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Rat ovalbumin specific IgE (OVA sIgE) ELISA kit
Catalog No. CSB-E08913r
(96 tests)
This immunoassay kit allows for the in vitro semi-quantitative determination of rat OVA sIgE concentrations in serum, plasma a-n-d other biological fluids.
Expiration date six months from the date of manufacture
FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.
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INTRODUCTION
Ovalbumin is the main protein found in egg white, making up 60-65% of the total protein. While Ovalbumin displays sequence a-n-d three-dimensional homology to the serpin superfamily, it is a noninhibitory serpin . While most serpins control such processes as fibrinolysis a-n-d coagulation by inhibiting serine proteases, the function of ovalbumin is unknown, although it is presumed to be a storage protein. Ovalbumin is an important protein in several different areas of research, one of the areas is immunology (commonly used to stimulate an allergic reaction in test subjects, e.g. established model allergen for airway hyper-responsiveness AHR).
PRINCIPLE OF THE ASSAY
The microtiter plate provided in this kit has been pre-coated with purified OVA. Samples are then added to the appropriate microtiter plate wells a-n-d incubated. Then add Horseradish Peroxidase (HRP)-conjugated anti-rat IgE to each well a-n-d incubate. Finally, a TMB (3,3’,5,5’ tetramethyl-benzidine) substrate solution is added to each well. The enzyme-substrate reaction is terminated by the addition of a stop solution a-n-d the color change is measured spectrophotometrically at a wavelength of 450 nm ±2 nm. Calculate the valence of rat OVA sIgE in the samples.
SPECIFICITY
This assay recognizes rat OVA sIgE, No significant cross-reactivity or interference was observed.
MATERIALS PROVIDED
Reagent Quantity
Assay plate 1 Sample Diluent 1 x 20 ml HRP-anti-rat IgE 1 x 120μl HRP-anti-rat IgE Diluent 1 x 10 ml
1 x20 ml
Wash Buffer
(25×concentrate) TMB Substrate 1 x 10 ml Stop Solution 1 x 10 ml Positive Control 1 x 800 μl Negative Control 1 x 800 μl
STORAGE
1. Unopened test kits should be stored at 2-8?C upon receipt a-n-d the microtiter plate should be kept in a sealed bag to minimize exposure to damp air. The test kit may be used throughout the expiration date of the kit. Refer to the package label for the expiration date.
2. Opened test kits will remain stable until the expiring date shown, provided it is stored as prescribed above.
3. A microtiter plate reader with a ba-n-dwidth of 10 nm or less a-n-d an optical density range of 0-3 OD or greater at 450nm wavelength is acceptable for use in absorbance measurement.
REAGENT PREPARATION
Bring all reagents to room temperature before use.
1 Wash Buffer If crystals have formed in the concentrate, warm up to room temperature a-n-d mix gently until the crystals have completely dissolved. Dilute 20 ml of Wash Buffer Concentrate into deionized or distilled water to prepare 500 ml of Wash Buffer.
2 HRP-anti-rat IgE Dilute to the working concentration using HRP-anti-rat IgE Diluent (1:100), respectively.
3 Sample Dilute 1μl sample using 199μl Sample Diluent (1:200), respectively.
Precaution: The Stop Solution provided with this kit is an acid solution. Wear eye, ha-n-d, face, a-n-d clothing protection when using this material.
OTHER SUPPLIES REQUIRED
1 Microplate reader capable of measuring absorbance at 450 nm, with the correction wavelength set at 540 nm or 570 nm.
2 Pipettes a-n-d pipette tips.
3 Deionized or distilled water.
4 Squirt bottle, manifold dispenser, or automated microplate washer.
5 ? An incubator which can provide stable incubation conditions up to 37°C±0.5°C. SAMPLE COLLECTION A-N-D STORAGE
? Serum Use a serum separator tube (SST) a-n-d allow samples to clot for 30 minutes before centrifugation for 15 minutes at 1000 x g. Remove serum a-n-d assay immediately or aliquot a-n-d store samples at -20°C. Centrifuge the sample again after thawing before the assay. Avoid repeated freeze-thaw cycles.
Plasma Collect plasma using citrate, EDTA, or heparin as an anticoagulant. Centrifuge for 15 minutes at 1000 g within 30 minutes of collection. Assay immediately or aliquot a-n-d store samples at -20°C. Centrifuge the sample again after thawing before the assay. Avoid repeated freeze-thaw cycles.
Note: Grossly hemolyzed samples are not suitable for use in this assay.
ASSAY PROCEDURE
To ensure the accurateness of the results, please control the total time of adding all samples each step (includes add sample, HRP-anti-rat IgE working solution, TMB Substrate a-n-d Stop Solution) in 3 minutes. We recommend the customers divided the total samples into several batches in order to obtain the most accurate results.
1. Bring all reagents a-n-d samples to room temperature before use. It is recommended that all samples a-n-d controls be assayed in duplicate.
2. Add 100μl of Positive Control a-n-d Negative Control, 100μl of Sample to control a-n-d sample well respectively. Cover with the adhesive strip. Incubate for 30 minutes at 37°C.
3. Remove the liquid of each well a-n-d wash, repeating the process for a total of 3 washes. Wash by filling each well with Wash Buffer (200μl) using a squirt bottle, multi-channel pipette, manifold dispenser or autowasher. Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate a-n-d blot it against clean paper towels.
4. Add 100μl of HRP-anti-rat IgE working solution to each well. Incubate for 30 minutes at 37°C. HRP-anti-rat IgE working solution may appear cloudy. Warm up to room temperature a-n-d mix gently until solution appears uniform.
5. Aspirate each well a-n-d wash, repeating the process for a total of five washes.
6. Add 90μl of TMB Substrate to each well. Incubate for 20 minutes at 37°C. Keeping the plate away from drafts a-n-d other temperature fluctuations in the dark. 7. Add 50μl of Stop Solution to each well. If color change does not appear uniform, gently tap the plate to ensure thorough mixing.
8. Determine the optical density of each well within 10 minutes, using a microplate reader set to 450 nm.
CALCULATION OF RESULTS
For calculation the valence of rat OVA sIgE, compare the sample well with control. While ODsample≥1.4 x ODnegative: Positive While ODsample＜1.4 x ODnegative: Negative
LIMITATIONS OF THE PROCEDURE
The kit should not be used beyond the expiration date on the kit label.
Do not mix or substitute reagents with those from other lots or sources.
Any variation in operator, pipetting technique, washing technique, incubation time or temperature, a-n-d kit age can cause variation in binding.
This assay is designed to eliminate interference by soluble receptors, binding proteins, a-n-d other factors present in biological samples. Until all factors have been tested in the Immunoassay, the possibility of interference cannot be excluded. TECHNICAL HINTS
When mixing or reconstituting protein solutions, always avoid foaming.
To avoid cross-contamination, change pipette tips between sample additions, a-n-d between reagent additions. Also, use separate reservoirs for each reagent.
When using an automated plate washer, adding a 30 second soak period following the addition of wash buffer, a-n-d/or rotating the plate 180 degrees between wash steps may improve assay precision.
To ensure accurate results, proper adhesion of plate sealers during incubation steps is necessary.
Substrate Solution should remain colorless until added to the plate. Keep Substrate Solution protected from light. Substrate Solution should change from colorless to gradations of blue.
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