CSB-E08913r Rat Ovalbumin Specific IgE (OVA sIgE) ELISA Kit Instructions

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 and other biological fluids.

Expiration date six months from the date of manufacture

FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.

INTRODUCTION

Ovalbumin is the main protein found in egg white, making up 60-65% of the total protein. While Ovalbumin displays sequence and three-dimensional homology to the serpin superfamily, it is a noninhibitory serpin. While most serpins control such processes as fibrinolysis and 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, eg 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 and encouraged. Then add Horseradish Peroxidase (HRP) -conjugated anti-rat IgE to each well and incubate. 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 and 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 and 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 and 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 and 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-nd, face, and 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 and 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 AND STORAGE

? Serum Use a serum separator tube (SST) and allow samples to clot for 30 minutes before centrifugation for 15 minutes at 1000 x g. Remove serum and assay immediately or aliquot and 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 and 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 and 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 and samples to room temperature before use. It is recommended that all samples and controls be assayed in duplicate.

2. Add 100μl of Positive Control and Negative Control, 100μl of Sample to control and sample well respectively. Cover with the adhesive strip. Incubate for 30 minutes at 37 ° C.

3. Remove the liquid of each well and 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 and 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 and mix gently until solution appears uniform .

5. Aspirate each well and 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 and 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, and kit age can cause variation in binding.

This assay is designed to eliminate interference by soluble receptors, binding proteins, and 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, and 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, and / 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.