AgraQuant F.A.S.T. Casein Sandwich ELISA Order #: COKAL1248F Casein According to EU Directive 2003/89/EG the addition of bovine milk has to be labelled. For the detection of bovine milk in foodstuffs, sensitive detection systems are required. Approximately 80% of bovine milk proteins are caseins which are composed of -, - and -caseins. These heat-stable allergens represent the main fraction of bovine milk proteins. Bovine milk is one of the most important allergenic food ingredients, especially for children. Very low amounts of bovine milk can cause allergic reactions, which may lead to anaphylactic shock in severe cases. Because of this, milk allergic persons must strictly avoid the consumption of milk or foods containing milk. In particular, the risk of hidden milk proteins in foods such as sausage, cookies, ready meals or beverages represent a critical problem for milk allergic persons. Short Instruction: Dispense 150 L sample extract or standards into transfer wells (blue) Transfer 100 L sample extract or standards into the antibody coated wells (colourless) using a multichannel pipette and incubate for 10min Wash 5 times with wash buffer Tap dry washed wells Pipette 100 L conjugate solution into the antibody coated wells and incubate for 10min, wash the wells afterwards as described in step 3 & 4 Pipette 100 L substrate solution into the wells and incubate for 10min in the dark Add 100 L stop solution into the antibody coated wells Read Results at 450 nm with an ELISA reader Page 1 of 8
Performance Characteristics: Number of determinations: 48 (incl. Standards) Extraction time: approx. 1min Assay time: approx. 30min LOD: Foodstuffs: 0.2 ppm Casein Swabs: 0.02 g/swab Casein Sponge: 0.2 g/sponge Casein Rinse water: 20 g/l Casein LOQ: Foodstuffs: 1 ppm Casein Swabs: 0.1 g/swab Casein Sponge: 1 g/sponge Casein Rinse water: 100 g/l Casein Range: 1-25 ppm Casein Sample Preparation / Extraction Allow all the reagents to reach room temperature (20-25 C) before use. Wash buffer: Dilute the wash buffer concentrate 1:20 (transfer contents of bottle to 1 L screwcap bottle, top up to the 1,000 ml mark with distilled or deionised water and shake), and store at 2-8 C. Before starting the test, allow approx. 50 ml of the wash buffer to reach room temperature for each strip of 8 wells. I.Procedure for foodstuffs: Carefully homogenize a representative amount of the sample to be analysed (e.g. melt and stir chocolate). 1. Bring the required amount of distilled or deionised water (20 ml per sample plus a reserve) to the boil in a kettle, or heat to 80-100 C using a water-bath. 2. Accurately weigh 1 g of sample into a 50 ml centrifuge tube and add one capsule each of extraction additives 1 and 2. 3. Add 20 ml hot, but not boiling water from Step 1. Tightly close the centrifuge tube and shake vigorously for 15 seconds (until sample and capsules are dissolved). 4. Transfer an aliquot to a 1.5 or 2 ml centrifuge tube and centrifuge at 8,000 g for 5 min, use the supernatant for testing. Alternatively, filter through a pleated filter paper and use the filtrate in the test. If Page 2 of 8
required, dilute with specially prepared sample dilution buffer (=sample dilution factor F). Leave the sample extracts to cool down to room temperature before carrying out the ELISA. Sample dilution buffer: Make as described in Steps 1 4 with both extraction additives, but without sample material. II.Procedure for environmental samples (swabs): Sampling 1. Moisten swab with water and rub on the surface using horizontal movements, turning the swab while doing so. Repeat procedure with vertical movements. 2. Break off or cut off swab and transfer to a 10 ml graded centrifuge tube. Extraction 1. Bring the required amount of distilled or deionised water (at least 20 ml) to the boil in a kettle, or heat to 80-100 C in a water-bath. 2. Add one capsule each of extraction additives 1 and 2 per 20mL water, and shake vigorously for 15 seconds (until the capsules are dissolved). 3. Add 2 ml of the extraction buffer thus prepared into the 10 ml centrifuge tube containing the swab, close tightly and shake vigorously for 15 seconds. 4. Transfer the liquid into a 2 ml reaction vial and centrifuge at 8,000 g for 5 min. Alternatively, filtrate through a pleated filter. If required, dilute with the extraction buffer from Step 2 (=sample dilution factor F). Leave the sample extracts to cool down to room temperature before using in ELISA. III.Procedure for environmental samples (sponge): Sampling 1. Moisten dry sponges with water and rub on the surface using horizontal and vertical movements. Sponges that are moist can be used immediately. 2. Transfer the sponge to a 50 ml graded centrifuge tube. Note: The sponge may need to be cut into small pieces (e.g. with a knife or scissors). Page 3 of 8
Extraction 1. Bring the required amount of distilled or deionised water (20 ml per sample plus a reserve) to the boil in a kettle, or heat to 80-100 C in a water-bath. 2. Add one capsule each of extraction additives 1 and 2 to the centrifuge tube containing the sponge. 3. Top up to the 20 ml mark with the hot, but not boiling water from Step 1 (or add 20 ml). Tightly close the centrifuge tube and shake vigorously for 15 seconds. 4. Transfer an aliquot to a 1.5 or 2 ml reaction vial and centrifuge at 8,000 g for 5 min. Alternatively, filter through a pleated filter paper. If required, dilute with specially prepared sample dilution buffer (=sample dilution factor F). Leave the sample extracts to cool down to room temperature before using in ELISA. Sample dilution buffer: Make as described in Steps 1 4 with both extraction additives, but without sponge. IV.Procedure for rinse water: Sample preparation Dilute the rinse water (ph 4-9) 1:2 in a suitable vial using sample dilution buffer (make as described in Section III., but without sponge), e.g. dispense 0.5 ml rinse water into the vial, add 0.5 ml sample dilution buffer and shake. Dilute with the same buffer if required (= sample dilution factor F). Assay Procedure in Detail To guarantee comparable incubation times, use no more than three colourless or blue strips of 8 (24 wells each) for each test assay. 1. Insert the number of colourless wells and wells marked blue required for the standards and samples into the frame. 2. Dispense 150 μl of the standard solution or sample extract (room temperature) prepared from the previous section into the wells marked blue. Using the multichannel pipette, transfer 100 µl into the colourless wells and leave to incubate at 20-25 C for 10 min. Page 4 of 8
3. Empty the wells and remove any residual liquids by knocking them out well over laboratory-grade absorbent tissue. Washing procedure: Pipette 300 μl wash buffer into the wells, then empty the wells. Repeat this procedure four times (wash 5 times all together). Finally, thoroughly knock out residual quantities onto laboratory-grade absorbent tissue. 4. Pipette 100 μl conjugate solution (green cap) into the wells and leave to incubate at 20-25 C for 10 min. Then wash 5 times as described in Step 3. 5. Pipette 100 μl substrate solution (blue cap) into the wells and leave to incubate at 20-25 C in the dark for 10 min. 6. Add 100 μl of stop solution (red cap), then measure the absorbance at 450 nm. Interpretation of results I.Interpretation for foodstuffs: Evaluation should be done using a 4-parameter curve-fit. Dilute samples with absorbance values (A 450 nm ) > standard 5 with sample dilution buffer to within the range of the standard curve. Evaluation must account for the sample dilution factor (F) and the sample weight as shown in Equation (1). The result shows the sample s casein content. Conversion to the skim milk powder content is done based on Equation (2). (1) casein [mg/kg (l)] = value from standard curve F sample weight [g (ml)] (2) skim milk powder [mg/kg (l)] = casein [mg/kg (l)] 3.5 II.Interpretation for environmental samples (swabs): The swab s absolute casein content is calculated using Equation (3). Conversion to the skim milk powder content is done based on Equation (4). (3) casein [µg/swab] = value from standard curve F 0.1 (4) skim milk powder [µg/swab] = casein [µg/swab] 3.5 Page 5 of 8
III.Interpretation for environmental samples (sponge): The sponge s absolute casein content is calculated using Equation (5). Conversion to the skim milk powder content is done based on Equation (6). (5) casein [µg/sponge] = value from the standard curve F (6) skim milk powder [µg/sponge] = casein [µg/sponge] 3.5 IV.Interpretation for rinse water: The casein content is calculated using Equation (7). Conversion to the skim milk powder content is done based on Equation (8). (7) casein [µg/l] = value from standard curve F 100 (8) skim milk powder [µg/l] = casein [µg/l] 3.5 Materials supplied 48 x wells (blue) for sample transfer, in a bag 48 x wells coated with antibodies, in a bag 1 x frame for the wells 5 x standards 1, ready to use (white caps): 0, 1, 5, 10, 25 mg/kg (ppm) 1 x wash buffer concentrate, 20 x conc. (50 ml) 1 x conjugate solution (green cap), ready to use 1 x substrate solution (blue cap), ready to use 1 x stop solution (red cap), ready to use 1 x extraction additive 1 (big capsule) 1 x extraction additive 2 (small capsule) 1 The sample s casein content can be read off the standard curve. The concentrations given already include the dilution factor that results from sample preparation (1:20). Page 6 of 8
Materials required but not supplied Homogenizer, impact mill, mortar grinder etc. for sample preparation Water boiler or water-bath (80-100 C) 50 ml and 15 ml graduated centrifuge tubes 1.5 or 2 ml centrifuge tubes Microcentrifuge > 8,000 g for 1.5 or 2 ml tubes Micropipettes and pipette tips (20-200 μl and 200-1000 μl) Multichannel pipette and pipette tips (100-300 µl) Microtiter plate photometer (450 nm) Screw-cap bottles (1 L) for storing the wash buffer Distilled or deionised water Technical and Background Information AgraQuant F.A.S.T. Casein is a sandwich enzyme immunoassay (ELISA) for the detection and quantitative determination of milk casein (native and processed) in foods, environmental samples and rinse waters. The test is based on the antigen-antibody reaction. The wells in the microtiter plate are coated with casein-specific antibodies, to which the standards or sample extracts are added. The milk protein is now bound in an antigen-antibody complex. Non-bound quantities are removed by washing. Next, a horseradish peroxidase coupled antibody (conjugate) is added which in turn binds to the casein protein. Excess conjugate is removed once more by washing. Addition of a colourless substrate starts off the blue staining. The stop solution added next will cause the colour to change from blue to yellow. The absorbance measured at 450 nm is proportional to the sample s casein content. Precautions The test kit components can be used until the expiry date given on the package if stored at 2-8 C; do not freeze. Keep unused wells in the foil bag, together with the desiccant, and ensure that it is kept closed. The substrate is sensitive to light. Avoid direct exposure to light. Never swap around reagents from kits of different lot numbers. Page 7 of 8
Indication of reagents no longer being suitable for use: The substrate solution is bluish even before it is added to the reagents in the wells. The absorbance (A 450 nm ) of standard 5 is less than 0.6. Carefully follow the instructions when conducting the test. Skin contact with scalding hot water can cause burns. Wear heat protection gloves if required. The stop solution contains 1 mol/l sulphuric acid (H290, H315, H319; P280, P302+P352, P305+P351+P338, P309, P310, P406). For further information please contact: Romer Labs UK Ldt. Tel: +44 845 519 50 10 Block 5, The Heath Business & e-mail: enquiry@romerlabs.com Technical Park Runcorn, Cheshire WA7 4QX, UK www.romerlabs.com Warranty The user assumes all risk in using Romer Labs, Inc. products and services. Romer Labs, Inc. will warrant that its products and services meet all quality control standards set by Romer Labs, Inc., and Romer Labs, Inc. will, at its option, repair or replace any product, components, or repeat services which prove to be defective in workmanship or material within product specific warranty periods or expiration dates and which our examination shall disclose to our satisfaction to be defective as such. This warranty is expressly in lieu of all other warranties, expressed or implied, as to description, quality, merchantability, fitness for any particular purpose, productiveness, or any other matter. Romer Labs, Inc. shall be in no way responsible for the proper use of its products. Romer Labs, Inc. hereby disclaims all other remedies, warranties, guarantees or liabilities, expressed or implied, arising by law or otherwise, and it shall have no liability for any lost profits or damage, direct, indirect or otherwise, to person or property, in connection with the use of any of its products or services. This warranty shall not be extended, altered or varied except by a written instrument signed by an authorized representative of Romer Labs, Inc. Page 8 of 8