Instructions for Use

Transcription

1 Mouse Ovalbumin-Specific IgE ELISA Kit Catalogue code: MCA2475KZZ FOR INVITRO USE AND RESEARCH USE ONLY NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES Instructions for Use Intended Use This ELISA kit is a sandwich enzyme immunoassay designed for the quantitative determination of ovalbumin-specific IgE in mouse serum or plasma. Summary & Explanation Ovalbumin (OVA) is commonly used to produce mouse models of allergic asthma. As a potent allergen it induces Th2 responses, and the production of IgE. IgE binds mast cells and basophils through Fc receptors present on the cells. Cross-linking of the cell-bound IgE causes an inflammatory response initiated by activation of the mast cells and basophils. Initially a response against pathogens, following improvements in general health, the IgE inflammatory response is now more commonly associated with allergy. Principle of the Method This assay is based on a sandwich ELISA using a rat anti-mouse IgE monoclonal antibody and HRP-conjugated Ovalbumin. The standards are ovalbumin-specific mouse IgE. Performance Assay range Between 2.7ng/ml and Standard F (the concentration of which is batch-specific), in mouse serum or plasma. Sensitivity The limit of detection of the Ovalbumin-specific mouse IgE is typically < 3ng/ml. Bio-Rad recommend that each individual laboratory should determine their own range and limit of detection. QIFUMCA2475KZZ Mouse Ovalbumin-Specific IgE Kit Insert IFU Page 1 of 5

2 Contents of the Mouse Ovalbumin- Specific IgE Kit Item Quantity Antibody-coated 96 well microtiter plate 1 Pre-dispensing 96 well microtiter plate 1 Ovalbumin-specific Mouse IgE standards, 6 lyophilized Assay Buffer (30ml ready to use) 1 Wash Buffer Concentrate (90ml) 1 Detection Conjugate (15ml ready to use) 1 HRP Substrate (15ml ready to use) 1 Stop Solution (15ml ready to use) 1 Reagents and Equipment required but not supplied Distilled or Deionised water Plate reader with 450nm and 620nm (or 630nm) reading capability Pipettes for dispensing up to 500µl, and disposable tips Multi-channel pipette for dispensing µl Plate sealers or aluminum foil Paper towels Preparation of Reagents This ELISA kit is a sandwich enzyme immunoassay designed for the quantitative determination of ovalbumin-specific mouse IgE in mouse serum and plasma. NOTE: Bring all reagents to room temperature before use. Item Antibody-coated microtiter plate Pre-dispensing microtiter plate Assay Buffer Ovalbuminspecific IgE Standards Preparation Just before use, cut and remove the seal from enough antibodycoated wells required for the samples and standards. Decant the contents of each well, then tap the wells upside-down on a paper towel to remove the contents completely. Store unused wells with the seal intact at +4 C. Prepare standards and samples in this plate before transferring them to the antibody-coated plate to run the ELISA. For samples containing a high concentration of mouse IgE, dilute using the assay buffer provided. For example: 5-fold dilution: 20µl of sample + 80µl of buffer; 10-fold dilution: 15µl of sample + 135µl of buffer. Reconstitute standards A-F by adding 0.5ml of purified water. Leave for 15 minutes and then mix gently to fully dissolve the contents. Once resuspended, the closed vials can be stored at +4 C for one week. For longer term storage, freeze at -20 C; before use thaw the vial, bring to room temperature and mix well. The standards can be freeze-thawed up to 3 times without effect. QIFUMCA2475KZZ Mouse Ovalbumin-Specific IgE Kit Insert IFU Page 2 of 5

3 Wash Buffer Concentrate Detection Conjugate HRP Substrate Stop Solution Prepare the required amount of wash buffer (assuming each well requires 2mls wash buffer for this assay) by diluting the wash buffer concentrate 10-fold with purified water. Unused diluted wash buffer can be stored at +4 C for up to one week. To avoid contamination avoid contact with metals or exposure to light. Pipette the required amount of solution into a clean tube and do not return any residual solution back to the original bottle Sample collection and storage Serum - Use a serum separator tube (SST) and allow samples to clot for 30 minutes before centrifugation for 15 minutes at approximately 1000 x g. Remove serum and assay immediately or aliquot and store samples at -20 C or -80 C. Plasma - Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples for 15 minutes at 1000 x g at +4 C within 30 minutes of collection. Store samples at -20 C or -80 C. Avoid repeated freeze-thaw cycles. Note: Serum and plasma samples to be used within 7 days may be stored at +4 C, otherwise samples must be stored at -20 C ( 3 months) or -80 C ( 6 months) to avoid loss of bioactivity and contamination. Avoid freeze-thaw cycles. When performing the assay slowly bring samples to room temperature. It is recommended that all samples be assayed in duplicate. DO NOT USE HEAT-TREATED SPECIMENS. Assay Protocol Calculate the number of wells required (samples must be tested at least in duplicate) and include 12 wells for a standard curve. Carefully pipette 150µl of buffer per well into the pre-dispensing microplate. Dispense 15µl of the sample or standard solution into the predispensing microplate and agitate the plate. Keep the plate at room temperature for 5-10 minutes. From the pre-dispensing microplate, add 110µl of diluted standards to the wells designated for the standard curve on the antibody-coated plate. Add samples (diluted in buffer) to the other wells, all in duplicate using a multichannel- pipette. Incubate the plate covered with aluminum foil (or sealed with a plate sealer) for 60 minutes at room temperature. Remove the plate sealer and wash the wells three times with wash buffer (300µl per well). Aspirate the last wash and gently tap the plate upside down on a paper towel. Do not allow the wells to dry. QIFUMCA2475KZZ Mouse Ovalbumin-Specific IgE Kit Insert IFU Page 3 of 5

4 Add 100µl of the antibody-enzyme conjugate to each well and agitate the plate. Incubate the plate covered with aluminum foil (or sealed with a plate sealer) for 30 minutes at room temperature. Remove the plate sealer and wash the wells three times with wash buffer (300µl per well). Aspirate the last wash and gently tap the plate upside down on a paper towel. Do not allow the wells to dry. Add 100µl of substrate to each well and agitate the plate. Protect the plate from light and incubate for 30 minutes at room temperature. Add 100µl of Stop solution to each well and agitate the plate. Protect the plate from light. Read the plate within 20 minutes. Use 450nm as the main wavelength (620 or 630nm reference wavelength). Precaution The stop solution suggested for use with this kit is an acid solution. Wear eye, hand, face and clothing protection when using this material. For product support or advice please contact: Technical inquiries can be made by telephone, fax, or simply choose the most convenient option. Please provide as many details as possible, so that we can deal with your request quickly and efficiently. Region Phone Fax North America antibody_tech_us@bio-rad.com United Kingdom antibody_tech_uk@bio-rad.com Other Countries antibody_tech_uk@bio-rad.com QIFUMCA2475KZZ Mouse Ovalbumin-Specific IgE Kit Insert IFU Page 4 of 5

5 Bio-Rad Mouse Ovalbumin-Specific IgE ELISA Quick Guide Prepare standard/samples in pre-dispensing microplate. Keep plate at room temperature for 5-10 minutes. Add 110µL of standard/samples to pre-coated plate. Incubate for one hour at room temp, then wash three times. Add 100µL of antibody-enzyme conjugate to each well. Incubate for 30 minutes at room temp, then wash three times. Add 100µL of substrate to each well. Incubate for 30 minutes at room temp. Add 100µL of Stop solution to each well. Read the plate at 450 nm within 20 minutes (using 620 or 630nm as the reference wavelength). QIFUMCA2475KZZ Mouse Ovalbumin-Specific IgE Kit Insert IFU Page 5 of 5