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Porcine Immunoglobulin E
（IgE）ELISA Kit
（96T）
? This immunoassay kit allows for the in vitro quantitative determination of porcine
IgE concentrations in serum and plasma,.
? Expiration date six months from the date of manufacture
? FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.
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PRINCIPLE OF THE ASSAY
The microtiter plate provided in this kit has been pre-coated with
porcine IgE. Standards or samples are then added to the
appropriate microtiter plate wells with Horseradish Peroxidase
（HRP） -conjugated antibody preparation specific for porcine IgE，
mix well and incubated. The more the amount of porcine IgE in
samples, the less HRP-conjugated antibody preparation specific
for porcine IgE bound by pre-coated porcine IgE. Then a TMB
（3,3',5,5' tetramethyl-benzidine） substrate solution is added to
each well. And the color develops in opposite to the amount of
porcine IgE in the sample. The color development is stopped and
the intensity of the color is measured.
DETECTION RANGE
0.05ng/ml-12.5 ng/ml. The standard curve concentrations used
for the ELISA’s were 12.5 ng/ml, 3.12 ng/ml, 0.78ng/ml,
0.19ng/ml, 0.05ng/ml.
SPECIFICITY
This assay recognizes porcine IgE. No significant cross-reactivity
or interference was observed.
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SENSITIVITY
The minimum detectable dose of porcine IgE is typically less
than 0.05ng/ml.
The sensitivity of this assay, or Lower Limit of Detection （LLD）
was defined as the lowest protein concentration that could be
differentiated from zero.
MATERIALS PROVIDED
Reagent Quantity
Assay plate 1
Standard 6 x 0.5 ml
Sample Diluent 2 x 20 ml
HRP- conjugate 1 x 6 ml
Wash Buffer
1 x 20 ml
（25×concentrate）
TMB Substrate 1 x 10 ml
Stop Solution 1 x 10 ml
Standard S1 S 2 S3 S4 S5 S6
Concentration
（ng/ml） 0 0.05 0.19 0.78 3.12 12.5
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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. The
test kit may be used throughout the expiration date of the kit,
provided it is stored as prescribed above. Refer to the
package label for the expiration date.
2. Opened test plate should be stored at 2-8?C in the aluminum
foil bag with desiccants to minimize exposure to damp air. The
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 compley dissolved. Dilute 20 ml of Wash
Buffer Concentrate into deionized or distilled water to prepare
500 ml of Wash Buffer.
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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
minutes at 1000 g. Remove serum and assay immediay 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 immediay or aliquot and
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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
Bring all reagents and samples to room temperature before use. It is
recommended that all samples, standards, and controls be assayed in duplicate.
All the reagents should be added directly to the liquid level in the well. The
pipette should avoid contacting the inner wall of the well.
1. Recommend to dilute the serum and plasma samples with
Sample Diluent（1:1000） before test. The suggested 1000-fold
dilution can be achieved by adding 5μl sample to 95μl of
Sample Diluent. Complete the 1000-fold dilution by adding
5μl of this solution to 245μl of Sample Diluent. The
recommended dilution factor is for reference only. The
optimal dilution factor should be determined by users
according to their particular experiments.
2. Add 50μl of Standard or Sample per well. Add 50μl of HRPconjugate
to each well immediay. Mix well with the pipette
or shake the plate gently for 60 seconds.
3. Then incubate for 40 minutes at 37° C.
4. Aspirate each well and wash, repeating the process five
times for a total of five washes. Wash: Fill each well with
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Wash Buffer （200μl） and let it stand for 2 minutes, then
remove the liquid by flicking the plate over a sink. The
remaining drops are removed by patting the plate on a paper
towel. Complete removal of liquid at each step is essential to
good performance.
5. 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.
6. Add 50μl of Stop Solution to each well when the last four
wells containing the lowest concentration of standards
develop obvious blue color. If color change does not appear
uniform, gently tap the plate to ensure thorough mixing.
7. Determine the optical density of each well within 15 minutes,
using a microplate reader set to 450 nm.
CALCULATION OF RESULTS
Using the professional soft "Curve Exert 1.3" to make a standard curve is
recommended, which can be downloaded from our web.
Average the duplicate readings for each standard, control, and
sample and divide the average zero standard optical density.
Create a standard curve by reducing the data using computer
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software. As an alternative, construct a standard curve by
plotting the absorbance ratio for each standard on the x-axis
against the concentration on the y-axis and draw a best fit curve
through the points on the graph. The data may be linearized by
plotting the porcine IgE concentrations versus the ratio and the
best fit line can be determined by regression analysis. This
procedure will produce an adequate but less precise fit of the
data. If samples have been diluted, the concentration read from
the standard curve must be multiplied by the dilution factor.
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.
? If samples generate values higher than the highest standard,
dilute the samples with the appropriate Diluent and repeat the
assay.
? 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
? Centrifuge vials before opening to collect contents.
? When mixing or reconstituting protein solutions, always avoid
foaming.
? To avoid cross-contamination, change pipette tips between
additions of each standard level, 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.
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? Substrate Solution should remain colorless or light blue until
added to the plate. Keep Substrate Solution protected from
light. Substrate Solution should change from colorless or light
blue to gradations of blue.
? 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.