IgE ELISA Kit

Produktnummer ABIN649044

Produktdetails IgE ELISA Kit

Target: IgE

Reaktivität: Human

Nachweismethode: Colorimetric

Methodentyp: Sandwich ELISA

Applikation: ELISA

Verwendungszweck: Immunoenzymometric sequential assay (TYPE 4): The essential reagents required for an immunoenzymometric assay include high affinity and specificity antibodies (enzyme and immobilized), with different and distinct epitope recognition, in excess, and native antigen. In this procedure, the immobilization takes place during the assay at the surface of a microplate well through the interaction of streptavidin coated on the well and exogenously added biotinylated monoclonal anti-IgE antibody. Upon mixing monoclonal biotinylated antibody, and a serum containing the native antigen, reaction results between the native antigen and the antibody, forming an antibody-antigen complex. Simultaneously, the complex is deposited to the well through the high affinity reaction of streptavidin and biotinylated antibody. After a suitable incubation period, the antibody-antigen bound fraction is separated from unbound antigen by decantation or aspiration. Another antibody (directed at a different epitope) labeled with an enzyme is added. Another interaction occurs to form an enzyme labeled antibody-antigen-biotinylated-antibody complex on the surface of the wells. Excess enzyme is washed off via a wash step. A suitable substrate is added to produce color measurable with the use of a microplate spectrophotometer. The enzyme activity on the well is directly proportional to the native antigen concentration. By utilizing several different serum references of known antigen concentration, a dose response curve can be generated from which the antigen concentration of an unknown can be ascertained.

Analytische Methode: Quantitative

Bestandteile: A. Human Serum References (1. 0 ml/vial). Six vials of human serum based reference calibrators at concentrations of 0 (A), 5 (B), 25 (C), 50 (D), 150 (E) and 400 (F) IU/ml. Store at 2-8°C. A preservative has been added. (The Calibrators are standardized against WHO's 2ndIRP 75/502 for IgE). B. IgE Biotin Reagent (13 ml/vial). One vial of biotinylated anti-human IgE mIgG reagent presented in a protein-stabilized matrix. A preservative has been added. Store at 2-8°C. C. IgE Enzyme Reagent (13 ml/vial). One vial of anti-human IgE-HRP incorporated complex in a protein-stabilized matrix. A preservative has been added. Store at 2-8°C. D. Streptavidin Plate (96 wells). One 96-well microplate coated with streptavidin and packaged in an aluminum bag with a drying agent. Store at 2-8°C. E. Wash Solution Concentrate (20ml). One vial containing a surfactant in buffered saline. A preservative has been added. Store at 2-30°C. F. Substrate A (7. 0ml/vial). One bottle containing tetramethylbenzidine (TMB) in acetate buffer. Store at 2-8°C. G. Substrate B (7. 0ml/vial). One bottle containing hydrogen peroxide (H2O2) in acetate buffer. Store at 2-8°C. H. Stop Solution (8. 0ml/vial). One bottle containing a strong acid (1N HCl). Store at 2-8°C. Product Instructions: Note 1: Do not use reagents beyond the kit expiration date. Note 2: Opened reagents are stable for 60 days when stored at 2-8°C. Note 3: Above reagents are for a single 96-well microplate.

Benötigtes Material: 1. Pipette capable of delivering 25 & 50µl volumes with a precision of better than 1. 5%. 2. Dispenser(s) for repetitive deliveries of 0. 100ml and 0. 350ml volumes with a precision of better than 1. 5%. 3. Microplate washers or a squeeze bottle (optional). 4. Microplate Reader with 450nm and 620nm wavelength absorbance capability. 5. Absorbent Paper for blotting the microplate wells. 6. Plastic wrap or microplate cover for incubation steps. 7. Vacuum aspirator (optional) for wash steps. 8. Timer. 9. Quality control materials.

Anwendungsinformationen

Applikationshinweise: Precautions: All products that contain human serum have been found to be non-reactive for Hepatitis B Surface Antigen, HIV 1&2 and HCV Antibodies by FDA licensed reagents. Since no known test can offer complete assurance that infectious agents are absent, all human serum products should be handled as potentially hazardous and capable of transmitting disease. Good laboratory procedures for handling blood products can be found in the Center for Disease Control / National Institute of Health, Biosafety in Microbiological and Biomedical Laboratories, 2nd Edition, 1988, HHS Publication No. (CDC) 88-8395.

Probenmenge: 25 μL

Plattentyp: Pre-coated

Aufbereitung der Reagenzien:

1. Wash Buffer: Dilute contents of wash concentrate to 1000ml with distilled or deionized water in a suitable storage container. Store at room temperature (20-27°C) for up to 60 days. 2. Working Substrate Solution: Pour the contents of vial labeled Solution A into the vial labeled Solution B. Place the yellow cap on the mixed reagent for easy identification. Mix and label accordingly. Store at 2-8°C. Note: Do not use the working substrate if it looks blue.

Probennahme: The specimens shall be blood, serum in type and the usual precautions in the collection of venipuncture samples should be observed. For accurate comparison to established normal values, a fasting morning serum sample should be obtained. The blood should be collected in a plain redtop venipuncture tube without additives or anti-coagulants. Allow the blood to clot for samples. Centrifuge the specimen to separate the serum from the cells. Samples may be refrigerated at 2-8°C for a maximum period of five days. If the specimen(s) cannot be assayed within this time, the sample(s) may be stored at temperatures of -20 °C for up to 30 days. Avoid repetitive freezing and thawing. When assayed in duplicate, 0. 050ml of the specimen is required.

Ergebnisberechnung:

A dose response curve is used to ascertain the concentration of IgE in unknown specimens. 1. Record the absorbance obtained from the printout of the microplate reader. 2. Plot the absorbance for each duplicate serum reference versus the corresponding IgE concentration in IU/ml on linear graph paper (do not average the duplicates of the serum references before plotting). Draw the best-fit curve through the plotted points. 4. To determine the concentration of IgE for an unknown, locate the average absorbance of the duplicates for each unknown on the vertical axis of the graph, find the intersecting point on the curve, and read the concentration (in IU/ml) from the horizontal axis of the graph (the duplicates of the unknown may be averaged as indicated). Note: Computer data reduction software designed for IEMA (ELISA) assays may also be used for the data reduction.

Beschränkungen: Nur für Forschungszwecke einsetzbar

Handhabung

Handhabung: Before proceeding with the assay, bring all reagents, serum references and controls to room temperature (20-27°C). 1. Format the microplates' wells for each serum reference, control and patient specimen to be assayed in duplicate. Replace any unused microwell strips back into the aluminum bag, seal and store at 2-8°C. 2. Pipette 0. 025 ml (25µl) of the appropriate serum reference, control or specimen into the assigned well. 3. Add 0. 100 ml (100µl) of the IgE Biotin Reagent to each well. It is very important to dispense all reagents close to the bottom of the coated well. 4. Swirl the microplate gently for 20-30 seconds to mix and cover. 5. Incubate 30 minutes at room temperature. Discard the contents of the microplate by decantation or aspiration. If decanting, tap and blot the plate dry with absorbent paper. 7. Add 300µl of wash buffer (see Reagent Preparation Section), decant (tap and blot) or aspirate. Repeat two additional times for a total of three washes. An automatic or manual plate washer can be used. Follow the manufacturer's instruction for proper usage. If a squeeze bottle is employed, fill each well by depressing the container (avoiding air bubbles) to dispense the wash. Decant the wash and Repeat two additional times. 8. Add 0. 100 ml (100µl) of the IgE Enzyme Reagent labeled antibody to each well. DO NOT SHAKE THE PLATE AFTER ENZYME ADDITION. 9. Cover and incubate 30 minutes at room temperature. 10. Discard the contents of the microplate by decantation or aspiration. If decanting, blot the plate dry with absorbent paper. 11. Add 350µl of wash buffer (see Reagent Preparation Section), decant (tap and blot) or aspirate. Repeat two additional times for a total of three washes. An automatic or manual plate washer can be used. Follow the manufacturer's instruction for proper usage. If a squeeze bottle is employed, fill each well by depressing the container (avoiding air bubbles) to dispense the wash. Decant the wash and Repeat two additional times. 12. Add 0. 100 ml (100µl) of working substrate solution to all wells (see Reagent Preparation Section). Always add reagents in the same order to minimize reaction time. DO NOT SHAKE THE PLATE AFTER SUBSTRATE ADDITION. 13. Incubate at room temperature for 15 minutes. 14. Add 0. 050ml (50µl) of stop solution to each well and gently mix for 15-20 seconds. 15. Read the absorbance in each well at 450nm (using a reference wavelength of 620-630nm to minimize well imperfections) in a microplate reader. The results should be read within 30 minutes of adding the stop solution.

Lagerung: 4 °C/-20 °C

Details zu IgE

Target: IgE

Abstract: IgE Produkte

Synonyme: IGH2 ELISA Kit, IgE ELISA Kit, Gm900 ELISA Kit, immunoglobulin heavy chain (epsilon polypeptide) ELISA Kit, immunoglobulin heavy constant epsilon ELISA Kit, Immunoglobulin heavy constant epsilon ELISA Kit, Ighe ELISA Kit, IGHE ELISA Kit

Hintergrund: Summary and Explanation of the test: Allergic reactions, which are becoming more widespread, are usually diagnosed on the basis of medical history and clinical symptoms. In vitro and in vivo testing, however, play a key role in confirming clinical suspicions and tailoring treatment. The measurement of immunoglobulin E (IgE) in serum is widely used in the diagnosis of allergic reactions and parasitic infections. Many allergies are caused by the immunoglobulins of subclass IgE acting as point of contact between the allergen and specialized cells. The IgE molecules (MW 200,000) bind to the surface of the mast cells and basophillic granulocytes. Subsequently the binding of allergen to cell-bound IgE causes these cells to release histamines and other vasoactive substances. The release of histamines in the body results initiates what is commonly known as an allergic reaction. Before making any therapeutic determination it is important, however, to know whether the allergic reaction is IgE mediated or non-IgE mediated. Measurement of total IgE in serum sample, along with other supporting diagnostic information, can help to make that determination. Measurement of total circulating IgE may also be of value in the early detection of allergy in infants and as a means of predicting future atopic manifestations. Before deciding on any therapy it is important to take into consideration all the relevant clinical information as well as information supplied by specific allergy testing. IgE levels show a slow increase during childhood, reaching adult levels in the second decade of life. In general, the total IgE levels increase with the allergies a person has and the number of times of exposure to the relevant allergens. Significant elevations may be seen in the sensitized individuals, but also in cases of myeloma, pulmonay aspergillosis, and during the active stages of parasitic infections. In this method, IgE calibrator, patient specimen or control is first added to a streptavidin coated well. Biotinylated monoclonal antibody (specific for IgE) is added and the reactants mixed. Reaction between the IgE antibodies and native IgE forms complex that binds with the streptavidin coated to the well. The excess serum proteins are washed away via a wash step. Another enzyme labeled monoclonal antibody specific to IgE is added to the wells. The enzyme labeled antibody binds to the IgE already immobilized on the well through its binding with the biotinylated monoclonal antibody. Excess enzyme is washed off via a wash step. A color is generated by the addition of a substrate. The intensity of the color generation is directly proportional to the concentration of the IgE in the sample. Intended Use: The Quantitative Determination of Immunoglobulin E (IgE) Concentration in Human Serum by a Microplate Enzyme Immunoassay. Q. C. Parameters: In order for the assay results to be considered valid the following criteria should be met: The absorbance (OD) of calibrator A should be lower than 0. 1. The absorbance (OD) of calibrator F should be greater than 1.3. 2. Four out of six quality control pools should be within the established ranges.