Antibodies of class IgE against food allergens Test instruction for the EUROLINE Food
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1 ORDER-NO. Antibodies of class IgE against food allergens Test instruction for the EUROLINE Food ANTIBODIES AGAINST DP E food allergens IgE IG-CLASS SUBSTRATE FORMAT test-strips coated with allergens 16 x 01 (16) Indications: lip, tongue and/or throat burning and itching, diarrhoea, vomiting, abdominal pain, asthma, erythema or urticaria caused by allergic reactions. Principle of the test: The EUROLINE test kit provides a semi-quantitative in vitro assay for human IgE antibodies to food allergens in serum or plasma. The test kit contains test strips coated with parallel lines of 20 different allergen extracts. The test strips are first moistened and then incubated in the first reaction step with patient serum. If samples are positive, specific antibodies of class IgE will bind to the allergens. To detect the bound antibodies, a second incubation is carried out using an enzyme-labelled monoclonal anti-human IgE (enzyme conjugate), which is capable of promoting a color reaction. Contents of a test kit: Descritption Format Symbol 1. Test strips coated with the allergens: f1, f75, f2, f45, f4, f5, f9, f14, f13, f17, f20, f49, f84, f237, f25, f31, f35, f85, f3, f23 16 x 1.STRIPS. 2. Enzyme conjugate Alkaline phosphatase-labelled anti-human-ige (mouse), ready 1 x 20 ml.conjugate. for use 3. Universal buffer 10x concentrated 1 x 100 ml.buffer 10x. 4. Substrate solution Nitrobluetetrazoliumchloride/5-Bromo-4-chloro-3- indolylphosphate (NBT/BCIP), ready for use 1 x 30 ml.substrate. 5. Evaluation protocol 1 Protocol 6. Incubation tray 2 x 8 channels TRAY 7. Plastic foil 1 sheet 8. Instruction booklet 1 booklet Lot storage temperature In-vitro diagnostics unopened usable until Storage and stability: The test kit must be stored at a temperature between +2 C to +8 C. Do not freeze. Unopened, all test kit components are stable until the indicated expiry date. Waste disposal: Patient samples, controls and incubated test strips should be handled as infectious waste. Other reagents do not need to be collected separately, unless stated otherwise in official regulations.
2 Preparation and stability of the reagents Note: The individual reagents of one lot are matched with one another and should not generally be swapped with reagents of another lot. All reagents must be brought to room temperature (+18 C to +25 C) around 30 minutes before use. After first use, the reagents are stable until the indicated expiry date if stored at +2 C to +8 C and protected from contamination, unless stated otherwise below. - Coated test strips: Ready for use. Open the package with the test strips only when the strips have reached room temperature to prevent condensation on the strips. After removement of the strips the package should be sealed tightly and stored at +2 C to +8 C. - Enzyme conjugate: Ready for use. Mix thoroughly before using. - Universal buffer: The universal buffer is supplied as a 10x concentrate. For the preparation of the ready for use universal buffer shake the bottle (50 ml). The amount required should be removed from the bottle using a clean pipette and diluted 1:10 with deionised or distilled water. Due to the special membrane used for the present EUROLINE the ready for use universal buffer is used for the dilution of patient samples, the dilution of the enzyme conjugate and the washing of the test strips. For the incubation of 1 test strip 2.0 ml buffer concentrate should be diluted with 18.0 ml deionised or distilled water. The ready for use universal buffer should be used on the same working day. - Substrate solution: Ready for use. Close bottle immediately after use, as the contents are sensitive to light. Warning: Some of the reagents are poisonous (buffer, substrate solution). Avoid contact with skin. Preparation and stability of the patient samples Sample material: Human serum or EDTA, heparin or citrate plasma. Stability: Patient samples to be investigated can generally be stored at +2 C to +8 C for up to 14 days. Diluted samples should be incubated within one working day. Sample dilution: The patient samples to be investigated are used undiluted. Should only a limited amount of the sample be available, the serum or plasma can be diluted 1:11 with the pre-diluted universal buffer. The diluted sample is left to incubate overnight. For example: dilute 100 µl sera in 1,0 ml pre-diluted universal buffer and mix thoroughly by vortexing. (sample pipettes are not suitable for mixing). 2
3 Incubation Pretreat: Serum incubation (1 st step): Washing: Place the required amount of test strips in the incubation tray. Fill each of the channels with 1.0 ml universal buffer and incubate the test strips for 5 minutes. Afterwards aspirate off all the liquid. Fill each channel with 1.0 ml of undiluted serum sample and incubate at room temperature for 60 minutes on a rocking shaker. (Alternatively 1:11 diluted sera can be incubated over night at room temperature on a rocking shaker. Cover the incubation tray to prevent evaporation) Aspirate off the liquid from each channel and wash for 3 x 5 minutes with 1.0 ml universal buffer on a rocking shaker. Conjugate incubation Pipette 1.0 ml enzyme conjugate (alkaline phosphatase conjugated anti- (2 nd step): human IgE, monoclonal) into each channel and incubate for 60 minutes at room temperature on a rocking shaker. Washing: Aspirate off the liquid from each channel and wash for 3 x 5 minutes with 1.0 ml universal buffer on a rocking shaker. Chromogen/substrate Pipette 1.0 ml chromogen/substrate solution into each channel. incubation (3 rd step): Incubate for 10 minutes at room temperature on a rocking shaker. Stopping: Evaluate Aspirate off the liquid from each channel and wash each strip 3 x with deionised or distilled water. Place test strip on the evaluation protocol, air dry and evaluate. Note: If an automated incubation system is used, the incubation volume can be increased to 1.5 ml depending on the cannel volume of the incubation trays used. 3
4 EUROLINE Food Incubation protocol Pre-treatment: Put the blot strip into the incubation channel containing 1.0 ml universal buffer 5 min Rocking Shaker 1a. Sample incubation: Aspirate off, pipette 1.0 ml of undiluted serum sample into the incubation channel 1b. Sample incubation: Aspirate off, pipette 1.0 ml of 1:11 diluted serum sample into the incubation channel 60 min Rocking shaker over night Washing: 3 x 5 min Aspirate off, wash 3 x 5 min with 1 ml universal buffer each 2. Incubation with conjugate: Aspirate off, pipette 1.0 ml of enzyme conjugate into the incubation channel 60 min Rocking shaker Washing: 3 x 5 min Aspirate off, wash 3 x 5 min with 1 ml universal buffer each 3. Incubation with substrate: Aspirate off, pipette 1.0 ml of chromogen/substrate solution into the incubation channel 10 min Rocking shaker Stopping: Aspirate off, rinse with water Evaluate Fix the test strips to the evaluation protocol, air dry and evaluate 4
5 Interpretation of results Handling: Place the wet strips after incubation onto the plastic foil (enclosed for visual evaluation, for digital evaluation the protocol is to be previously entered into the EUROLineScan-Program) on the evaluation protocol and align with the marks. Carefully dab the strips with absorbent paper. After drying the strips stick to the plastic foil. Incubated strips that are still moist show a background colouring that disappears when they are completely dry. Therefore the evaluation of the strips is only to take place after the strips have completely dried. For visual evaluation distinclty recognizable bands on the dried blot strips which correspond with marks on the evaluation matrix are entered in the evaluation protocol. For digital evaluation instuctions described in the EUROLineScan user s manual are to be followed. Attention: Three indicator bands are located on the lower end of the strip. The incubation was performed correctly if different colour reactions are visible on these control bands. A semiquantitative evaluation of these bands is possible by visual comparison of the signal with the indicator bands. Some samples might display a dark background staining of the membrane and white bands at the position of the antigens. These unspecific reactions have to be interpreted as negative. Missing indicator bands indicate improper incubation. The incubation should be repeated with new reagents. Allergens: The test strips include the following allergens: f1 f75 f2 f45 f4 f5 f9 f14 f13 f17 f20 f49 f84 f237 f25 f31 f35 f85 f3 f23 In 1 In 2 In 3 position abbr. allergen 1 f1 egg white 2 f75 egg yolk 3 f2 milk 4 f45 yeast 5 f4 wheat flour 6 f5 rye flour 7 f9 rice 8 f14 soybean 9 f13 peanut 10 f17 hazelnut 11 f20 almond 12 f49 apple 13 f84 kiwi 14 f237 apricot 15 f25 tomato 16 f31 carrot 17 f35 potato 18 f85 celery 19 f3 codfish 20 f23 shrimp a In 1 indicator band 1 b In 2 indicator band 2 c In 3 indicator band 3 Interpretation of results: 5
6 In order to evaluate the signals, the band positions and intensity of staining must be taken into consideration. By comparing the incubated test strips with the printed evaluation strip, the allergens against which IgE antibodies are present can be identified. The signals can be divided into 4 classes which correspond to the bands: Class Result Characteristics 0 negative; no clinical significance no band + low allergen-specific IgE concentration; partial clinical significance weak band signal ++ moderate allergen-specific IgE concentration; often with clinical symptoms clear band signal +++ high allergen-specific IgE concentration; clinical symptoms in most cases intense band signal When using the digital evaluation system EUROLineScan the intensity of the bands are calculated in EAST classes of 0-6. EAST is the abbreviation for Enzyme-Allergo-Sorbent-Test and is with respect to the concentration grades identical to the well-known RAST system (Radio-Allergo-Sorbent-Test) used in allergy diagnostics. The classes can be divided into the following concentrations: Class Concentration [ku/l] Result 0 < 0,35 ku/l No specific antibodies detected 1 0,35 ku/l 0,7 ku/l 2 0,7 ku/l 3,5 ku/l 3 3,5 ku/l 17,5 ku/l 4 17,5 ku 50 ku/l Very weak antibodies detected, frequently no clinical symptoms where sensitisation is present. Weak antibodies detected, existing sensitisation, frequently with clinical symptoms in the upper range of class. Significant level of antibodies detected. Clinical symptoms usually present. High level of antibodies detected. Almost always with clinical symptoms ku/l 100 ku/l Very high antibody titre. 6 > 100 ku/l Very high antibody titre. Test characteristics Measurement range: The EUROLINE is a semi-quantitative method. The measurement range is given in the EAST system in the classes from 0-6. Cross reactions: Due to the similar structure of the allergens, e.g., similarities in chemical substances or botanical relations, cross reactions may occur. The specific IgE antibodies that have developed in a patient also attach to identical epitopes of the homologous protein allergen. 6
7 Examples for cross-reactivities between airborne allergens and food allergens pollinosis grass birch mugwort ragweed plantain latex associated food allergy tomato, potato, carrot, celery, garlic, onion, wheat, rice, green pea, peanut, apple, peach, orange, watermelon, melon, kiwi hazelnut, walnut, apple, pear, carrot, celery, potato, orange, kiwi celery, carrot, spices, green bean, mustard, hazelnut watermelon, melon, cucumber, banana melon avocado, potato, banana, tomato, chestnut, kiwi Interference: Haemolytic, lipaemic and icteric sera up to a concentration of 5 mg/ml for haemoglobin, of 20 mg/ml for triglycerides and of 0.4 mg/ml bilirubin showed no effect on the analytical results of the present EUROLINE. Inter- and intra-assay variation: The inter-assay variation was determined by multiple analyses of characterised samples over several days. The intra-assay variation was determined by multiple analyses of characterised samples on one day. In every case, the intensity of the bands was within the specified range. This EUROLINE displays excellent inter- and intra-assay reproducibility. Clinical significance An allergy is a hypersensitivity to foreign substances which are normally harmless but which produce a violent reaction in the allergy sufferer. Allergies are not exclusively due to airborne allergens such as pollen, dust and molds but as well can be caused by ingested food. The most common foods causing allergic reactions are peanuts, soy, wheat, shellfish, fish, milk, eggs and tree nuts. A food allergy is an IgE mediated reaction which leads to symptoms within hours of having ingested the food. Possible symptoms are lip, tongue and throat burning or itching, nausea, abdominal cramps, diarrhoea and erythema. Major symptoms can even include asthma, shortness of breath, fast heart rate, panic and confusion. Sometimes in cases where nuts, shellfish, fish and peanuts have been eaten anaphylaxis can occur. Some people may also experience allergy symptoms since conserved plant allergens can cause IgE antibodies to crossreact with allergens from plant derived food or other non-food allergens. For example, a person who is allergic to birch tree pollen may develop allergies to apples, carrots, celery, hazelnuts, potatoes or kiwi (for further details see list below). Furthermore, there exist adverse reactions to food which are not IgE mediated. These sensitivities are thought to be IgG mediated and involve a delayed pattern of reactions to food, usually occurring h after eating. The symptoms are more generalised and tend to be chronic or recurrent and persist for days. This in-vitro test in designed to detect specific IgE antibodies to identify food allergies. 7
8 Literature 1. Salkie ML. Role of clinical laboratory in allergy testing. Clin Biochem; 27/5: (1994). 2. Janeway CA, Traves P. Allergie und Hypersensibilität. Immunologie; Spektrum Akademischer Verlag (1997). 3. Eigenmann PA, Calza AM. Diagnosis of IgE-mediated food allergy among Swiss children with atopic dermatitis. Pediatr Allergy Immunol; 11/2: (2000). 4. Bock SA, Munoz-Furlong A, Sampson HA. Fatalities due to anaphylactic reactions to foods. J. Allergy Clin Immunol; 107/1: (2001). 5. Chandra RK. Food allergy and nutrition in early life: implications for later health. Proc Nutr Soc; 59/2: (2000). 6. Pascual CY, Crespo JF, Perez PG, Esteban MM. Food allergy and intolerance in children and adolescents, an update. Eur J Clin Nutr; 54 Suppl 1: (2000). 7. Sicherer SH, Morrow EH, Sampson HA. Dose-response in double-blind, placebo-controlled oral food challenges in children with atopic dermatitis. J Allergy Clin Immunol; 105/3: (2000). 8. Valenta R, Kraft D. Type I allergic reactions to plant-derived food: A consequence of primary sensitization to pollen allergens. J Allergy Clin Immunol; 97/4: (1996). 9. Caballero T, Martin-Esteban M. Association between pollen hypersensitivity and edible vegetable allergy: A review. Invest Allergol Clin Immunol; 8/1: 6-16 (1998). 10. Beezhold DH, Sussmann GL, Liss GM, Chang NS. Latex allergy can induce clinical reactions to specific food. Clin Exp Allergy; 26/4: (1996). 11. Dixon HS. Treatment of delayed food allergy based on specific immunoglobulin G RAST testing. Otolaryngol Head Neck Surg; 123 (1 Pt 1): (2000). DP_3410_A_UK_C01.doc :18
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