V.1/May/2012 Gluten (Gliadin) Lateral Flow IIR For the quick detection of wheat protein contaminations in foods and food-processing equipments 20 Test Sticks For Laboratory Use Only Please read full descriptions in this manual before use. Manufactured by: Morinaga Institute of Biological Science, Inc. (MIoBS) 2-1-16 Sachiura, Kanazawa-ku, Yokohama 236-0003, Japan E-mail: info@miobs.com Website: http://www.miobs.com/english/index.html
Intended Use Gluten (Gliadin) Lateral Flow IIR determines quickly whether or not wheat proteins reside in foods, including unprocessed foodstuffs and processed products (TABLE 1). The kit is also applicable to swab tests for the contamination on surfaces of food-processing equipments. The kit detects gluten (gliadin) specifically, as a representative of wheat proteins, by the use of anti-gliadin antibodies. The novel extraction solution provided with the kit enhances the extraction recovery yield of wheat proteins from test samples, even from extensively processed foods, leading to more reliable results than ever. For the best results, please follow the instructions exactly. TABLE 1: Performance characteristics Sample preparation time Chromatographic run time Detection limit 30 min 15 min 5 µg wheat proteins/g food (5 ppm) Kit Storage 1. Store the kit at 2-8 C (35-46 F), and DO NOT FREEZE. 2. Do not use the kit after the expiration date indicated on the box. Kit Components Kit components are listed in TABLE 2. TABLE 2: Kit components Mark Component Amount A Reagent A (10X concentrate) 1 bottle (50 ml) B Reagent B (10X concentrate) 1 bottle (50 ml) C Reagent C (10X concentrate) 1 bottle (50 ml) D Reagent D 1 bottle (25 ml) E Reagent E 1 bottle () F Test stick 20 packs (1 stick/pack) Materials required but not provided Homogenizer/blender Balance Volumetric pipettes and volumetric cylinders Vortex mixer Water bath Centrifuge (for 3000 x g) Funnel and paper filter Polypropylene centrifugation tubes (15 and 50 ml) with screw caps Micropipettes and disposable tips (ranging 0.1-1 ml) Polypropylene test tubes (1-) ph test paper
Warnings and Precautions 1. Operators with allergy to bovine serum albumin (BSA) should pay special attentions when handle the kit, because the kit contains BSA as a basic component. In case of allergic symptoms, seek medical aids promptly. 2. Wear suitable protective clothes, goggles and gloves when handle the kit. 3. All operations should be performed under contamination-free conditions for reliable results. Make sure to avoid cross-contaminations via equipments, devices, tubes, containers, pipette tips, etc., and the use of disposable materials are recommended. 4. Do not combine reagents from different lots. 5. Pipette exact volumes of reagents and samples for accurate results. 6. Duplicated tests are recommended for one sample to ensure the results. Note: The extensive denaturation of wheat proteins during processing can lead gliadin unreactive to the antibody and/or scarcely soluble, which may result in false-negative results. Thus, possibilities cannot be ruled out in such processed foods that denatured wheat proteins are still contained even though the test result with this kit shows wheat-protein free. Reagent Preparation Prior to use, bring all reagents to room temperature (20-25 C/68-77 F) and agitate the contents gently to make them homogeneous. Reagent A may form precipitates during storage under refrigeration. If this is the case, dissolve the precipitates completely before use by warming to 30-37 C (86-99 F). Although Reagent C may become milky during storage under refrigeration, it exhibits full performance and no deterioration can be detectable when used as it is. Preparation of Sample Extraction Solution Prepare Sample Extraction Solution in a total volume of 20 ml per one test sample, by mixing Reagents A, B, C and distilled water as follows. Adjust the volume in proportion to the number of test samples. Sample Extraction Solution Reagent A Reagent B Reagent C Water* 14 ml (*Distilled or deionized water) Total Volume 20 ml Preparation of Diluting Solution Prepare Diluting Solution in a total volume of 1.0 ml per one test sample, by mixing Reagents D and E as follows. Adjust the volume in proportion to the number of test samples. Diluting Solution is recommended to be prepared after preparation of Sample Extract and used as promptly as possible. Diluting Solution Reagent D Reagent E Total Volume 0.9 ml 0.1 ml 1.0 ml
Sample Extraction and Preparation of Test Solution Solid and liquid food samples 1. Grind and mix up the test food sample to homogeneity with a contamination-free homogenizer/blender. 2. A disposable 50-mL polypropylene tube, containing 1.0 g of the homogenized sample and 19 ml of Sample Extraction Solution, is capped tightly and vortexed for 30 seconds. 3. Expose the capped tube to 100 C (212 F) in boiling water for 10 minutes. 4. Place the tube in running water to cool it down to ambient temperature (it takes some 10 minutes). 5. Vortex the tube for 30 seconds. 6. Check the fluid ph with ph test paper, and neutralize (ph 6-8) with HCl or NaOH, if required. 7. Centrifuge the tube at 3,000 g for 20 minutes at 20-30 C (68-86 F), and the supernatant is retained as Sample Extract. (Filter the supernatant with filter paper, if necessary.) 8. Dilute the Sample Extract (0.1 ml) by 10-fold with Diluting Solution (0.9 ml), and the resulting solution is referred to as Test Solution. Surface swab test samples 1. Wipe off a specified surface area (e.g. 10cm X 10cm) thoroughly with a water-moistened cotton swab. 2. Put the cotton swab into a disposable 15-mL polypropylene tube containing 3 ml of Sample Extraction Solution, immerse the cotton in solution, put the screw cap on tube, and vortex extensively to extract proteins from swab into Sample Extraction Solution. 3. Expose the capped tube to 100 C (212 F) in boiling water for 10 minutes. 4. Place the tube in running water to cool it down to ambient temperature (it takes some 10 minutes). 5. Vortex the tube for 30 seconds and filter if necessary. 6. Check the fluid ph with ph test paper, and neutralize (ph 6-8) with HCl or NaOH, if required. The resulting solution thus prepared is denoted Sample Extract. 7. Dilute the Sample Extract (0.1 ml) by 10-fold with Diluting Solution (0.9 ml), and the resulting solution is referred to as Test Solution. Note: False-negative results may occur when Test Solution contain very high concentrations of gliadin, due to the high-dose hook effect that is well known in one-step immunoassay systems. When the test sample is suspicious of high gliadin content, tests with Test Solution diluted much more than 10-fold are recommended. In this case, adjust the dilution appropriately by diluting Sample Extract with Sample Extraction Solution, and keep the dilution with Diluting Solution (10-fold) constant.
Chromatography Procedures 1. Place the test stick horizontally and apply a 200-µL aliquot of Test Solution to the sample application slot. 2. Leave it standing for 15 min at room temperature. 3. Judge the results according to the criteria described below. Judgment criteria A: A red-purple line in the test window together with red sign in the confirmation window indicates POSITIVE result. B: No red-purple line in the test window together with red sign in the confirmation window indicates NEGATIVE result. C: No red sign in the confirmation window indicates that the test was unsuccessful due to chromatographic failure. A. Positive B. Negative C. Unsuccessful Red sign Red-purple line Note: When no red sign is found in the confirmation window, repeat the test with a new stick. Samples with high viscosity may result in the unsuccessful chromatography. Warranty Morinaga Institute of Biological Science, Inc. makes no warranty of any kind, either expressed or implied, except that the materials from which its products are made are of standard quality. Buyer assumes all risk and liability resulting from the use of this product. There is no warranty of merchantability of the product, or of the fitness of the product for any purpose. Morinaga Institute of Biological Science, Inc. agrees to replace any defective product, but expressly disclaims liability for damages, including special or consequential damage, or expenses arising directly or indirectly from the use of this product.