Milk Protein Detection (beta-lacto) Residue Detection (In Food) (48 well)

Transcription

1 DIAGNOSTIC AUTOMATION, INC Craftsman Road, Suite D/E/F, Calabasas, CA Tel: (818) Fax: (818) See external label 2 C-8 C Σ=96 tests Cat # Milk Protein Detection (beta-lacto) Residue Detection (In Food) (48 well) Cat # INTENDED USE The Diagnostic Automation, INC. Milk Residue assay is an enzyme-linked immunosorbent assay (ELISA) that may be used to screen food products for the presence of this potential allergen caused by cross-contamination with dairy products. BACKGROUND Beta-Lactoglobulin is the major whey protein in ruminants and pigs but it is not found in the milk of many other species. (1) Milk from humans appears to be devoid of Beta-Lactoglobulin. No function has definitely been ascribed to this protein. The ability of Beta-Lactoglobulin to bind retinol, fatty acids and other hydrophobic substances suggest the biological function of this protein could be the transport of these substances to the newborn. (2) Human infants can develop an allergenic response to cow milk proteins. Beta-Lactoglobulin is the primary antigenic component that stimulates the immune hypersensitivity response in the infant. Symptoms are similar to lactose intolerance. (1) The Diagnostic Automation, INC. Milk Residue ELISA is a rapid and reliable test, which significantly reduces the time required to screen food products for the presence of Beta-Lactoglobulin. DAI Code # 3 1

2 PRINCIPLE OF PROCEDURE The Diagnostic Automation, INC. Milk Residue ELISA is a double antibody (sandwich) ELISA utilizing specific anti-beta-lactoglobulin antibodies coated onto microwells. After addition of the sample and the enzyme conjugate, a positive reaction (indicating the presence of Beta-Lactoglobulin) produces a deep blue color. Addition of the Stop Solution ends the assay and turns the blue color to yellow. The results may be read visually or with an ELISA reader. Note: Results are for screening purposes only and are not designed for quantitative results. REAGENTS SUPPLIED Test strips: microwells containing anti - Beta-Lactoglobulin antibodies- 48 test wells. Test strip holder: One (1) Negative Control: Two (2) vials containing 1.5 ml of a buffered base. Positive Control: Two (2) vials containing 1.5 ml of Beta-Lactoglobulin in a buffer to provide a control value of 5 ng/ml Enzyme Conjugate: One (1) bottle containing 6 ml of peroxidase conjugated anti-beta-lactoglobulin polyclonal antibodies with preservative. Chromogen: One (1) bottle containing 11 ml of stabilized tetramethylbenzidine (TMB). Wash Buffer Concentrate (20X): Two (2) bottles containing 25 ml each of concentrated buffer and surfactant with Thimerosal. Extraction Solution Concentrate (20x): Four (4) bottles containing 25 ml each of concentrated buffer extraction solution and surfactant with Thimerosal. Stop solution: One (1) bottle containing 11 ml of 1 M phosphoric acid. (CAUTION THIS SOLUTION IS ACIDIC) Additional Materials Required: Pipetter, 100 µl Disposable micropipette tips Data record sheets Disinfecting Solution Optional: Microelisa plate reader capable of reading bichromatic at 450/ nm (optional) PRECAUTIONS Do not use solutions if they precipitate or become cloudy. Exception: Wash concentrate may precipitate during refrigerated storage but will dissolve upon warming. Do not add azides to the samples or any of the reagents. Controls and some reagents contain a preservative. The ph of the samples should be in the range ph Always firmly reseal the foil bag containing the antibody coated strips, to prevent moisture degradation. STORAGE CONDITIONS Reagents, strips and bottled components: Store between 2 7 C. Squeeze bottle containing diluted wash buffer may be stored at room temperature. DAI Code # 3 2

3 REAGENT PREPARATION Wash Buffer Remove cap and add contents of one bottle to 475 ml DI water. Transfer contents of diluted wash buffer into a squeeze bottle (small tip bottle). Extraction Solution Remove cap and add contents of one bottle to 475 ml DI water. Transfer contents of diluted extraction solution into a Storage bottle. SAMPLE PREPARATION A representative sample must be taken from the product. Pre-Warm the Diluted Extraction Solution to 60 C FOR SOLID SAMPLES Weigh out 5 grams of sample into a suitable blender, a Stomacher type bag or a similar device. Add 50ml of the diluted Extraction Solution. A ratio of 1 part sample plus 10 volumes of the prepared Extraction Solution must be used. Blend or Stomach until the sample is homogenous and only minimal clumps are present. Complete mixing to remove lumps will help ensure consistent results. Allow to settle. Filter the extract through Filter paper #1 or similar. The filtrate is then the sample to be tested on the kit. FOR LIQUID SAMPLES Place 5 ml of sample into a suitable blender, bottle or similar device and add 45ml of the diluted Extraction Solution. A ratio of 1 part sample plus 9 volumes of the prepared Extraction Solution must be used for liquid samples. Shake or vortex for a minimum of 1 minute. Allow to settle. Filter if necessary (as for solid samples). Supernatant or filtrate is then the sample to be tested on the kit. TEST PROCEDURE Controls must be included each time the kit is run 1. Break off number of wells needed (number of samples plus 2 for controls) and place in strip holder. 2. Add 100 µl of the Neg. control to well #1 and 100 µl of Pos. control to well #2 (use both undiluted). 3. Add 100 µl of the test sample to the appropriate test well. 4. Incubate at room temperature for 10 minutes, then wash.* (Room Temperature should be C) 5. Add 2 drops of Enzyme Conjugate to each well. 6. Incubate at room temperature for 10 minutes, then wash.* Tap out plate against an absorbent towel. 7. Add 2 drops of Chromogen to each well. 8. Incubate at room temperature for 5 minutes. DO NOT WASH AT THIS STAGE 9. Add 2 drops of Stop Solution to each well. Mix wells by gently tapping strip holder. DAI Code # 3 3

4 10. Read results visually or on a spectrophotometer using a bichromatic reading, with the filters set at 450nm & nm. Zero the reader on air. Read Results within 2 hours of the addition of the Stop Solution. * Each washing consists of dumping the contents of the wells into an appropriate container with disinfecting solution (e.g. 3% bleach in water) and using the diluted wash buffer to fill to overflowing in each well, shaking out the contents and refilling the wells for a total of 5 times. Samples with sticky particulate matter may require more thorough washing than other samples. The potential exists for false positive results if the sample is not thoroughly washed from the well before addition of subsequent reagents. INTERPRETATION OF RESULTS Interpretation is based on the suggested extraction/dilution protocol Results are for screening purposes. The assay is not designed to yield quantitative results. Visual Compare the color of the sample well against the color of the positive control well. Any sample well that has a yellow color of the same or greater intensity than the positive control is suspected to contain Beta Lactoglobulin at a level above 5 ng/ml. If the sample well has less yellow color than the positive control well, the sample is likely to contain less Beta Lactoglobulin than 5 ng/ml. NOTE: The negative control, as well as some samples, may show some slight yellow color. The positive control should be a distinct yellow color. If there is no yellow color in the positive control, the test should be regarded as invalid and should be repeated. If the positive control again shows no color, then contact Diagnostic Automation, INC. immediately. ELISA Reader Zero the ELISA Reader on air. Read all wells using a bichromatic reading with filters at 450nm and nm. An Absorbance reading equal to or greater than the reading of the positive control well indicates the sample contains Beta Lactoglobulin equal to or greater than 5 ng/ml. An Absorbance reading less than the reading of the positive control indicates the sample contains less Beta Lactoglobulin than 5 ng/ml. Quality Control The use of a positive and negative control allows easy validation of kit stability. For a valid test, the controls should have the following OD readings: The 5 ng/ml positive control should be over 0.5 OD units The negative control must be under 0.15 OD units. Should the values fall outside these ranges, please contact Diagnostic Automation, INC.. Troubleshooting Problem: Negative control has substantial color development or OD value over 0.15 Correction: Washings were insufficient. Repeat test with more vigorous washings. DAI Code # 3 4

5 REFERENCES 1. Hurley, W.L. Milk Proteins and Protein Synthesis in Lactation Biology University of Illinois, Urbana-Champaign 2. Milk Science Home Page Beta-Lactoglobulin Date Adopted Reference No. DA-Milk Protein Detection (In Food)-2009 DIAGNOSTIC AUTOMATION, INC Craftsman Road, Suite D/E/F, Calabasas, CA Tel: (818) Fax: (818) ISO Revision Date: 7/15/09 DAI Code # 3 5