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Rat Estrogen (E) ELISA Kit 
 
 
 
?  This immunoassay kit allows for  the in vitro rapid detection of rat Estrogen
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
Estrogens are a group of steroid  compounds, named for their
importance in the estrous cycle, and functioning as the primary
female sex hormone, their name  comes from estrus/oistros
(period of fertility for female mammals) + gen/gonos = to
generate.
Estrogens are used as part of some oral contraceptives, in
estrogen replacement therapy for postmenopausal women, and
in hormone replacement therapy for trans women.
Like all steroid hormones, estrogens readily diffuse across the
cell membrane. Once inside the cell, they bind to and activate
estrogen receptors which in turn up-regulate the expression of
many genes. Additionally, estrogens have been shown to
activate a G protein-coupled receptor, GPR30.
PRINCIPLE OF THE ASSAY
This assay employs the competitive inhibition enzyme
immunoassay technique. The microtiter plate provided in this kit
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has been pre-coated with goat-anti-rabbit antibody. Standards or
samples are added to the appropriate microtiter plate wells with
an antibody specific for estrogen  and Horseradish Peroxidase
(HRP) conjugated estrogen, then incubated. Then substrate
solutions are added to the wells, respectively. 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 nm. The
concentration of estrogen in the samples is then determined by
comparing the O.D. of the samples to the standard curve.
DETECTION RANGE
The standard curve concentrations used for the ELISA’s were
1000pg/ml, 500 pg/ml, 200pg/ml, 60pg/ml, 20 pg/ml.
SPECIFICITY
This assay recognizes estrogen. No significant cross-reactivity or
interference was observed.
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MATERIALS PROVIDED
Reagent    Quantity
Assay plate  1
Standards (S1-S5)  5 x0.5ml
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
Stop Solution      1 x 7 ml
 
Standard  S1  S2  S3  S4  S5
Concentration
(pg/ml)
20  60  200  500  1000
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.
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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.
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.
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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. 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.
6. 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.
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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 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.
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?  Plasma   Collect plasma using citrate, EDTA, or heparin as
an anticoagulant. Centrifuge for 15 minutes at 1000 x 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
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. Standard need test in duplicate.  
2. Add 50μl of HRP-conjugate to each well (not to Blank well),
then 50μl Antibody to each well. Mix well and then incubate
for 2 hour at 37°C.  
3.  Fill each well with Wash Buffer (about 250μl), stay for 10
seconds and Spinning. Repeat the process for a total of three
washes. Complete removal of liquid at each step is essential
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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 when the first four
wells containing the highest concentration of standards
develop obvious blue color. 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 estrogen 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.
?  If samples generate values higher than the highest standard,
dilute the samples with the appropriate Diluent and repeat the
assay.
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? 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
Quantikine Immunoassay, the  possibility of interference
cannot be excluded.