Egg Protein Residue ELISA Kit Catalog Number KA3208 96 assays Version: 01 Intended for research use only www.abnova.com
Table of Contents Introduction... 3 Intended Use... 3 Background... 3 Principle of the Assay... 3 General Information... 5 Materials Supplied... 5 Storage Instruction... 5 Materials Required but Not Supplied... 5 Precautions for Use... 5 Assay Procedure... 7 Reagent Preparation... 7 Sample Preparation... 7 Assay Procedure... 7 Data Analysis... 9 Interpretation of Results... 9 Resources... 10 Reference... 10 Plate Layout... 11 KA3208 2 / 11
Introduction Intended Use The Egg Protein Residue ELISA Kit is an enzyme-linked immunosorbent assay (ELISA) that may be used to screen appropriate samples for the presence of Egg protein residues. This assay is a rapid and sensitive test, which significantly reduces the time required to screen for the presence of Egg residues. Background The Egg Protein Residue ELISA Kit is designed to provide a sensitive and convenient method for screening food samples for Egg protein residues. Ovomucoid comprises approximately 11% of egg white protein. This factor should be taken into consideration when assessing the potential total egg protein concentration and the potential allergenic issues in the sample being tested. Hen s egg is one of the more frequent causes of food hypersensitivity in infants and young children(1,2). Studies have shown that Ovomucoid (also known as trypsin inhibitor from chicken egg white) is a major allergen of egg whites.(3,4) Ovomucoid is not coagulated by heat, whereas the other potential allergenic components do coagulate.(5) Ovomucoid comprises approximate 11% of egg white protein. It has been demonstrated that Ovomucoid and not Ovalbumin, is the dominant allergen in egg white protein for humans. It also appears to be allergenic in minute quantities and because of its hardy physical characteristics, it may remain in the body in an allergenic state for years. (4) These properties. make Ovomucoid a suitable marker protein for the presence of egg residues (egg white) in food products. Principle of the Assay The Egg Protein Residue ELISA Kit uses anti-egg trypsin inhibitor antibodies coated onto microwells to absorb the trypsin inhibitor protein. Once the sample and enzyme conjugate are added to the microwells, a positive test yields a double antibody sandwich indicated by a deep blue color. Stop solution is added, and positive results change from blue to yellow. Results may be read visually or with an ELISA reader. The level of egg trypsin inhibitor will vary according to the ingredients and the manufacturing process. This test may not detect egg protein material that has been significantly altered by processes, such as fermentation and hydrolysis. This assay will detect residues of non-denatured egg trypsin inhibitor present in the sample, which indicates the presence of egg protein. A negative test result, however, cannot conclusively rule out the presence of egg protein. The supplied positive control serves to indicate the approximate levels of egg trypsin inhibitor present in the sample and not necessarily an indication of the total egg protein present. This factor KA3208 3 / 11
must be taken into consideration when assessing the potential total egg protein concentration and the allergenic issues associated with the sample being tested. Note: Results are for screening purposes only. KA3208 4 / 11
General Information Materials Supplied List of component Component Test strips: Microwells containing anti egg trypsin inhibitor antibodies in a strip holder. Negative Control: a buffered base. Positive Control: Egg trypsin inhibitor in a buffer to provide a control value of 125 ng/ml. Enzyme Conjugate: Peroxidase conjugated anti-egg trypsin inhibitor polyclonal antibodies with preservative. Chromogen: Stabilized tetramethylbenzidine (TMB). Wash Buffer Concentrate (20X): Concentrated buffer and surfactant with Thimerosal. Extraction Solution Concentrate (20x): Concentrated buffer extraction solution and surfactant with Thimerosal. Stop solution: 1 M phosphoric acid. Amount 48-wells 2 vials x 1.5 ml 2 vials x 1.5 ml 6 ml 11 ml 2 bottles x 25 ml 4 bottles x 25ml 11 ml Storage Instruction Reagents, strips and bottled components: Store between 2 7 C. Squeeze bottle containing diluted wash buffer may be stored at room temperature. Materials Required but Not Supplied Pipetter, 100 µl Disposable micropipette tips Optional: Microelisa plate reader capable of reading bichromatic at 450/620-650 nm (optional) Precautions for Use 1. Important note: Do not use reagents if they precipitate or become cloudy. Exception: Wash concentrate may precipitate during refrigerated storage but will dissolve upon warming. Controls and some reagents contain a preservative. The ph of the samples should be in the range ph 6.8 7.4 Do not pipette directly from the reagent bottles as this may contaminate these solutions. KA3208 5 / 11
Do not pour unused reagents back into their bottles. Pipette the required amount into a clean test tube for use. Always firmly reseal the foil bag containing the antibody coated strips, to prevent moisture degradation. KA3208 6 / 11
Assay Procedure 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 for 1 min. Allow to settle. Filter if necessary (as for solid samples). Supernatant or filtrate is then the sample to be tested on the kit. For Contact Surface Samples Place swab into a minimal volume of heated extraction buffer. Mix well. Assay Procedure Controls must be included each time the kit is run. Temperature should be in the 15 to 25 C range while running the assay. 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). KA3208 7 / 11
3. Add 100 µl of the test sample to the appropriate test well. 4. Incubate at room temperature for 30 minutes, then wash (see below for washing instructions) 5. Add 100 µl of Enzyme Conjugate to each well. 6. Incubate at room temperature for 15 minutes, then wash. 7. Add 100 µl of Substrate to each well. 8. Incubate at room temperature for 5 minutes. Note: DO NOT WASH AT THIS STAGE. 9. Add 100 µl of Stop Solution to each well. Mix wells by gently tapping strip holder. 10. Read results visually or on a spectrophotometer using a bichromatic reading, with the filters set at 450nm and 620-650nm. Zero the reader on air. Washing Instructions: Each washing set consists of dumping out the contents of the wells and using the diluted wash buffer to fill each well to overflowing, shaking out the contents and refilling the wells for a total of 3 to 5 times. After the final wash, slap test wells against an absorbent towel to remove any excess wash buffer. 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. KA3208 8 / 11
Data Analysis Interpretation of Results 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 the same or greater intensity than the positive control is suspected to contain egg protein at a level of 1,250 ng/ml or greater. If the sample well has less yellow color than the positive control well, the sample is likely to contain less egg protein than 1,250 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 significant yellow color in the positive control, the test should be regarded as invalid and the test should be repeated.. ELISA Reader Zero the ELISA Reader on air. Read all wells using a bichromatic reading with filters at 450nm and 620-650nm. An absorbance (OD) reading equal to, or greater than, the reading of the positive control well indicates the sample contains Egg protein of at least 1,250 ng/ml. An absorbance reading (OD) less than the reading of the positive control well, indicates the sample contains less than 1,250 ng/ml.. Troubleshooting Problem: Negative control has substantial color development. Correction: Washings were insufficient. Repeat test with more vigorous washings. KA3208 9 / 11
Resources Reference 1. Sampson. H.A., McCakill. C.C. Food hypersensitivity and atopic dermatitis: evaluation of 113 patients. J Pediatr 1985; 107:669-75. 2. Bock. S.A., Atkins.F.M. Patterns of food hypersensitivity during sixteen years of double-blind, placebo controlled food challenges. J Pediatr 1990;117:561-7. 3. Bleumink. E, Young. E., Studies on the atopic allergen in hen s egg.ii. Further characterization of the skin reactive fraction in egg-white immunoelectrophoresis studies. Int Arch Allergy 1971; 40:72-88 4. Bernhisel-Broadbent J, Dintzis. H.M., Dintzis. R.Z., Sampson.H.A.. Allergenicity and antigenicity of chicken egg ovomucoid (Gal d III) compared with ovalbumin (Gal d I) in children with egg allergy and in mice. J Allergy Clin Immunol 1994;93:1047-59. 5. Atsuo Urisu, Hitoshi Ando, Yutaka Morita, Eiko Wada, Takehiko Yasaki, Kazue Yamada, Katsuhide Komada, Shimpei Torii, Masahiro Goto, Toshio Wakamatsu. Allergenic activity of heated and ovomucoid-depleted egg white. J Allergy Clin Immunol 1997;100: 171-6 KA3208 10 / 11
Plate Layout A B C D E F G H 1 2 3 4 5 6 7 8 9 10 11 12 KA3208 11 / 11