AOAC Official Method
Aflatoxins B1, B2, and G1 in Corn,
Cottonseed, Peanuts, and Peanut Butter
Enzyme-Linked Immunosorbent
(ImmunoDot Screen Cup)
Screening Assay
First Action 1990
Final Action 1994
AOAC–IUPAC Method
(Applicable to screening aflatoxin B1, B2, and G1 contamination in
whole cottonseed and peanut butter at ‡ 20 ng/g and in corn and raw
peanuts at ‡ 30 ng/g. If false negative rate of <5% is required, per-
form duplicate ELISAs on same test sample extract. Reanalyze posi-
tive test samples by official, quantitative method.)
A. Principle
Antibodies specific to aflatoxins B1, B2, and G1 are immobi-
lized on a filter, and toxin (aflatoxin B1) is labeled with an enzyme
(horseradish peroxidase). Binding of toxin–enzyme conjugate by
immobilized antibodies is inhibited by addition of free toxin pres-
ent in test sample. Since fixed number of antibody reaction sites
are available, enzyme activity is proportional to amount of bound
toxin–enzyme conjugate. Antibody–toxin–enzyme complex con-
centration is inversely proportional to concentration of free toxin
added. Bound enzyme catalyzes oxidation of substrate to form
blue complex. Development of color indicates that test sample
contains aflatoxins at <20 ng/g; no color development indicates
that test sample contains aflatoxins at ‡ 20 ng/g.
B. Confidence Intervals
The following 95% confidence intervals, shown in Table ,
for correct identification of samples positive for aflatoxin contami-
nation were obtained in the collaborative study on this method.
C. Specificity of Antibodies
Antibodies have specific ability to bind structurally related com-
pounds, namely, aflatoxins B1, B2, and G1. Determine specificity of
purified rabbit anti-aflatoxin B1 polyclonal antibodies by direct
competitive ELISA method. Coat serially diluted antibodies on
microtiter plates. Prepare standard solutions of aflatoxins B1, B2, G1,
G2, and M1; zearalenone; T-2 toxin; and deoxynivalenol, and add to
individual microtiter wells. Then add solution of aflatoxin B1 conju-
gated to horseradish peroxidase to each well. Add substrate solution
of tetramethylbenzidine and hydrogen peroxide, and measure devel-
opment of color with scanner. Least color development indicates
highest reactivity of toxin-antibody reaction. Cross-reactivity to af-
latoxin B1 for antibody used in collaborative study of this method
was 100, 70, 75, and <10% for aflatoxins B1, B2, and G1,and G2, re-
spectively. All other toxins tested showed no cross-reactivity.
D. Sensitivity of ELISA Reagent
Calibrate aflatoxin B1 standard according to (see )
and –C (see ).
(a) Negative control test sample.—Follow procedure in enzyme
immunoassay for corn, J(a).
(b) Threshold-level standard.—Used to define lower limit of
determination. Dispense 100 m L working standard into test
tube. Add 350 m L methanol–buffer, E(d), (30 + 70), and mix.
Follow procedure in enzyme immunoassay for corn, J(a), steps
(2), (4)–(7).
(c) Positive control test sample.—Use working standard
solution; follow procedure for enzyme immunoassay for corn,
J(a), steps (2), (4)–(7).
Negative control test sample should develop blue color; positive
control test sample should have no color development. Threshold
standard should show no color development.
E. Reagents
Items (a)–(h) are available as ImmunoDot Screen (IDS) Cup, (In-
ternational Diagnostic Systems Corp., PO Box 799, St. Joseph, MI
49085, USA). Reagents from other suppliers can be used provided
requirements listed below are met.
(a) Antibody-coated solid support.—Antibody-coated filter ma-
terial attached to analytical cup made of porous polyethylene
( cm diameter, cm high, capacity 4 mL). Coated cup is speci-
fied by manufacturer to be stable for 6 months stored at 4–8°C.
(b) Aflatoxin–enzyme conjugate.—Aflatoxin B1–horseradish
peroxidase conjugate at toxin–enzyme molar ratio of 10–15:1.
Conjugate is specified by manufacturer to be stable for 6 months
at 4–8°C.
(c) Wash solution.—Phosphate–buffer saline solution. Dissolve
g NaH2PO4 × H2O, g K2HPO4× 3H2O, g NaCl, mL
Tween 20 (polyoxyethylene[20]sorbitan monolaurate), and 10 mg
thimerosal (ethylmercurithiosalicylic acid, sodium salt), in 900 mL
H2O, adjust pH to , and dilute to 1 L.
(d) Buffer.—% bovine serum albumin in phosphate buffered
saline solution containing % thimerosal.
(e) Substrate solution A.—Tetramethylbenzidine (TMB)
( g/L H2O), pH .
(f) Substrate solution B.—Hydrogen peroxide (% H2O2 in
% aqueous citric acid solution), pH (Kirkegaard and Perry
Laboratories, Inc., 2 Cessna Ct, Gaithersburg, MD 20879, USA).
(g) Methanol, hexane, and chloroform.—Reagent grade.
(h) Standard aflatoxin B1.—Approximately 28 m g as dry film.
F. Apparatus
Equipment specified is not restrictive; other suitable equipment
can be substituted.
(a) High-speed blender.—With 500 mL jar.
(b) Micropipet and tips.—Recommended range 100–1000 m L;
use with disposable polypropylene tips.
(c) Glass culture (test) tubes.—10 · 75 mm; 3 mL.
(d) Filters.—Whatman No. 4, or equivalent.
(e) Timer.—Graduated in 1 s intervals.
(f) Carborundum boiling chips.
Table Confidence intervals for correct identification
of samples positive for aflatoxin contamination
Sample Level, ppb 95% Confidence interval, %
Cottonseed 20 79–100
Peanut butter 20 72–99
Raw peanuts 30 78–100
Corn 30 73–99
G. General Instructions
Store all kit components at 4–8°C. Do not freeze. Before use, al-
low 1 h for cups and reagents to reach room temperature (23–29°C).
Use separate disposable pipet tip for each test solution to avoid
cross-contamination. Include one negative control with each group
(20 cups) of test samples. Negative control must be functioning
properly (must develop blue color in center of cup) for test to be
valid. Positive standard is provided for periodic checking or for
use with each group of test portions (must show no color in center of
cup). Threshold-level standard should also be used and must show
no color development. If color develops, repeat test. Color develop-
ment in more than 2 tests indicates a defective kit.
Reagents are stable 6 h at room temperature. To ensure shelf life of
kit components, promptly return reagents to refrigerator after use.
Because of difficulty in monitoring 1 min timing intervals, run 1 cup
at a time. As proficiency is gained, analysts can run 3 cups successively
spaced at convenient time intervals for making observations.
H. Extraction of Test Portion
(Note: Test portion extraction and cup testing should be per-
formed during same day.)
(a) Corn, raw peanuts, and whole cottonseed.—Weigh 50 g
test portion into blender jar. Add 100 mL CH3OH–H2O (8 + 2).
Blend 3 min at high speed. Filter mixture and recover filtrate. Al-
ternatively, let mixture stand 10–15 min and recover supernatant
liquid. (Note: For cottonseed, to obtain results applicable to whole
cottonseed when analysis is performed on ground cottonseed meal
[about 50% by weight of whole cottonseed], dilute extract in ratio
1:1 with extraction solvent, CH3OH–H2O [8 + 2].)
(b) Peanut butter.—Weigh 50 g test portion into blender jar. Add
100 mL hexane and 250 mL CH3OH–H2O (55 + 45). Blend 3 min at
high speed. Filter mixture and transfer filtrate to separatory funnel.
Let layers separate for 10 min. Place 20 mL lower layer in 150 mL
beaker. Add minimum of 15 boiling chips and heat in steam bath or
on hot plate. Boil 3 min and let cool.
I. Preparation of Aflatoxin B1 Standard Solutions
(a) Stock solution.—Add 3 mL CHCl3 to vial containing 28 m L
aflatoxin B1 standard (ca 9 ng/m L). Cap vial, mix contents, and store
vial in refrigerator.
(b) Working solution.—Dispense 300 m L stock standard solution
into vial. Add 2400 m L methanol (1 ng/ m L), mix, and store solution
in refrigerator. Prepare daily. Dispense 10 m L diluted standard
(1 ng/m L) into test tube. Add 300 m L methanol and 700 m L buffer,
E(d). Prepare £2 h before use. Proceed as for diluted test extract
[Enzyme Immunoassay, J(a), steps (2), (4)–(7)].
J. Enzyme Immunoassay
(a) Corn, raw peanuts, and whole cottonseed.—(1) Allow 1 h for
all reagents to reach room temperature (23–29°C).
(2) Prepare fresh substrate in small culture (test) tube by mixing
500 m L (10 drops) substrate solution A with 500 m L (10 drops) sub-
strate solution B for each cup being used. Do not combine substrate
solution A with substrate solution B more than 15 min before use.
{Note: Run 1 negative control cup and 1 positive standard cup
each day to ensure that all reagents are functional. Thresh-
old-level standard should be run with each set of new reagents
[follow steps (4)–(7)]. Negative control cup should be run by ap-
plying 100 m L (2 drops) buffer to center of cup. For positive stan-
dard cup, apply working standard to cup as in (4). For both cups pro-
ceed with steps (5)–(7).}
(3) Add 200 m L test extract to 400 m L buffer, E(d), (600 m L total).
(4) Thoroughly mix diluted test extract and apply one 150 m L
aliquot to center of cup. Using timer, wait 1 min and then add second
150 mL aliquot of diluted test extract. Using timer, wait additional
1 min before proceeding to next step. (In summary, two 150 m L
aliquots of diluted test extract are applied to cup—150 m L at a time
with 1 min wait between each addition and before proceeding to next
step.)
(5) Apply 100 m L (2 drops) enzyme solution to center of cup.
Using time, wait 1 min.
(6) Wash with mL (30 drops) wash solution added dropwise. If
more than 1 cup is being used, wash successively with 500 m L
(10 drops) per cup 3 times.
(7) Add entire contents of substrate solution mL (20 drops
mixture) from each test tube to each cup. (Start time as soon as sub-
strate mixture is added to cup.) Wait 1 min and immediately observe
disk (center of cup) for blue color development (negative) or no
color development (positive) (see Interpretation of Results, K).
(b) Peanut butter.—(1) Allow 1 h for all reagents to reach room
temperature (23–29°C).
(2) Prepare fresh substrate solution in small culture (test) tube by
mixing 500 mL (10 drops) substrate solution A with 500 m L (10
drops) substrate solution B for each cup being used. Do not combine
substrate solution A with substrate solution B more than 15 min be-
fore use.
(3) Add 500 m L test extract to 500 m L buffer, E(d), (1000 m L total).
(4) Thoroughly mix diluted test extract and apply one 200 m L
aliquot to center of cup. Using timer, wait 1 min and add second 200
mL aliquot of diluted test extract. Using timer, wait additional 1 min
and then add third 200 m L aliquot of diluted test extract before pro-
ceeding to next step. (In summary, three 200 m L aliquots of diluted
test extract are applied to cup—200 m L at a time with 1 min wait be-
tween each addition and before proceeding to next step.) Proceed as
for corn, etc., steps (5)–(7) above.
K. Interpretation of Results
Observe disk (center of cup) for blue color or no color develop-
ment at exactly 1 min after adding substrate A and B mixture.
Negative.—If disk (center of cup) turns light blue or darker, test
sample contains total aflatoxins B1, B2, and G1 at <20 ng/g (cotton-
seed, peanut butter).
Positive.—If no blue color is observed in disk (center of cup) and
disk remains completely white (no color change) for at least 1 min,
test sample contains total aflatoxins B1, B2, and G1 at ‡ 20 ng/g.
Negative control.—Negative control cup must develop blue color
in center of cup.
Positive control standard.—Positive standard cup must re-
main completely white (no color change) for at least 1 min.
Threshold-level standard.—Cup must remain completely white
(no color change) for 1 min.
[Note: Development at 1 min of any blue color, however faint,
means aflatoxin levels are <20 ppb (negative). False negative rate
may be higher for corn and raw peanuts at this cutoff level. If false
negative rate of <5% is required, duplicate ELISAs must be per-
formed on the test sample extract; otherwise, <30 ng/g is considered
negative for these conditions.]
Reference: JAOAC 72, 957 (1989).
Revised: March 1996
