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Rat ovalbumin specific IgE
(OVA sIgE) ELISA kit
(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.
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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
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, 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 and incubated. Then add Horseradish
Peroxidase (HRP)-conjugated anti-rat IgE to each well and
incubate. Finally, a TMB (3,3’,5,5’ tetramethyl-benzidine)
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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
Wash Buffer
1 x 20 ml
(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
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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 bandwidth 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.
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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, hand, face, and clothing protection when using this material.
OTHER SUPPLIES REQUIRED
? Microplate reader capable of measuring absorbance at 450
nm, with the correction wavelength set at 540 nm or 570 nm.
? Pipettes and pipette tips.
? Deionized or distilled water.
? Squirt bottle, manifold dispenser, or automated microplate
washer.
? 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
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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.
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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.
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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.
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? 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.
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? Stop Solution should be added to the plate in the same order
as the Substrate Solution. The color developed in the wells
will turn from blue to yellow upon addition of the Stop Solution.
Wells that are green in color indicate that the Stop Solution
has not mixed thoroughly with the Substrate Solution.
? 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.