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MaxSignal Total Aflatoxin ELISA Test Kit Manual
MaxSignal Total Aflatoxin ELISA Test Kit Manual
MaxSignal® Total Aflatoxin ELISA Test Kit is intended for laboratory use only, unless otherwise indicated. This product
is NOT for clinical diagnostic use. MaxSignal is a registered trademark of Bioo Scientific Corporation (BIOO).
MaxSignal® Total Aflatoxin ELISA Test Kit Manual – 1030-02
GENERAL INFORMATION
GENERAL INFORMATION
Product Description
MaxSignal® Total Aflatoxin ELISA Test Kit is a competitive enzyme immunoassay for the quantitative
analysis of Aflatoxin B1, B2, M1, G1 and other aflatoxins in cereals, feed, meat, milk, peanuts and
pistachios. Aflatoxins are toxic secondary metabolites of the fungi species Aspergillus flavus, A.
parasiticus and A. nominus. Aflatoxins are believed to be the most carcinogenic naturally occurring
substances. Aflatoxin B1 is the most potent of all of the aflatoxins. Depending on the toxicity of these
mycotoxins in the countries of the EU equal limits are valid for aflatoxins, 2 ppb for aflatoxin B1 and 4 ppb
for all aflatoxins in total in cereals. Aflatoxins are most commonly found in peanuts, corn, and oil seeds
such as cotton. Aflatoxins have also been found to contaminate wheat, sorghum, Brazil nuts, legumes
and rice as well as many other commercially important foods. As these toxins are heat stable, they are
very difficult to destroy once formed.
MaxSignal® Total Aflatoxin ELISA Test Kit enables international and government regulatory agencies,
food manufacturers and processors, as well as quality assurance organizations, to detect Aflatoxin B1
and other aflatoxins in cereals and other foods and to satisfy customer concerns about food safety. The
unique features of the kit are:
High recovery (>80%), rapid (10 - 40 minutes), and cost-effective extraction methods.
High sensitivity (0.05 ng/g or ppb).
A quick ELISA assay (less than 2 hours regardless of number of samples).
High reproducibility.
Procedure Overview
The method is based on a competitive colorimetric ELISA assay. The toxin of interest has been coated
in the plate wells. During the analysis, sample is added along with the primary antibody specific for the
target toxin. If the target is present in the sample, it will compete for the antibody, thereby preventing the
antibody from binding to the toxin attached to the well. The secondary antibody, tagged with a
peroxidase enzyme, targets the primary antibody that is complexed to the toxin coated on the plate
wells. The resulting color intensity, after addition of substrate, has an inverse relationship with the target
concentration in the sample.
Specificity (Cross-Reactivity)
Analytes Cross-Reactivity (%)
Aflatoxin B1 100
Aflatoxin B2 76
Aflatoxin M1 79
Aflatoxin M2 33
Aflatoxin G1 55
Aflatoxin G2 6
BIOO makes no warranty of any kind, either expressed or implied, except that the materials from which its
products are made are of standard quality. There is no warranty of merchantability of this product, or of the
fitness of the product for any purpose. BIOO shall not be liable for any damages, including special or
consequential damage, or expense arising directly or indirectly from the use of this product.
SAMPLE PREPARATION
SAMPLE PREPARATION
Be sure samples are properly stored. In general, samples should be refrigerated at 2-4°C for no more
than 1-2 days. Freeze samples to a minimum of -20°C if they need to be stored for a longer period.
Frozen samples can be thawed at room temps (20 – 25C / 68 – 77F) or in a refrigerator before use.
Meat/Liver/Kidney
1. Grind and mix a representative sample (according to accepted sampling techniques).
2. Weigh out 5 g of the ground sample and place into a suitable container.
3. Add 25 mL of 70 % methanol * and shake for 20 minutes with a shaker.
4. Centrifuge sample for 10 minutes at 4,000 rpm.
5. Dilute 1 mL of the obtained supernatant with 1 mL of distilled or deionized water.
6. Use 50 µL of the diluted supernatant per well in the test.
*Note: Dilution Factor: 10. The sample size may be increased if required, but the volume
of methanol/water must be changed accordingly, for example, if a 10 g sample is to be
used, 50 mL of 70% methanol should be used. If the aflatoxin concentration is expected
to be high, the sample must be diluted. Please remember that the samples to be pipetted
into the microtiter wells must be in 35% methanol (35% methanol, 65% water).
Milk
1. For fat-free milk, dilute the milk sample with 35% methanol (1:9) (e.g. 20 L of milk + 180 L of
35% methanol). Take 50 L of the diluted sample per well for the assay.
2. For regular milk with fat, centrifuge the milk sample at 4,000 rpm for 5 minutes, discard the upper
fat layer. Dilute the sample with 35% methanol (1:9) (e.g. 20 L of milk + 180 L of 35% methanol).
Take 50 L of the diluted sample per well for the assay.
Note: Dilution factor: 10.
Preparation protocols for samples other than above can be made available upon request.
Please contact your local distributor or write us at foodfeedsafety@biooscientific.com.
BIOO FOOD AND FEED SAFETY • WWW.BIOOSCIENTIFIC.COM 4
MaxSignal® Total Aflatoxin ELISA Test Kit Manual – 1030-02
1. Add 50 L of each Aflatoxin B1 Standards in duplicate into different wells ( Add standards to
plate only in the order from low concentration to high concentration).
2. Add 50 L of each sample in duplicate into different sample wells.
3. Add 100 L of Antibody #1 and mix well by gently rocking the plate manually for 1 minute.
4. Incubate the plate for 30 minutes at room temperature (20 – 25C / 68 – 77F).
5. Wash the plate 3 times with 250 L of 1X Wash Solution. After the last wash, invert the plate and
gently tap the plate dry on paper towels ( Perform the next step immediately after plate
washings. Do not allow the plate to air dry between working steps).
6. Add 150 L of 1X Antibody #2 solution. Incubate the plate for 30 minutes at room temperature (20
– 25C / 68 – 77F) ( Avoid direct sunlight and cold bench tops during the
incubation. Covering the microtiter plate while incubating is recommended).
7. Wash the plate 3 times with 250 L of 1X Wash Solution. After the last wash, invert the plate and
gently tap the plate dry on paper towels ( Perform the next step immediately after plate
washings. Do not allow the plate to air dry between working steps).
8. Add 100 L of TMB substrate. Time the reaction immediately after adding the substrate. Mix the
solution by gently rocking the plate manually for 1 minute while incubating ( Do not put any
substrate back to the original container to avoid any potential contamination. Any
substrate solution exhibiting coloration is indicative of deterioration and should be
discarded. Covering the microtiter plate while incubating is recommended).
9. After incubating for 15 minutes at room temperature (20 – 25C / 68 – 77F), add 100 L of Stop
Buffer to stop the enzyme reaction.
BIOO FOOD AND FEED SAFETY • WWW.BIOOSCIENTIFIC.COM 5
MaxSignal® Total Aflatoxin ELISA Test Kit Manual – 1030-02
10. Read the plate as soon as possible following the addition of Stop Buffer on a plate reader with 450
nm wavelength ( Before reading, use a lint-free wipe on the bottom of the plate to ensure
no moisture or fingerprints interfere with the readings).
Use the mean relative absorbance values for each sample to determine the corresponding
concentration of the tested drug in ng/mL from the standard curve. A special program with Excel
functionality, MaxSignal® ELISA Analysis Program in Excel, is available upon request to evaluate the
MaxSignal® ELISA test results. Please contact your local distributor or techsupport@biooscientific.com
for further information.
The following figure is a typical Aflatoxin B1 standard curve. The sample detection and quantification
limit for this kit are calculated as below.
For example, the dilution factor for feed sample is 10, therefore, the detection limit for feed sample is
0.5 ng/g or ppb (0.05 ng/g x dilution factor 10) and the quantification limit is 1 ng/g or ppb (0.1 ng/g x
dilution factor 10).
90
Relative Absorbance (%)
80
70
60
50
40
30
20
10
0.01 0.10 1.00 10.00
Standard ng/mL (LN)
TROUBLESHOOTING TROUBLESHOOTING
No Color Development or No Signals with Standards
Possible Causes Recommended Action
Reagents were used in the wrong
Follow the protocol carefully and repeat the assay.
order or a step was skipped.
Wrong antibodies were used, or Make sure that the antibodies used are the ones that came with the kit. All
antibody #2 was prepared antibodies are kit- and lot-specific. Make sure that the antibody #2 and diluent
incorrectly or has deteriorated. are mixed in correct volumes.
TMB substrate has deteriorated. Use a new set of BIOO TMB substrate.