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Rat Prostaglandin E2 (PGE2)
ELISA Kit
This immunoassay kit allows for the in vitro quantitative determination of rat
PGE2 concentrations in tissue homogenates.
Expiration date six months from the date of manufacture
FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.
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INTRODUCTION
One of the prostaglandins, a group of hormone-like substances
that participate in a wide range of body functions such as the
contraction and relaxation of smooth muscle, the dilation and
constriction of blood vessels, control of blood pressure, and
modulation of inflammation. Prostaglandin E2 (PGE-2) is
released by blood vessel walls in response to infection or
inflammation that acts on the brain to induce fever. The enzyme
mPGES-1 is involved in the production of PGE2 and is an
important "switch" for activating the fever response.
PRINCIPLE OF THE ASSAY
The microtiter plate provided in this kit has been pre-coated with
an goat-anti-rabbit antibody. Standards or samples are then
added to the appropriate microtiter plate wells with a
HRP-conjugated PGE2 and antibody preparation specific for
PGE2 and incubated. Then substrate solutions are added to
each well. The enzyme-substrate reaction is terminated by the
addition of a sulphuric acid solution and the color change is
measured spectrophotometrically at a wavelength of 450 nm ± 2
3
nm. The concentration of PGE2 in the samples is then
determined by comparing the O.D. of the samples to the
standard curve.
DETECTION RANGE
0.4 pg/ml-100 pg/ml. The standard curve concentrations used for
the ELISA’s were 100 pg/ml, 25 pg/ml, 6.25 pg/ml, 1.55 pg/ml,
0.4 pg/ml.
SPECIFICITY
This assay recognizes recombinant and natural rat PGE2. No
significant cross-reactivity or interference was observed.
SENSITIVITY
The minimum detectable dose of rat PGE2 is typically less than
0.25 pg/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.
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MATERIALS PROVIDED
Standard S 1 S 2 S 3 S 4 S 5
Concentration
(pg/ml)
0.4 1.55 6.25 25 100
Reagent Quantity Storage
Unopened kit Store at 2 - 8° C. Do not use beyond kit expiration date.
Coated plate 1
May be stored for up to 1 month
at 2 - 8° C. Try to keep it in a
sealed aluminum foil bag,and
avoid the damp
Standards
(S1-S5)
5 x 0.5 ml
Antibody 1 x 6 ml
HRP-conjugate 1 x 6 ml
Wash Buffer
1 x 15 ml
(20×concentrate)
Substrate A 1 x 7 ml
Substrate B 1 x 7 ml
Opened kit
Stop Solution 1 x 7 ml
May be stored for up to 1 month
at 2 - 8° C.
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TECHNICAL HINTS
1. Bring all reagents and plate to room temperature for at least
30 minutes before use. Unused wells need store at 2-8℃and
avoid sunlight.
2. Wash Buffer If crystals have formed in the concentrate,
warm to room temperature and mix gently until the crystals
have completely dissolved. Dilute 15 ml of Wash Buffer
Concentrate into deionized or distilled water to prepare 300 ml
of Wash Buffer.
3. 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.
4. 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.
5. To ensure accurate results, proper adhesion of plate sealers
during incubation steps is necessary. Sealers can not be
reused.
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6. 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.
7. 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.
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.
SAMPLE COLLECTION AND STORAGE
Tissue Homogenates The preparation of tissue
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homogenates will vary depending upon tissue type. Heart
and lung tissue from one rat was rinsed with 1X PBS to
remove excess blood, homogenized in 20 mL of 1X PBS and
stored overnight at -20° C. After two freeze-thaw c ycles were
performed to break the cell membranes, the homogenates
were centrifuged for 5 minutes at 5000 x g. The supernate
was assayed and removed immediately. Alternatively, 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
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. Set a Blank well without any solution. Add 50µl of Standard or
Sample per well. Standards need test in duplicate.
2. Add 50µl of HRP-conjugate to each well (not to Blank well),
then add 50µl Antibody to each well. Mix well and then
incubate for 1 hour at 37°C.
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3. Fill each well with Wash Buffer (about 200µl), stay for 10
seconds and Spinning. Repeat the process for a total of three
washes. 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 50µl of Substrate A and Substrate B to each well, mix
well. Incubate for 15 minutes at 37°C. Keeping the plate away
from drafts and other temperature fluctuations in the dark.
5. Add 50µl of Stop Solution to each well. If color change does
not appear uniform, gently tap the plate to ensure thorough
mixing.
6. Determine the optical density of each well within 10 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, Blank, and
sample and subtract the optical density of Blank. Create a
standard curve by reducing the data using computer software
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capable of generating a four parameter logistic (4-PL) curve-fit.
As an alternative, construct a standard curve by plotting the
mean absorbance for each standard on the y-axis against the
concentration on the x-axis and draw a best fit curve through the
points on the graph. The data may be linearized by plotting the
log of the PGE2 concentrations versus the log of the O.D. 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.
It is important that the Calibrator Diluent selected for the
standard curve be consistent with the samples being
assayed.
If samples generate values higher than the highest standard,
dilute the samples with the appropriate Calibrator Diluent and
repeat the assay.
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Any variation in Standard Diluent, 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
Quantikine Immunoassay, the possibility of interference
cannot be excluded.
PRECISION
Intra-assay Precision (Precision within an assay)
One sample whose concentration between the highest and the
second standard were tested twenty times on one plate to
assess.
CV%<8%
Inter-assay Precision (Precision between assays)
One sample whose concentration between the highest and the
second standard were tested in thirty-five separate assays to
assess
CV%<10%
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RECOVERY
The recovery of PGF2 spiked to three levels throughout the
range of the assay in the tissue homogenates and cell culture
supernates samples was evaluated.
Sample Type Average % Recovery Range
Rat Tissue
Homogenates(n
=6)
90 84 - 102%
Samples were spiked then diluted prior to assay as directed in
Sample Preparation.
PLATE LAYOUT
Use this plate layout as a record of standards and samples
assayed.
