Quantitative, 10 Minute Deoxynivalenol (DON) Test Kit User’s Guide
A guide for using the DON Test Kit to quantitatively measure deoxynivalenol in wheat, barley, oats and corn products.
Ver. 1.3 Part Number: 7000135 IMPORTANT NOTICE: All kit components must be stored at 2-8C. Allow kit components to warm up to room temperature before use in the ELISA procedure. Manufactured By: Strategic Diagnostics Inc. 111 Pencader Drive Newark, DE 19702 USA Phone: 302.456.6789 www.sdix.com
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INTRODUCTION
Deoxynivalenol (DON or vomitoxin) is a mycotoxin produced by certain members of the Fusarium species, most commonly Fusarium graminearum. In wheat this fungus results in wheat head blight (scab, or pink scab) and in corn pink ear rot. Ingestion of this toxin is known to cause nausea, feed refusal, weight loss, diarrhea, immunosuppression, and renal toxicity. Toxic effects are observed in both humans and animals, to differing degrees. The FDA has established advisory levels for DON as follows: 1 ppm in finished wheat products for human consumption; 10 ppm in grain for cattle and chickens, not to exceed 50% of diet; 5 ppm for in grain for swine, not to exceed 20% of diet; and 5 ppm for grain for all other animals, not to exceed 40% of diet. The Myco test kit for DON is a 10 minute ( two 5 minute incubations ) competitive enzyme-linked immunosorbent assay. The 48 well microtiter plate test will detect the quantitative presence of DON in wheat, wheat flour, barley, malted barley, oats, corn and other grains and grain products from a range of 0 to 3.0 ppm. The principles of the test are based on the ability of free toxin, extracted from the sample, to compete with the enzyme-toxin conjugate (enzyme-labeled toxin supplied in the test kit ) for antibody binding sites on the test wells. After a brief washing step, substrate is added to the wells which reacts with the bound enzyme conjugate to develop a blue color. The darker the blue color the lower the levels of toxin. The lighter the blue color the higher the levels of toxin. The test results may be read qualitatively visually or read in a microwell reader at 650 nm to provide quantitative results. The concentration of the toxin is determined by comparison to a standard curve. Up to 38 samples may be run using the 48 well test kit. Due to the speed of the test the maximum amount of wells run at one given time is 24 or two strips of 12. Running the test this way will provide a maximum amount of 19 sample wells. The antibody used in the test kit is specific for DON and is cross-reactive to
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15-O-Acetyl-4-deoxynivalenol (107%) and 3 Acetyldeoxynivalenol (121%).
Reagents and Materials Provided
1. 48 Antibody-coated microtiter wells ( 4 strips of 12 ) in foil pouch 2. 48 red-marked mixing wells in poly bag 3. 5 vials each containing 2 ml of DON calibrators corresponding to 0, 0.25, 0.5, 1.0 and 3.0 ppm of DON. Note: The standards are adjusted so that they compensate for the dilution of the DON during the extraction step, no mathematical correction is required. 4. 1 vial containing 8 ml of DON-HRP Enzyme Conjugate 5. 1 vial containing 8 ml of Substrate 6. 1 vial containing 8 ml of Stop Solution 7. 1 vial containing 25 ml of 20X Wash Concentrate 8. 4 multi-channel pipette reservoirs 9. 1 set of instructions Materials Required But Not Provided 1. Laboratory quality distilled water 2. 100 ml graduated cylinder 3. Glassware with 125 ml capacity, for sample extraction 4. Whatman #1 filter paper or filter syringe (3000099/3000100) 5. Filter funnel 6. 100 L pipette with disposable tips 7. 50-200 L multi-channel 12 position pipette and disposable tips 8. Grain mill 9. 500 ml plastic squeeze bottle 10. Balance for weighing 10 to 50 grams 11. Microwell strip reader with 650 nm filter 12. Timer 13. Blender 14. Weigh boats
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15. Micro-well strip holder
PRECAUTIONS
1. Each reagent is optimized for use with the DON quantitative plate test kit. Do not substitute reagents from any other manufacturer into the test kit. Do not combine reagents from other SDI DON kits with different lot numbers. 2. Dilution of reagents or samples not called for in the procedure may result in inaccurate results. 3. Do not use reagents after expiration date. 4. Store refrigerated at 2º-8ºC (36º-46ºF). 5. Reagents should be brought to room temperature (18º-30ºC (64º-86ºF) prior to use. Avoid prolonged storage at room temperature. 6. Deoxynivalenol is a very toxic substance. Dispose of all liquids in a plastic container containing household bleach ( minimum 10% ). All labware should be soaked for at least 1 hour in a 30% solution of household bleach. Avoid contact of skin and mucous membranes with reagents and sample extracts by wearing gloves and protective apparel. If contact of liquids to skin occurs, flush with water immediately. 7. Use caution when handling stop solution. Avoid contact with skin and mucous membranes. If contact should occur, flush with water immediately. 8. Do not mix different reagents in the reagent reservoirs – this may cause cross contamination. Dedicate a reservoir to a specific reagent. 9. The test is developed with very rapid reagent reactions. Please follow all directions and complete all steps in an expeditious manner to avoid inaccurate results.
SAMPLE PREPARATION
Sampling error is generally the greatest source of variability in an analytical procedure. Because mycotoxins do not have a uniform distribution in grains, following proper sampling
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guidelines is a key component to an accurate test result. If you do not have a defined sampling protocol we suggest you follow the protocol below or the GIPSA published guidelines. Extracted samples must be used within 48 hrs. or stored at 4C for future use. 1. Collect a representative sample. 2. Grind samples using a mill to pass a 20 mesh sieve and thoroughly mix prior to sub-sampling. Samples not being immediately analyzed should be stored refrigerated. A 500 gram sub-sample is then mixed and a 20 gram sample obtained for testing . 3. Weigh 20 grams of ground sample and combine with 100 ml of water in a clean blender container with tight fitting lid. 4. Blend the sample/water for 2 minutes. 5. Allow sample to stand for 2 – 3 minutes to allow slurry to settle. 6. Filter a minimum of 15 ml of the extract through a Whatman #1 filter or SDI filter syringe. Collect the extract into a clean container – it is now ready for testing. Note: For GIPSA testing, a 50 g sub-sample should be blended for 2 minutes with 250 mL of water (steps 3-4)
WASH SOLUTION PREPARATION
1. Transfer contents of Wash Concentrate vial to a 500 ml plastic squeeze bottle and add 475 ml Lab grade water. Swirl to mix.
TEST PREPARATION
NOTE: Allow all reagents to warm to room temperature, 18º30ºC (64º- 86ºF) before running tests. Running calibrators and samples in duplicate will improve assay precision and accuracy.
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1. An example of a microtiter strip with 12 wells is as follows. The calibrators are identified as 0, 0.25, 0.5, 1.0, and 3.0 ppm, the samples are identified as S1 through S7. Calibrators and samples may be run in duplicate to increase accuracy and precision:
0
0.25
0.5
1.0
3.0
S1
S2
S3
S4
S5
S6
S7
2. 3.
4.
5.
6.
7.
Place the appropriate number of mixing wells and clear test wells into a microwell holder. Be sure to re-seal unused test wells in the resealable bag with desiccant. Dispense 100 L of Enzyme Conjugate into each red colored mixing well using a pipette. Dispense 100 L of each calibrator and sample into the appropriate mixing wells using an adjustable or fixed 100 L volume pipette. Use a clean pipette tip for each use. Using a multi-channel pipette, mix the contents of the wells by repeatedly filling and emptying the tips into the mixing wells. Using the multi-channel pipette, transfer 100 L of each reaction mixture directly into all corresponding clear test wells. Discard the mixing wells into an appropriate waste container. Let reaction mixture incubate for exactly 5 minutes. Mix the solution in the wells by gently swirling the plate on a flat surface for the first 15 seconds. After the 5 minutes, dump the contents of the wells into an appropriate waste container. Using either a 500 ml squeeze bottle, multi-channel pipette or automated washing method, vigorously wash each well by overfilling. Dispense wash solution and repeat the vigorous wash for a total of four washes. After the last wash, invert strip of wells and tap on absorbent paper to remove residual wash solution. Wipe
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8.
9. 10.
11.
12. 13.
excess liquid from the bottom surface of the well strip(s) to avoid reader interference. Measure enough Substrate Solution to be dispensed into a clean reagent reservoir for use with the multi-channel pipette. For 1 to 7 samples measure 2.0 ml of Substrate and dispense into the reagent reservoir. For 8 to 20 samples measure 3.0 ml and dispense into a clean reservoir. Place the multi-channel pipette tips (12) into the reservoir and withdraw the Substrate Solution uniformly into each pipette tip. Dispense 100 L of Substrate Solution into each test well using the multi-channel pipette. After use change tips. Let Substrate Solution incubate for exactly 5 minutes. Mix the solution in the wells by gently swirling the plate on a flat surface for the first 15 seconds. Measure enough Stop Solution to be dispensed into a clean reagent reservoir for use with the multi-channel pipette. For 1 to 7 samples measure 2.0 ml of Stop Solution and dispense into the reagent reservoir. For 8 to 19 samples measure 3.0 ml and dispense into a clean reservoir. Place the multichannel pipette tips (12) into the reservoir and withdraw the Stop Solution uniformly into each pipette tip. Dispense 100 L of Stop Solution into each test well using the multi-channel pipette. For quantitative results, read and record the optical density of the wells at 650 nm using a microwell strip or plate reader. Make sure the well bottoms are clean and dry before placing into reader. Results should be read and recorded no more than 20 minutes after test completion. Follow microwell strip or plate reader instructions.
EXTENDED ASSAY RANGE DILUTION PROCEDURE
Some samples may read above the top calibrator of 3.0 ppm. If it is necessary to accurately quantitate the level of DON
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contamination of the commodity, a dilution of the sample extract must be made and the sample re-run. 1. Run samples as described in this document. Determine which are above the range of the assay (>3.0 ppm). 2. Dilute these samples with water. One hundred (100) L of the sample extract is diluted with 400 L of water. A 100 L sample of this dilution is run in the assay as if it were a normal sample. 3. The evaluation of these samples involves a mathematical compensation for the dilution factor as described below. DATA INTERPRETATION Test Results Quantitative: Average the absorbance readings for duplicate reference standards and unknowns. Plot a standard curve of the mean absorbance (OD) vs. DON concentration of the Standards on the graph paper provided (log/log). Draw a best-fit line through the Standards. An EXCEL spreadsheet can be provided for the quantitative analysis by contacting the SDI Technical Service Department. Interpolate the unknown sample concentrations from the reference standard curve. Alternatively, commercial data reduction software may be used to determine DON levels from the data generated. Qualitative: A less precise and accurate visual interpretation can be achieved by comparing the color of the samples to those of the standards. The concentration of DON in the well is inversely proportional to the intensity of color of the well. For instance, if the blue
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color in the sample well is lighter than that of the 1.0 ppm DON standard then the DON concentration of the sample is greater than 1.0 ppm. Qualitative interpretation is not recommended for samples requiring high accuracy and precision.
TECHNICAL SUPPORT
For questions on the procedure or general technical support please contact: Attn. Strategic Diagnostics Inc. Technical Service 111 Pencader Drive Newark, DE 19702 USA U.S. Only: 800-544-8881 Phone: 302-456-6789 Fax: 302-456-6782 E-mail: techservice@sdix.com OR Attn: SDI Europe Ltd Technical Services - Europe Hamlet House , 80a High Street Alton, Hants GU34 1ET UK Phone: 44 (0)1420-561001 Fax: 44 (0) 1420-561786 Email: europe@sdix.com
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each application protocol to in their individual applications. SDI MAKES NO OTHER WARRANTY, EXPRESSED OR IMPLIED. THERE IS NO WARRANTY OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE.
SDI's sole obligation with respect to the foregoing warranties shall be, at its option, to either replace or to refund the purchase price of the Product(s) or part thereof that proves defective in materials or workmanship within the warranty period, provided the customer notifies SDI promptly of any such defect. SDI SHALL NOT BE LIABLE FOR ANY DIRECT, INDIRECT OR CONSEQUENTIAL DAMAGES RESULTING FROM ECONOMIC LOSS OR PROPERTY DAMAGES SUSTAINED BY BUYER OR ANY CUSTOMER FROM THE USE OF THE PRODUCT
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