GLUTEN RAPID KIT Lateral Flow Devices for Qualitative Analysis of Gluten Proteins Product: GLTN-1005 Storage at 4-8 C (35-45 F) UPDATED FEBRUARY 2013
Gluten Rapid LFD Analysis Note: This kit format has NO extra control line (called the Hook line) Research shows that pure wheat, barley or rye will not produce erroneous results as with other methods. Intended Use: This assay is intended for screening for Gluten residue down to 5ppm in raw ingredients, finished products, CIP and surface swab testing. The dynamic range of this method has been shown to be between 1ppm and >100,000ppm for Gluten protein derived from Wheat, Barley or Rye or their cultivars. The upper limit of detection has not been reached even with pure toxic grains. The antibodies used in this assay have been shown not to react with: Oat, Soya, Rice, Millett, Corn, Sorghum, Egg, Flax seed, Amaranth, Spices, Herbs, Potato, etc. As with all methods for food analysis, this test should be validated with the specific matrix being evaluated to prove that there are no interfering substances, thereby making the test inaccurate. REQUIRED EQUIPMENT: Clean, bright flat surface @ room temperature (~21 C) HELPFUL EQUIPMENT: Pipettes (100µl & 1000µl, Vortex, Microcentrifuge) Storage Instructions: Store kit at 4-8 C (35-45 F). DO NOT FREEZE ANY KIT COMPONENTS Kit Reagents should not be exposed to prolonged UV light No quality is guaranteed after expiry of kit. Store in clean dry place. WHEN RUNNING TEST: Measure out desired allotment of Buffer. Keep remaining Buffer in fridge. WAIT until LFD, Buffer and Sample have reached Room temperature Before use. Once the Total Gluten LFD cartridge has been removed from sealed pouch the LFD should be used within 30min. Note: Prolonged exposure to excess moisture can render the test strip ineffective. 1 GTLN-1005
CIP Prep: MEASURE OUT DESIRED ALLOTMENT OF BUFFER AND BRING TO ROOM TEMPERATURE BEFORE USE. KEEP REMAINING BUFFER IN FRIDGE CIP: Measure 800µl of Extraction/Running buffer (provided) (1 drop from a transfer pipette = 25µl) into a microcentrifuge tube (provided) and using the same pipette, add 200µl of CIP solution, (for raw ingredient or finished product, see page 4) and mix well for approx. 30 sec. This is your Extracted Mixed Sample. *ph of sample should not be >10 or <5. If ph is above or below these levels, further dilute in buffer (i.e. extra 1:1 until it falls within these parameters). If analyzing more than one product, be sure to mark your pipette and to use a separate pipette for each sample to avoid cross contamination. (Proceed to page 5) Note: For Chocolate Samples See Amendment A (page 7) ALWAYS USE A NEW, UNUSED PIPETTE WHEN PIPETTING FROM THE EXTRACTION SOLUTION BOTTLE. NEVER USE A "DIRTY" PIPETTE OR PREVIOUSLY USED PIPETTE! 2 GLTN-1005
Swab Prep: MEASURE OUT DESIRED ALLOTMENT OF BUFFER AND BRING TO ROOM TEMPERATURE BEFORE USE. KEEP REMAINING BUFFER IN FRIDGE Swab Samples: Remove a single microcentrifuge tube from kit and mark it appropriately for future reference. Add 500µl (0.5ml) of extraction buffer into your pre-marked microcentrifuge tube, then take a clean swab and dip the tip into the microcentrifuge tube wetting the tip with buffer. Gently wick excess liquid from the tip by pressing the swab tip lightly on the side of the tube. Now take wetted swab and survey the surface you want to test by moving the swab back and forth on the area of interest. Take the swab and insert it back to the premarked tube and swirl swab several times to release any residues that might be on the surface of the tube into the buffer. (Proceed to page 5) Note: For Chocolate Samples See Amendment A (page 7) 3 GLTN-1005
Raw Ingredients and Finished Products Prep: MEASURE OUT DESIRED ALLOTMENT OF BUFFER AND BRING TO ROOM TEMPERATURE BEFORE USE. KEEP REMAINING BUFFER IN FRIDGE LIQUID: Raw Ingredients or Finished product: -Remove a single microcentrifuge tube from kit and mark it appropriately. -Measure 900µl of Extraction/Running buffer into previously marked tube. Then add 100µl of a well representative sample. -Mix well (Proceed to page 5). Note: If sample is too viscous and does not run well on LFD, centrifuge, or further dilute before proceeding to page 5. NON-liquid samples: -Grind sample into a fine powder and add about 0.1g of sample to a pre-marked microcentrifuge tube or add the powdered sample to just above the conical bottom of the microcentrifuge tube. -Then add 0.9ml of extraction buffer. -Mix well by hand for ~30 sec (or vortex if available for ~30 sec) -Allow the tube to sit for ~1-2 min so as to dissolve any gluten into the extraction buffer then re-mix sample. -Let tube sit until the non-liquid portion settles to the bottom of the centrifuge tube and upper liquid is mostly clear (or if centrifuge is available run for ~20 sec @ 5K) (If you would like to use a centrifuge and don t have one please call ET and we can provide you one at low cost) (Proceed to page 5). Note: For Chocolate Samples See Amendment A (page 7) 4 GLTN-1005
Procedure: 1. Remove one Gluten LFD from the package and place on a clean, dry, flat surface. WAIT UNTIL LFD REACHES ROOM TEMPERATURE BEFORE USE 2. Measure 100µl (1 drop from a transfer pipette = 25µl) using the same transfer pipette used for the extracted & mixed sample. NOTE: If testing a Dry Sample extract the 100µl from the middle layer of the settled or centrifuged sample. 3. Add the 100µl to the sample dispense area (round opening) at one end of the cartridge and start your timer. 5 GLTN-1005
Results and Interpretations: Read LFD 5 minutes after introduction of sample. A sample is considered to be positive for Gluten allergen when 2 lines form on the LFD. The top line (the control line), will be to the left of the letter C. The second line (the test line), will appear to the left of the letter T on the LFD. If both lines (the Test Line (T) and the Control Line (C)) are present at this reading, this may indicate a high concentration of Gluten protein, >10ppm, however, reading this strip again at 11mins is required for confirmation. Read LFD again at 11 minutes (+/- 1min) after introduction: At this point: -A sample is considered to be negative for Gluten allergen when only the top line forms on the LFD. The line will appear to the left of the Letter C on the LFD. -A sample is considered to be positive for Gluten allergen when both lines form on the LFD (See above) >5ppm. -If the LFD test does not develop the top line, the individual LFD should be considered defective. Any reading after 15 minutes from the initial introduction of the sample into the LFD should be considered null and void. A reading at this time cannot be interpreted and can lead to erroneous results. 6 GLTN-1005
Amendment A: MEASURE OUT DESIRED ALLOTMENT OF BUFFER AND BRING TO ROOM TEMPERATURE BEFORE USE. KEEP REMAINING BUFFER IN FRIDGE CHOCOLATE: LIQUID: Raw Ingredients or Finished product: Measure 500µl of warm (~60 C) Extraction/Running buffer (provided) and add it to 500µl of the liquid sample into a microcentrifuge tube (provided), mix well, then return to the beginning of page 4 (LIQUID: Raw Ingredients or Finished product). Note: If sample is too viscous and does not run well on LFD, centrifuge, or further dilute before returning to page 4. NON-liquid samples: grind sample into a fine powder and add about 0.5g of sample to the micro-centrifuge tube, then add 500µl of warm (~60 C) Extraction/Running buffer (provided) and mix well, allowing the chocolate to melt. *Return to the beginning of page 4 (LIQUID: Raw Ingredients or Finished product)* Flours or ingredients containing high concentrations of gums (xanthium, etc.) might not flow efficiently across the test strip. If this is the case, take 0.3g of sample and dilute it with 600ml of 60% EtOH(Sigma-Aldrich #277649 or LabChem #LC22204 (diluted) or equivalent) mix for 30 sec or centrifuge for 20 sec. Then take 100µl from the liquid layer and follow directions from the Liquid Prep on page 4. TROUBLESHOOTING: Q. Sample fails to migrate across the strip within the first 5 min. A. The sample might need to be centrifuged if this was not already done in the prep. If the sample was centrifuged then further dilute the sample 1:1 with buffer (Note: this will reduce the sensitivity to ~10ppm for some matrices) Q. A red dot appears on the test line but all else is clear. A. Sometiimes sample particulates manage to pass around the filter in the cassette, simply re-centrifuge the sample and take a new test strip from the kit and repeat the test. 7 GLTN-1005