FinTest TM IgG4 Screen 88 ELISA Kit
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1 FinTest TM IgG4 Screen 88 ELISA Kit Cat. No.:DEIA6227 Pkg.Size:96T Intended use The Kit is for the quantitative determination of IgG4 antibodies against 88 Food Allergens in human serum, plasma and capillary blood. General Description Lucretius once said that one man s food may be another man s poison. Individuals with allergies and other types of food sensitivities react adversely to certain foods and food ingredients that others can consume with no problems. Many different types of reactions are involved in these individual adverse reactions. Adverse food reactions may include IgE and non-igemediated primary immunological sensitivities, non immunological intolerances(such as an enzyme deficiency or reactions to certain chemicals), as well as secondary sensitivities. Adverse immune reactions to foods which are not IgE mediated are often called food intolerances. The absence of IgE does not make them any less real; instead, other immune mechanisms, such as IgG or IgG4 antibodies are involved. IgG or IgG4- mediated adverse reactions to food are characterized by less severe reactions, are much more common (affecting approx. 45 % of the population) and have delayed onset (2 to 72 hours) after ingestion of an offending substance. Avoiding ingestion of such food (exclusion or rotation diet) is the best treatment to decrease symptoms. There are several evidences for the involvement of IgG or IgG4 in food "intolerances e.g.: Allergic reactions may occur independently of antigen-specific IgE Subsequent decrease of IgG when the offending food is removed from the diet Specific serum IgG has been reported in cases of celiac disease, dermatitis, or atopic eczema, as well as in diseases with increased intestinal permeability, and inflammatory bowel disease (IBD) This evidence leads to the recommendation that IgG or IgG4-specific testing should be considered in cases where the patient shows unclear and chronic disorders, and in cases where classical diagnostics show no evidence. Principle Of The Test The FinTestTM IgG4 Screen 88 ELISA test kit is based on the principle of the enzyme immunoassay (EIA). 88 different allergens and 8x reference antigen for standards and controls are bound on the surface of a microtiter plate (see page before last). Diluted patient sample or ready-to-use standards and controls are pipetted into the wells of the microtiter plate. A binding between the IgG4 antibodies of the sample and the immobilized allergens takes place. After one hour incubation at 37, the plate is rinsed with diluted wash solution, in order to remove unbound material. Then ready-to-use anti-human-igg4-ap conjugate is added and incubated for 30 minutes at 37. After a further washing step, the substrate (PNPP) solution is pipetted and incubated for 60 minutes at 37, inducing the development of a yellow dye in the wells. The colour development is terminated by the addition of a stop solution. The resulting dye is measured spectrophotometrically at the wavelength of 405 nm. The concentration of the IgG4 antibodies is directly proportional to the intensity of the colour. Reagents And Materials Provided 1. 1 x 12 x 8 - MTP Microtiter Plate: Break apart strips. Coated with 88 different nutritional allergens and 8 x reference allergen (Standards and Controls). Ready to use. 1
2 2. 1 x 15 ml - IgG Enzyme Conjugate IgG4: Ready to use. Contains goat-a-human-igg4-ap in proteinacious buffer solution x 6 x 0.5 ml - Calibrator A - F 0.35, 0.70, 3.5, 17.5, 50, 100 U/mL: Ready to use. Contains: IgG4 antibodies against reference allergen, human plasma diluted with PBS/BSA, 0.05% sodium azide x 2 x 0.5 ml - Control 1+2: Ready to use. Contains: IgG4 antibodies. Addition of 0.05% sodium azide x 40 ml - Diluent Buffer Ready to use. Contains: Tris/BSA buffer, <0.1% sodium azide x 60 ml - Wash Buffer, Concentrate (10x) Contains: PBS + Tween x 15 ml - Substrate Solution: Ready to use. Contains: Paranitrophenylphosphate (PNPP) x 15 ml - PNPP Stop Solution: Ready to use.1 M sodium hydroxide x FOIL Adhesive Foil x Distribution Plan Materials Required But Not Supplied 1. Micropipettes (Multipette Eppendorf or similar devices, < 3% CV). Volumes: 25;100; 500;1000 µl 2. 8-Channel Micropipettor with reagent reservoirs 3. Tubes (1 x 10 ml) for sample dilution 4. Wash bottle, automated or semi-automated microtiter plate washing system 5. Microtiter plate reader capable of reading absorbance at 405 nm (reference wavelength nm) 6. Bidistilled or deionised water 7. Incubator, Paper towels, pipette tips and timer Storage The kit is shipped at ambient temperature and should be stored at 2-8. Keep away from heat or direct sun light. The storage and stability of specimen and prepared reagents is stated in the corresponding chapters. The microtiter strips are stable up to the expiry date of the kit in the broken, but tightly closed bag when stored at 2 8. Specimen Collection And Handling Serum, Plasma The usual precautions for venipuncture should be observed. It is important to preserve the chemical integrity of a blood specimen from the moment it is collected until it is assayed. Do not use grossly hemolytic, icteric or grossly lipemic specimens. Samples appearing turbid should be centrifuged before testing to remove any particulate material. Storage: 2-8 /2days; -20 />2days. Keep away from heat or direct sun light. Avoid repeated freeze-thaw cycles. Capillary blood Storage: 2-8 /2days, Keep away from heat or direct sun light. Avoid repeated freeze-thaw cycles. Reagent Preparation 1. Preparation of Components Table one. 2
3 2. Dilution of Samples Table two. Assay Steps 1. For each patient sample prepare one Microtiter Plate. 2. Pipette 100 µl of each Standard, Control and diluted serum, plasma or capillary blood into the respective wells. The reference wells are located at the first eight positions (Standards A-F; Controls G-H) of strip number Cover plate with adhesive foil. Incubate 60 min at Remove adhesive foil. Discard incubation solution. Wash plate 3 x with 300 µl of diluted Wash Buffer. Remove excess solution by tapping the inverted plate on a paper towel. 5. Pipette 100 µl of Enzyme Conjugate into each well. 6. Cover plate with adhesive foil. Incubate 30 min at Remove adhesive foil. Discard incubation solution. Wash plate 3 x with 300 µl of diluted Wash Buffer. Remove excess solution by tapping the inverted plate on a paper towel. 8. For adding of Substrate and Stop Solution use, if available, an 8-channel Micropipettor. Pipetting should be carried out in the same time intervals for Substrate and Stop Solution. Use positive displacement and avoid formation of air bubbles. 9. Pipette 100 µl of PNPP Substrate Solution into each well. 10. Cover plate with adhesive foil. Incubate 60 min at Stop the substrate reaction by adding 100 µl of PNPP Stop Solution into each well. Briefly mix contents by gently shaking the plate. 12. Measure optical density with a photometer at 405 nm (Reference-wavelength: nm) within 60 min after pipetting of the Stop Solution. Quality Control The test results are only valid if the test has been performed following the instructions. Moreover the user must strictly adhere to the rules of GLP (Good Laboratory Practice) or other applicable standards/laws. All kit controls must be found within the acceptable ranges as stated on the vial labels. If the criteria are not met, the run is not valid and should be repeated. Each laboratory should use known samples as further controls. It is recommended to participate at appropriate quality assessment trials. In case of any deviation the following technical issues should be proven: Expiration dates of (prepared) reagents, storage conditions, pipettes, devices, incubation conditions and washing methods. Evaluation The ready-to-use standards of the FinTest IgG4 Screen 88 ELISA test kit are defined and expressed in arbitrary units (U/mL). This results in an exact and reproduce quantitative evaluation. Consequently for a given patient follow-up controls become posse. For a quantitative evaluation the absorptions of the standards are graphically drawn against their concentrations. From the resulting reference curve the concentration values for each patient sample can then be extracted in relation to their absorptions. It is also posse to use automatic computer programs. Interpretation of Results 3
4 The evaluation can be performed either in units per ml (U/mL) or in classes. This classification scheme has no established clinical significance and is only provided to give the physician a relative ranking of the IgG4 response. Figure one. The above figure for OD measurements contains only an example, which was achieved under in-house temperature and environmental conditions. The described data constitute consequently no reference values which have to be found in each laboratory accordingly. Reproducibility The intra-assay coefficient of variation of FinTest IgG4 Screen 88 ELISA was assessed by ten-fold determination of positive serum sample to less than 10%. Precautions 1. Before starting the assay, read the instructions completely and carefully. Use the valid version of the package insert provided with the kit. Be sure that everything is understood. 2. In case of severe damage of the kit package please contact or your supplier in written form, latest one week after receiving the kit. Do not use damaged components in test runs, but keep safe for complaint related issues. 3. Obey lot number and expiry date. Do not mix reagents of different lots. Do not use expired reagents. 4. Follow good laboratory practice and safety guidelines. Wear lab coats, disposable latex gloves and protective glasses where necessary. 5. Reagents of this kit containing hazardous material may cause eye and skin irritations. See MATERIALS SUPPLIED and labels for details. Material Safety Data Sheets for this product are available on the - Homepage or upon request directly. 6. Chemicals and prepared or used reagents have to be treated as hazardous waste according to national biohazard and safety guidelines or regulations. 7. Avoid contact with Stop solution. It may cause skin irritations and burns. 8. Some reagents contain sodium azide (NaN3 ) as preservatives. In case of contact with eyes or skin, flush immediately with 4
5 water. NaN3 may react with lead and copper plumbing to form explosive metal azides. When disposing reagents, flush with a large volume of water to avoid azide build-up. 9. All reagents of this kit containing human serum or plasma have been tested and were found negative for anti-hiv I/II, HBsAg and anti-hcv. However, a presence of these or other infectious agents cannot be excluded absolutely and therefore reagents should be treated as potential biohazards in use and for disposal. REFERENCES 1. Aas K: The diagnosis of hypersensitivity to ingested foods. Clinical Allergy 1978; 8: AMA Council on Scientific Affairs, In Vitro Testing for Allergy. Report II of the Allergy Panel Council on Scientific Affairs. JAMA, 1987, 258(12):
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