BIOO FOOD AND FEED SAFETY

Deoxynivalenol (DON) ELISA Test Kit Manual

Catalog #: 1064
Reference #: 1064-01

BIOO Scientific Corp.

2010

TABLE OF CONTENTS
GENERAL INFORMATION ...................................................................... 1 Product Description.................................................................................. 1 Procedure Overview.................................................................................. 1 Kit Contents, Storage and Shelf Life......................................................... 2 Sensitivity (Detection Limit) ..................................................................... 2 Specificity (Cross-Reactivity).................................................................... 2 Required Materials Not Provided With the Kit......................................... 3 Warnings and Precautions ........................................................................ 3 SAMPLE PREPARATION .......................................................................... 4 Cereal/Feed............................................................................................... 4 DON ELISA TEST KIT PROTOCOL ....................................................... 5 Reagent Preparation................................................................................. 5 ELISA Testing Protocol............................................................................. 5 DON Concentration Calculations ............................................................ 6 TROUBLESHOOTING ............................................................................... 7 No Color Development or No Signals with Standards ............................. 7 Low Optical Density (OD) Readings........................................................ 7 High Background or High Optical Density (OD) Readings..................... 7 High Intra-Plate Variance ........................................................................ 8 High Inter-Plate Variance......................................................................... 8 One or More of the Standard Curve Points Are Out of Range ................. 8
MaxSignal Deoxynivalenol (DON) 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 Deoxynivalenol (DON) ELISA Test Kit Manual 1064-01

GENERAL INFORMATION GENERAL INFORMATION

Product Description
Deoxynivalenol (DON), also known as vomitoxin, belongs to the trichothecene group of mycotoxins and is formed by fungi of the genus Fusarium. DON occurs predominantly in grains such as wheat, barley, oats, rye, and maize. Due to their high cytotoxic and immunosuppressive properties these toxins pose a risk to human and animal health. MaxSignal Deoxynivalenol (DON) ELISA Test Kit enables government agencies, food manufacturers and quality assurance organizations to detect DON in various sample types and to satisfy customer concerns about food safety. MaxSignal Deoxynivalenol (DON) ELISA Test Kit provides a unique extraction method, which is accurate, rapid, cost effective and easy to use. The features of the kit are: The extraction of DON from grain samples such as wheat, barley, oats, rye, and maize can be finished with a high recovery rate of 75 - 95%. A quick ELISA assay (less than 1.5 hours regardless of number of samples). High reproducibility.

Procedure Overview
The method is based on a competitive colorimetric ELISA assay. The DON antibody has been coated in the plate wells. During the analysis, sample is added along with the DON-horseradish peroxidase (DON-HRP) conjugate. If the DON residue is present in the sample, it will compete for the DON antibody, thereby preventing the DON-HRP from binding to the antibody attached to the well. The resulting color intensity, after addition of the HRP substrate (TMB), has an inverse relationship with the DON residue concentration in the sample.

BIOO FOOD AND FEED SAFETY WWW.BIOOSCIENTIFIC.COM

MaxSignal Deoxynivalenol (DON) ELISA Test Kit Manual 1064-01

Kit Contents, Storage and Shelf Life

MaxSignal Deoxynivalenol (DON) ELISA Test Kit has the capacity for 96 determinations or testing of 42 samples in duplicate (assuming 12 wells for standards). Return any unused microwells to the foil bag and reseal them with the desiccant provided in the original package. Store the kit at 2-8C*. The shelf life is 12 months when the kit is properly stored.
Kit Contents DON-Ab-coated Plate DON Standards: Negative control (white cap tube) 2.5 ng/mL (yellow cap tube) 5ng/mL (orange cap tube) 10 ng/mL (pink cap tube) 25 ng/mL (purple cap tube) 50 ng/mL (blue cap tube) 1000 ng/mL (spiking, optional, red cap tube) DON-HRP Conjugate 10X Sample Extraction Buffer ** 20X Wash Solution ** Stop Buffer ** TMB Substrate **
*

Amount 1 x 96-well plate (8 wells x 12 strips) 1.8 mL 1.8 mL 1.8 mL 1.8 mL 1.8 mL 1.8 mL 1.8 mL 8 mL 25 mL 28 mL 20 mL 12 mL

Storage 2-8C

2-8C

2-8C * 2-8C 2-8C 2-8C 2-8C

If you are not planning to use the kit for over 1 month, store DON-HRP Conjugate at -20C preferably or in a freezer. ** Components with the same BIOO part Nos within their expiration dates are interchangeable among BIOO kits.

Sensitivity (Detection Limit)

Detection Limit (ng/g or ppb) Sample Type Cereals/Feed 50

Specificity (Cross-Reactivity)
Analytes DON 3-Ac-DON 15-Ac-DON Triacetyl-DON T-2 Toxin T2 Triol Nivalenol Cross-Reactivity (%) 100 454 4.2 0.5 <0.1 <0.1 <0.1

BIOO FOOD AND FEED SAFETY WWW.BIOOSCIENTIFIC.COM

MaxSignal Deoxynivalenol (DON) ELISA Test Kit Manual 1064-01

Required Materials Not Provided With the Kit

Microtiter plate reader (450 nm) Incubator Tissue Mixer (e.g. Omni TissueMaster Homogenizer) Vortex mixer (e.g. Gneie Vortex mixer from VWR) 10, 20, 100 and 1000 L pipettes Multi-channel pipette: 50-300 L (Optional)

Warnings and Precautions

BIOO strongly recommends that you read the following warnings and precautions to ensure your full awareness of ELISA techniques and other details you should pay close attention to when running the assays. More information can also be found in Troubleshooting section. Periodically, optimizations and revisions are made to the kit and manual. Therefore, it is important to follow the protocol coming with the kit. If you need further assistance, you may contact your local distributor or BIOO at techsupport@biooscientific.com. The standards contain DON. Handle with particular care. Do not use the kit past the expiration date. Do not intermix reagents from different kits or lots except for components with the same part Nos within their expiration dates. HRP CONJUGATES AND PLATES ARE KIT-AND LOT-SPECIFIC. Try to maintain a laboratory temperature of 2025C (6877F). Avoid running assays under or near air vents, as this may cause excessive cooling, heating and/or evaporation. Also, do not run assays in direct sunlight, as this may cause excessive heat and evaporation. Cold bench tops should be avoided by placing several layers of paper towel or some other insulation material under the assay plates during incubation. Make sure you are using only distilled or deionized water since water quality is very important. When pipetting samples or reagents into an empty microtiter plate, place the pipette tips in the lower corner of the well, making contact with the plastic. Incubations of assay plates should be timed as precisely as possible. Be consistent when adding standards to the assay plate. Add your standards first and then your samples. Add standards to plate only in the order from low concentration to high concentration as this will minimize the risk of compromising the standard curve. Always refrigerate plates in sealed bags with a desiccant to maintain stability. Prevent condensation from forming on plates by allowing them equilibrate to room temperature (20 25C / 68 77F) while in the packaging.
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.

BIOO FOOD AND FEED SAFETY WWW.BIOOSCIENTIFIC.COM

MaxSignal Deoxynivalenol (DON) ELISA Test Kit Manual 1064-01

SAMPLE PREPARATION SAMPLE PREPARATION Be sure samples are properly stored. In general, samples should be refrigerated at 2-4C for no more than 1-2 days. Freeze samples to a minimum of -20C 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. 1. Preparation of 1X Sample Extraction Buffer: Mix 1 volume of 10X Sample Extraction Buffer with 9 volumes of distilled water.

Cereal/Feed
1. Weigh 5 g of ground cereals/feed sample, add 25 mL of distilled water. 2. Blend the sample by ultra-turrax (or equivalent) for two minutes or shake vigorously for three minutes (manually or with shaker). 3. Filter the extract through Whatman No. 1 filter (or equivalent). 4. Take out 100 L of filtered sample and add 300 L of 1 X Sample Extraction Buffer, mix well. 5. Use 100 L of the sample per well for the assay.
Note: Dilution factor: 20.

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

MaxSignal Deoxynivalenol (DON) ELISA Test Kit Manual 1064-01

DON ELISA TEST KIT PROTOCOL DEOXYNIVALENOL (DON) ELISA TEST KIT PROTOCOL

Reagent Preparation
IMPORTANT: All reagents should be brought up to room temperature before use (1 2 hours at 20 25C / 68 77F); Make sure you read Warnings and Precautions section on page 3. Solutions should be prepared just prior to ELISA test. All reagents should be mixed by gently inverting or swirling prior to use. Prepare volumes that are needed for the number of wells being run. Do not return the reagents to the original stock tubes/bottles. Using disposable reservoirs when handling reagents can minimize the risk of contamination and is recommended. Preparation of 1X Wash Solution
Mix 1 volume of the 20X Wash Solution with 19 volumes of distilled water.

ELISA Testing Protocol

Label the individual strips that will be used and aliquot reagents as the following example:
Component DON-HRP Conjugate 1X Wash Solution Stop Buffer TMB Substrate Volume per Reaction 50 L 2.0 mL 100 L 100 L 24 Reactions 1.2 mL 48 mL 2.4 mL 2.4 mL

1. Add 100 L of each DON Standard in duplicate into different wells ( Add standards to
plate only in the order from low concentration to high concentration).

2. Add 100 L of each sample in duplicate into different sample wells. 3. Add 50 L of DON-HRP Conjugate to each well and mix well by gently rocking the plate manually for 1 minute. 4. Incubate the plate for 45 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 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).

7. After incubating for 15 minutes at room temperature (20 25C / 68 77F), add 100 L of Stop Buffer to stop the enzyme reaction. 8. 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).

BIOO FOOD AND FEED SAFETY WWW.BIOOSCIENTIFIC.COM

MaxSignal Deoxynivalenol (DON) ELISA Test Kit Manual 1064-01

DON Concentration Calculations

A standard curve can be constructed by plotting the mean relative absorbance (%) obtained from each reference standard against its concentration in ng/mL on a logarithmic curve. Relative absorbance (%) = absorbance standard (or sample) x 100 absorbance zero standard

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 distributor or techsupport@biooscientific.com for further information. The following figure is a typical DON standard curve. The sample detection and quantification limit are calculated as below. Sample detection limit = (2.5 ng/g or ppb) x (dilution factor) Sample quantification limit = (5 ng/g or ppb) x (dilution factor) For example, the dilution factor for cereal sample is 20, therefore, the detection limit for cereal sample is 50 ng/g or ppb (2.5 ng/g x dilution factor 20) and the quantification limit is 100 ng/g or ppb (5 ng/g x dilution factor 20).

DO N Standard Curve

Relative Absorbance (%)

90 70 50 30 10 1.00 10.00 100.00 Standard ng/mL (LN)

BIOO FOOD AND FEED SAFETY WWW.BIOOSCIENTIFIC.COM

MaxSignal Deoxynivalenol (DON) ELISA Test Kit Manual 1064-01

TROUBLESHOOTING TROUBLESHOOTING

No Color Development or No Signals with Standards

Possible Causes
Reagents were used in the wrong order or a step was skipped. Wrong HRP conjugates were used. TMB substrate has deteriorated.

Recommended Action
Follow the protocol carefully and repeat the assay. Make sure that the HRP Conjugates used are the ones that came with the kit. All HRP Conjugates are kit- and lot-specific. Use a new set of BIOO TMB substrate.

Low Optical Density (OD) Readings

Possible Causes
Reagents were expired or mixed with a different lot number. Wash solution was prepared incorrectly. Too many wash cycles were used. Incubation times were too short. Lab temperature was too low. Reagents and plates were too cold. Reader was at wrong wavelength, or reader was malfunctioning. Excessive kit stress has occurred. Assay plates were compromised.

Recommended Action
Verify the expiration dates and lot numbers. Use the wash solution for the kit and make sure that it is prepared correctly. Make sure to use the number of washes per the protocol instruction. Time each plate separately to ensure accurate incubation times, follow protocol. Maintain the lab room temperature within 2025C (6877F). Do not run assays under air conditioning vents or near cold windows. Make sure plates and reagents are brought up to room temperature. Keep the kit components out of the kit box for at least 1 hour before starting the assay. Make sure the wavelength is 450 nm for the assay and read the plate again. Verify reader calibration and lamp alignment. Check records to see how many times the kit has cycled from the refrigerator. Check to see if the kit was left at extreme temperatures for too long. Always refrigerate plates in sealed bags with a desiccant to maintain stability. Prevent condensation from forming on plates by allowing them equilibrate to room temperature (20 25C / 68 77F) while in the packaging.

High Background or High Optical Density (OD) Readings

Possible Causes
Poor quality water was used in wash solution. Substrate solution has deteriorated. There was insufficient washing or poor washer performance. Reader was malfunctioning or not blanked properly. This is a high possibility if the OD readings were high and the color was light. Lab temperature was too high. Reagents were intermixed, contaminated or prepared incorrectly.

Recommended Action
If water quality is questionable, try substituting an alternate distilled water source to prepare the wash solution. Make sure the substrate is colorless prior to addition to the plate. Use the number of washes per the protocol instruction. Make sure that at least 250 L of wash solution is dispensed per well per wash. Verify the performance of the washer system; have the system repaired if any ports drip, dispense or aspirate poorly. Verify the readers performance using a calibration plate and check the lamp alignment. Verify the blanking procedure, if applicable, and reblank. Maintain the room temperature within 2025C (6877F). Avoid running assays near heat sources or in direct sunlight. Ensure that the correct reagents were used, that working solutions were prepared correctly and that contamination has not occurred.

BIOO FOOD AND FEED SAFETY WWW.BIOOSCIENTIFIC.COM

MaxSignal Deoxynivalenol (DON) ELISA Test Kit Manual 1064-01

High Intra-Plate Variance

Possible Causes
Inconsistent time was taken when adding standards, reagents or samples to the assay plate. Multichannel pipette was not functioning properly. There was inconsistent washing or washer system malfunctioning.

Recommended Action
Make sure all materials are set up and ready to use. Use a multichannel pipette to add reagents to multiple wells whenever possible. Do not interrupt while adding standards, reagents and samples. Verify pipette calibration and check that tips are on tight. Be sure all channels of the pipette draw and dispense equal volumes. Check performance of the wash system. Have the system repaired if any ports drip or dispense/aspirate poorly.

High Inter-Plate Variance

Possible Causes
Inconsistent incubation times occurred from plate to plate. Inconsistent washing occurred from plate to plate. Pipette was working improperly. Kit plates, reagents, standards and samples were at different temperatures.

Recommended Action
Time each plate separately to ensure consistent incubation times. Make sure to use the number of washes per the protocol instruction. Verify performance of the wash system and have the system repaired if any ports drip or dispense/ aspirate poorly. Check the pipette calibration. Verify that pipette tips are on tight before use and that all channels draw and dispense equal volumes. Make sure to allow sufficient time for kit plates, reagents, standards and samples come to room temperature (20 25C / 68 77F). Larger volumes will require longer equilibration time. If using a water bath to hasten equilibration, make sure it is maintained at room temperature; do not use a warm water bath to warm reagents, samples and kit standards. Carefully label each reagent to make sure the reagents are not intermixed. Kits with different expiration dates might generate different range of OD readings, however, the relative absorbance values may very well be comparable. In general, a value of less than 0.6 in zero standard reading may indicate certain degrees of deterioration of reagents.

Reagents used were intermixed from different kit lots, or the kits were of different expiration dates.

One or More of the Standard Curve Points Are Out of Range

Possible Causes
Standards were added in wrong order or recorded in wrong position. Standards were contaminated or intermixed with other standards. There was inconsistent washing or washer system malfunctioning. Inconsistent time was taken to add standards and reagents to plate. Multichannel pipette was not functioning properly.

Recommended Action
Follow the protocol and re-run the assay. Make sure the standards are applied and recorded correctly. Use a new set of standards. Add standards to plate only in the order from low concentration to high concentration. Perform washing consistently. Check performance of the wash system. Have the system repaired if any ports drip or dispense/aspirate poorly. Make sure all materials are set up and ready to use. Add standards to plate only in the order from low concentration to high concentration at undisrupted pace. Use a multichannel pipette to add reagents to multiple wells simultaneously. Verify pipette calibration and check that tips are on tight. Be sure all channels of the pipette draw and dispense equal volumes.

Bioo Scientific Corporation 3913 Todd Lane Suite 312 Austin, TX 78744 USA Tel: 1.888.208.2246 Fax: (512) 707-8122

Made in USA BIOO Food & Feed Safety Products info@biooscientific.com foodfeedsafety@biooscientific.com www.biooscientific.com
8

BIOO FOOD AND FEED SAFETY WWW.BIOOSCIENTIFIC.COM