Instructions for Morinaga Acrylamide EIA Kit

Transcription

1 V.3/ Apr/2014 Instructions for Morinaga Acrylamide EIA Kit - For the quantitative determination of acrylamide in foods - Please read before use 96 Assays Sachiura , Kanazawa-ku, Yokohama , JAPAN 13

2 INTRODUCTION The Morinaga Acrylamide EIA kit enables simultaneous determinations of the acrylamide content in multiple test food samples (up to 24 food samples when triplicate determinations are performed) within a short period of time (approx. 4 h except for sample extraction). Since an antibody to acrylamide cannot be raised in animals by immunization simply using acrylamide, an acrylamide derivative (3-CTBA; 3-[(2-carbamoylethyl)thio] benzoic acid) was employed as a hapten. For this reason, this kit must derivatize acrylamide quantitatively to 3-CTBA using a large excess of 3-MBA (3-mercaptobenzoic acid) prior to ELISA. The antibody recognizes 3-CTBA specifically and does not cross-react with intact acrylamide or 3-MBA. Cross-reactivity was found to be only 0.3% and 0.6% for metacrylamide and acrylonitrile, respectively, the highest found among the structural analogs examined. The kit permits a sensitive and specific assay of acrylamide with a detection limit of 0.4 ng/ml (RSD=30%) and a quantification range of ng/ml (RSD<20%). The results of ELISA are highly correlated with those of conventional methods (GC/MS/MS and LC/MS/MS), and show satisfactory accuracy, within 20% of authentic values when examined with certified reference materials. The kit uses no organic solvents at all, and is eco-friendly. Since the assay employs a competitive ELISA method, in principle, the triplicate determination protocol is recommended for reliable results. It is also recommended for calculation that computer software capable of 4-parameter logistic curve fitting be used. KIT CONTENTS Label Name of Component Quantity Content A 3-CTBA-coated Microplate Module (8 wells/module) 2 packs 6 modules/ pack B Acrylamide Standard (500 ng/ml) 2 vials 1.1 ml/vial C 3-MBA (3-Mercaptobenzoic Acid) 2 vials 48 mg/vial D 1 N Sodium Hydroxide Solution 1 vial 2.4 ml E Rabbit Anti-3-CTBA Antibody 2 bottles 3.5 ml/bottle F Enzyme-conjugated Goat Anti-rabbit IgG Antibody 2 bottles 6.5 ml/bottle G Enzyme Substrate (TMB Solution) 1 bottle 13 ml H Stop Solution (1 N Sulfuric Acid) 1 bottle 13 ml I Sample Dilution Buffer 1 bottle 13 ml J Washing Buffer (20 Concentrate) 1 bottle 50 ml Frame for Mounting the Microplate Module 1 piece Microplate Cover 1 piece 14

3 MATERIALS REQUIRED FOR SAMPLE EXTRACTION AND ELISA, BUT NOT INCLUDED IN THE KIT FOR SAMPLE EXTRACTION 1. Gravimetric balance (precision better than ±0.01 g) 2. Homogenizer, e.g. POLYTRON (generator shaft Type DA3012/2EC, Kinematica GmbH, Kriens/Luzern, Switzerland) 3. Grinder/blender, e.g. MILLCER (Type IFM-800DG, Iwatani Co. Ltd., Tokyo, Japan) 4. Centrifugation tubes (capacity greater than 20 ml) 5. Graduated pipette (20 ml) 6. Polypropylene tubes (5-10 ml) 7. Centrifuge (centrifugal force of more than 3,000 g, preferably 12,000 g) 8. Solid-phase extraction cartridges (ISOLUTE Multimode 500 mg/3 ml, Biotage Japan Ltd.) 9. Micropipette (dispensing range of µl) 10. Vortex mixer FOR ELISA 1. Micropipettes (dispensing ranges of µl and µl) 2. Multi-channel micropipette (8 or 12 channels, dispensing range of µl) 3. Graduated cylinder (1,000 ml) 4. Polypropylene tubes (1.5 ml) 5. Polypropylene tubes with air-tight cap (1.5 ml) 6. Microplate shaker 7. Pipette reservoir 8. Vortex mixer 9. Air-bath incubators for 25ºC (77ºF) and 37ºC (99ºF) 10. Microplate reader (for 96 wells) It is strongly recommended to use a microplate reader (primary wavelength: 450 nm; secondary wavelength: nm) connected to a computer having software for 4-parameter logistic curve-fitting. 15

4 REAGENT PREPARATION 1. ACRYLAMIDE EXTRACTION FROM THE TEST FOOD SAMPLE 1) Homogenize the test food sample (entire content of one serving package in one portion) by a method appropriate to the nature of the food (i.e. solids or liquids). A solid matrix should be ground as finely as possible to facilitate effective acrylamide extraction. Insufficient grinding/homogenization of the test food sample will lead to underestimation/poor reproducibility, and extensive pulverization/mixing is essential for reliable results. 2) Weigh exactly 1.00 g of the homogenized test food sample and place it in a 50 ml centrifugation tube, then add 19.0 ml distilled water using a graduated pipette. 3) Acrylamide is extracted from the test food sample into water by vigorous agitation of the mixture using an appropriate homogenizer/blender. Typically, extraction with a POLYTRON homogenizer at 15,000 rpm for 2 min at 20-30ºC (68-86ºF) with generator shaft Type DA3012/2EC (Kinematica GmbH, Kriens/Luzern, Switzerland) will suffice. Alternatively, three consecutive off-and-on cycles of extraction at 30-sec intervals using a MILLCER (Type IFM-800DG, Iwatani Co. Ltd., Tokyo, Japan), a safety food processor like a Waring blender, is also applicable. 4) Centrifuge the tube containing the homogenate in step 3 at 3,000 g (preferably 12,000 g) for 20 min at 20-30ºC (68-86ºF), and retain the supernatant. 5) Condition a solid-phase extraction cartridge (ISOLUTE Multimode 500 mg/3 ml; Biotage Japan Ltd.) before use by successive washings with 1 ml aliquots of methanol (once) and water (twice). The conditioning is facilitated by pushing the fluids out of the cartridge using a syringe, with a rubber sealant sandwiched at the interface between cartridge and syringe to prevent air leakage (see Figure 1). To the conditioned cartridge, apply a 1.0 ml aliquot of the supernatant in step 4 (discard the effluent during application), then elute with 3.0 ml distilled water. The eluate (3.0 ml), collected in a polypropylene tube, is vortexed thoroughly, and is defined as the test food sample extract. Figure 1 Syringe with a rubber sealant 16

5 2. SERIAL DILUTIONS OF THE ACRYLAMIDE STANDARD (CALIBRATOR) Dilute Acrylamide Standard (500 ng/ml) [B] with distilled water serially by a factor of 3 using separate 1.5 ml polypropylene tubes, as indicated below. This set of standard acrylamide solutions (0-500 ng/ml) is used for ELISA calibration. Acrylamide Concentration (ng/ml) Acrylamide Standard ( L) 600 Distilled Water ( L) Total Volume ( L) MBA SOLUTION The total amount of 3-MBA [C] is dissolved in 1 ml of 1 N Sodium Hydroxide Solution [D] by vortexing, to make a 48 mg/ml solution (light-yellow colored). Use this solution promptly. Avoid any eye or skin contact with this solution. 4. WASHING BUFFER Dilute 50 ml of Washing Buffer (20 concentrate) [J] by a factor of 20 with distilled water in a graduated cylinder, to make up 1,000 ml of the working Washing Buffer solution. 5. OTHER COMPONENTS Components described below are ready-to-use. [A] 3-CTBA-coated Microplate Module [E] Rabbit Anti-3-CTBA Antibody [F] Enzyme-conjugated Goat Anti-rabbit IgG Antibody [G] Enzyme Substrate (TMB Solution) [H] Stop Solution (1 N Sulfuric Acid) [I] Sample Dilution Buffer 17

6 ELISA PROCEDURES For reliable results, it is strongly recommended in this ELISA to perform triplicate determinations on all samples, including acrylamide standards and test food sample extracts. This kit is supplied, and should be kept, under refrigeration (2-8ºC, 36-46ºF) until use. After unpacking the kit, let all the individual components stand at the ambient temperature for equilibration before unsealing, in order to avoid moistening them with condensation. Once unsealed, however, they should be used immediately. DERIVATIZATION REACTION 1. Each of the acrylamide standards for calibration (0, 0.69, 2.06, 6.17, 18.5, 55.6, 167, 500 ng/ml) and test food sample extracts (after the solid-phase extraction treatment as described under REAGENT PREPARATION) is dispensed as a 200 L aliquot into separate polypropylene tubes (1.5 ml), followed by the addition of a 40 L aliquot of 3-MBA solution. Then cap all tubes tightly and mix the contents well by vortexing. 2. Incubate the tubes at 37ºC (99ºF) for 2 h to derivatize the acrylamide to 3-CTBA (3-[(2-carbamoylethyl)thio] benzoic acid). In this step, acrylamide is quantitatively converted to 3-CTBA by reacting with a large excess of 3-MBA. 3. After the incubation, each tube receives an 240 L aliquot of Sample Dilution Buffer [I], to make up a total volume of 480 L. The resulting solutions thus prepared are defined as the derivatized samples and subjected to ELISA. FIRST ANTIBODY REACTION 1. Fix 3-CTBA-coated Microplate Modules [A] to the Plate Frame, and then place the above described derivatized samples, including the acrylamide standards and test food sample extracts, in the wells in 50 L aliquots. 2. Place a 50 L aliquot of Rabbit Anti-3-CTBA Antibody [E] in all wells, and mix the solutions in each well thoroughly for 20 sec with a microplate shaker. It is an absolute prerequisite for obtaining accurate and reproducible results that a constant amount of the antibody is dispensed quickly (e.g. within 3 min) by using a well-calibrated multi-channel micropipette (8 or 12 channels), so as to minimize well-to-well differences in both the antibody concentration and reaction time. Although it is not necessarily required to replace micropipette-tips each time, it is important to avoid cross-contamination between neighboring rows. 3. Cover the plate with the plastic Microplate Cover, and incubate at 20-30ºC (68-86ºF) for 1 h. In this step, the antibody binds to the 3-CTBA immobilized on the solid-phase surface to form 18

7 3-CTBA/Rabbit anti-3-ctba antibody complex. If extra 3-CTBA is present in solution in the well along with the antibody, 3-CTBA s on the solid-phase surface and in solution will compete for the antibody. Thus, the higher the 3-CTBA concentration in the derivatized samples, the less antibody will bind to the solid phase. SECOND ANTIBODY REACTION 1. After completing the first antibody reaction, discard the solution inside the wells and wash wells 6 times with 300 L/well of fresh Washing Buffer. In each wash cycle, remove the wash fluid from wells as completely as possible by tapping the plate on a pile of clean paper towels. 2. Dispense a 100 L aliquot of Enzyme-conjugated Goat Anti-rabbit IgG Antibody [F] into each well. 3. Cover the plate with the plastic Microplate Cover, and incubate at 20-30ºC (68-86ºF) for 30 min. In this step, Enzyme-conjugated Goat Anti-rabbit IgG Antibody reacts with Rabbit Anti-3-CTBA Antibody on the solid phase, to form 3-CTBA/Rabbit Anti-3-CTBA Antibody/Enzyme-conjugated Goat Anti-rabbit IgG Antibody complex. ENZYME REACTION 1. After the second antibody reaction is over, discard the solution inside the wells and wash wells 6 times with 300 L/well of fresh Washing Buffer. In each wash cycle, remove the wash fluid from wells as completely as possible by tapping the plate on a pile of clean paper towels. 2. Dispense a 100 L aliquot of Enzyme Substrate (TMB Solution) [G] into each well quickly, using a multi-channel micropipette. 3. Cover the plate with the plastic Microplate Cover, and incubate at 20-30ºC (68-86ºF) for 30 min. During the incubation, a blue color will be developed by the action of the enzyme bound to the surface of the well. 4. Stop the enzyme reaction by adding a 100 L aliquot of the Stop Solution (1 N Sulfuric Acid) [H] into each well. The color will turn to yellow. 5. After stopping the enzyme reaction, measure the absorbance of each well at 450 nm (the primary wavelength) and nm (the secondary wavelength) with a 96-well microplate reader. Measure the absorbance within 30 min after stopping the enzyme reaction. 6. Calculate the acrylamide concentration in test food sample extracts. 19

8 SUMMARY OF ELISA PROCEDURES 1. Addition of a 200 L aliquot of Acrylamide Standards (0 and ng/ml) and test food sample extracts after solid-phase extraction, in separate 1.5 ml polypropylene tubes. 2. Addition of a 40 L aliquot of 48 mg/ml 3-MBA solution to all tubes. 3. Standing for 2 h at 37ºC (99ºF). 4. Addition of an 240 L aliquot of Sample Dilution Buffer [I], to make up a total volume of 480 L (defined as derivatized samples ). 5. Dispensing a 50 L aliquot of the derivatized samples, including the acrylamide standards and test food sample extracts, into ELISA microplate wells. 6. Dispensing a 50 L aliquot of Rabbit Anti-3-CTBA Antibody [E] to all wells. 7. Standing for 1 h at 20-30ºC (68-86ºF). 8. Six-fold washing of the well with each 300 L aliquot of the Washing Buffer. 9. Dispensing a 100 L aliquot of Enzyme-conjugated Goat Anti-rabbit IgG Antibody [F] into all wells. 10. Standing for 30 min at 20-30ºC (68-86ºF). 11. Six-fold washing of the well with each 300 L aliquot of the Washing Buffer. 12. Dispensing a 100 L aliquot of Enzyme Substrate [G] into all wells. 13. Light-protected standing for 30 min at 20-30ºC (68-86ºF). 14. Dispensing a 100 L aliquot of Stop Solution [H] into all wells. 15. Measurement of absorbance of all wells at 450 nm (the primary wavelength) and nm (the secondary wavelength) with a 96-well microplate reader. 16. Calculation of the acrylamide concentration in test food sample extracts (see page 21). 20

9 CALCULATION OF ACRYLAMIDE CONCENTRATION 1. Calculate the average absorbance of triplicate determinations (Measuring wavelength: 450 nm, subtracting a reference wavelength between 600 and 650 nm, inclusive) for each level of acrylamide standards (0, 0.69, 2.06, 6.17, 18.5, 55.6, 167, 500 ng/ml) as well as for test food sample extracts of unknown acrylamide concentrations. The calculation of B/B 0 described below employs those average absorbance values exclusively. 2. The absorbance for null acrylamide concentration is defined as B 0, and others as B. Calculate the ratio (B/B 0 ) for all acrylamide standards and test food sample extracts. B/B 0 = Absorbance for acrylamide standards ( ng/ml) or test food sample extracts Absorbance for null concentration of acrylamide standard (0 ng/ml) 3. Draw a semi-logarithmic graph of the calibration curve, where the vertical (linear) and horizontal (logarithmic) axes are B/B 0 and concentration of acrylamide standard, respectively, using the built-in graphing software of the microplate reader. For computer processing of the data, the 4-parameter curve fit (cubic regression) or a more suitable curve-fitting device is recommended. A typical calibration curve is shown in Figure The acrylamide concentration in the test food sample extract is interpolated from the calibration curve using B/B 0 values of each sample. 5. The acrylamide content in the test food sample (expressed as ng/g food) is calculated from the acrylamide concentration in the test food sample extract (expressed as ng/ml) according to the following equation. Acrylamide content in the test food sample (ng/g food) = Acrylamide concentration in the test food sample extract (ng/ml) 20 3 Dilution factor for the water-extraction step. Dilution factor for the solid-phase extraction step. 21

10 Figure 2 A typical calibration curve fitted to the 4-parameter logistic function. B/B0 =[ (a -d )/{1+(X/c)^b}]+d 22

11 WARNINGS AND PRECAUTIONS 1. This kit is for research use only. 2. Do not use the kit after its expiration date. 3. Do not use reagents from different lots in combination. 4. Wear suitable protection for eyes and skin, such as goggles and rubber gloves. 5. Avoid direct contact with the Acrylamide Standard (500 ng/ml) [B], Stop Solution (1 N Sulfuric Acid) [H] and Enzyme Substrate (TMB Solution) [G]. In case of contact, immediately flush eyes or skin with plenty of water and get medical advice. 6. Follow the local regulations on waste disposal. STORAGE CONDITIONS AND EXPIRY OF THE KIT 1. Store this kit at 2-8ºC (36-46ºF), and protect from light (NEVER freeze). 2. Use this kit before the expiration date. The expiration date is on the label (in the unsealed state). 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. TECHNICAL ASSISTANCE For further technical assistance or troubleshooting advice, contact Morinaga Institute of Biological Science, Inc. or your local distributor. Morinaga Institute of Biological Science, Inc. Telephone: Fax: info@miobs.com 23