method in which polyclonal antibodies are used.

ELISA Survey of Retail Grain-Based Food Products for Zearalenone and Aflatoxin B1

1987 ◽  
Vol 50 (6) ◽  
pp. 502-503 ◽  
Author(s):  
ROSCOE L. WARNER ◽  
JAMES J. PESTKA
Keyword(s):  
Wheat Flour ◽  
Immunosorbent Assay ◽  
Aflatoxin B1 ◽  
Food Products ◽  
Average Level ◽  
Enzyme Linked Immunosorbent Assay ◽  
Corn Meal ◽  
Breakfast Cereal ◽  
Snack Foods ◽  
Retail Grocery

Seventy-nine grain-based food products were purchased from mid-Michigan retail grocery outlets in 1985 and analyzed for the mycotoxins zearalenone and aflatoxin B1 by enzyme-linked immunosorbent assay. Twenty-two percent of these samples contained detectable zearalenone (limit ⩾2.5 μg/kg). Zearalenone was found in breakfast cereal, snack foods, popcorn, corn meal, and cake-muffin mixes representing 10, 11, 57, 78, and 20% of these samples, respectively. The average level of this toxin among the positive samples was 20 μg/kg with maximum levels of 120 and 130 μg/kg being found in samples of corn meal and popcorn, respectively. Zearalenone was not found in any of the wheat flour or baby foods samples. Detectable aflatoxin B1 (limit ⩾5.0 μg/kg) was not found in any of the 79 samples tested.

Determination of Aflatoxin B1 in Corn, Wheat, and Peanut Butter by Enzyme-linked Immunosorbent Assay and Solid Phase Radioimmunoassay

1981 ◽  
Vol 64 (5) ◽  
pp. 1077-1082 ◽  
Author(s):  
Ossama El-Nakib ◽  
James J Pestka ◽  
Fun S Chu
Keyword(s):  
Aflatoxin B1 ◽  
Solid Phase ◽  
Peanut Butter ◽  
Extraction Procedure ◽  
Tween 20 ◽  
Enzyme Linked Immunosorbent Assay ◽  
Coefficients Of Variation ◽  
Solid Phase Radioimmunoassay ◽  
Phosphate Buffered Saline

Abstract Determinations of aflatoxin B1 in corn, wheat, and peanut butter by an enzyme-linked immunoassay (ELISA) and a solid-phase radioimmunoassay (RIA) were compared. Samples spiked with 2.9-43.2 ppb B1 were subjected to AOAC extraction procedure 26.017 or 26.023. The extracts were concentrated, redissolved in methanol, diluted in phosphate-buffered saline with Tween 20, and directly analyzed for B1 by either ELISA or RIA. At 5.8 ppb or greater, recoveries for B1 in corn, wheat, and peanut butter samples were 80.0,86.6, and 94.8% by ELISA and 61.0, 93.3, and 110.0% by RIA, respectively. Recoveries greater than 120% were obtained for the wheat and peanut butter samples spiked with 2.9 ppb aflatoxin B1 by the RIA method but not by ELISA. Overall results indicated that ELISA gave more consistent data, relatively lower standard deviations, and lower coefficients of variation than did RIA. Analysis of 3 samples naturally contaminated with aflatoxins revealed that the ELISA data were comparable to those obtained by other established chemical methods.

scholarly journals Enzyme-linked immunosorbent assay for Pseudomonas aeruginosa exotoxin A

1979 ◽  
Vol 9 (6) ◽  
pp. 705-708
Author(s):  
W W Schultz ◽  
T J Phipps ◽  
M Pollack
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Horseradish Peroxidase ◽  
Immunosorbent Assay ◽  
Outer Layer ◽  
Solid Phase ◽  
Enzyme Linked Immunosorbent Assay ◽  
Exotoxin A ◽  
Microtiter Plates ◽  
Sandwich Method ◽  
Pseudomonas Aeruginosa Exotoxin

An enzyme-linked immunosorbent assay (ELISA) is described for Pseudomonas aeruginosa exotoxin A. A double antibody sandwich method was used, employing polyvinyl microtiter plates as the solid phase, a primary coat of monospecific rabbit antitoxin serum, an outer layer composed of a horseradish peroxidase-sheep antitoxin immunoglobulin G conjugate, and an ortho-phenylene-diamine substrate. Absorbance (optical density) of hydrolyzed end product was read spectrophotometrically at 492 nm. ELISA detected as little as 30 pg (0.3 ng/ml) of purified toxin, and absorbance was linear over a 20-fold or greater concentration range. Toxin was demonstrated in culture filtrates from 42 of 48 (88%) consecutive clinical P. aeruginosa isolates compared with 37 of 48 (77%) positive by hemagglutination inhibition. Results of the two assays correlated closely (r = 0.82, P less than 0.001). Specificity was confirmed by neutralizability of ELISA activity with monospecific antitoxin. ELISA was thus a sensitive, specific, and quantifiable technique for the assay of P. aeruginosa exotoxin A in both purified and crude culture materials.

A New Technique in Quantitative Immunohematology: Solid-Phase Kinetic Enzyme-Linked Immunosorbent Assay

Vox Sanguinis ◽  
1983 ◽  
Vol 45 (6) ◽  
pp. 440-448 ◽  
Author(s):  
S. Spitalnik ◽  
J. Cowles ◽  
M.T. Cox ◽  
D. Baker ◽  
J. Holt ◽  
...  
Keyword(s):  
Immunosorbent Assay ◽  
Solid Phase ◽  
New Technique ◽  
Enzyme Linked Immunosorbent Assay ◽  
A New Technique

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.

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