FOR RESEARCH USE ONLY; NOT FOR THERAPEUTIC OR DIAGNOSTIC APPLICATIONS! PLEASE READ THROUGH ENTIRE PROCEDURE BEFORE BEGINNING! Intended use
This immunoassay kit allows for the in vitro quantitative determination of rat E2
concentrations in serum, plasma and other biological fluids.
Introduction
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Test principle
The microtiter plate provided in this kit has been pre-coated with an antibody specific to
E2. Standards or samples are then added to the appropriate microtiter plate wells with a
biotin-conjugated polyclonal antibody preparation specific for E2. Next, Avidin conjugated
to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. Then a
TMB substrate solution is added to each well. Only those wells that contain E2,
biotin-conjugated antibody and enzyme-conjugated Avidin will exhibit a change in color.
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 E2 in the samples is then determined by comparing the O.D. of
Materials and components Other supplies required Sample collection and storage Serum - Use a serum separator tube and allow samples to clot for 30 minutes before
centrifugation for 20 minutes at approximately 1000 g. Remove serum and assay
immediately or aliquot and store samples at -20 or -80 .
Plasma - Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples
for 15 minutes at 1000 g at 2 - 8 within 30 minutes of collection. Store samples at
-20 or -80 . Avoid repeated freeze-thaw cycles.
Other biological fluids - Remove particulates by centrifugation and assay immediately or
aliquot and store samples at -20 or -80 . Avoid repeated freeze-thaw cycles.
Note: Serum and plasma to be used within 7 days may be stored at 2-8 , otherwise
bioactivity and contamination. Avoid freeze-thaw cycles. When performing the assay
slowly bring samples to room temperature.
Limitations of the procedure
1. The kit should not be used beyond the expiration date on the kit label.
2. Do not mix or substitute reagents with those from other lots or sources.
3. If samples generate values higher than the highest standard, further dilute the
samples and repeat the assay. Any variation in standard diluent, operator, pipetting
technique, washing technique,incubation time or temperature, and kit age can cause
4. This assay is designed to eliminate interference by soluble receptors, ligands, 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
5. Limited by the current condition and scientific technology, we can't completely conduct
the comprehensive identification and analysis on the raw material provided by
suppliers. So there might be some qualitative and technical risks to use the kit.
Reagent preparation
Bring all reagents to room temperature before use.
Wash Buffer - If crystals have formed in the concentrate, warm to room temperature and
mix gently until the crystals have completely dissolved. Dilute 30 mL of Wash Buffer
Concentrate into deionized or distilled water to prepare 750 mL of Wash Buffer.
Standard - Reconstitute the Standard with 0.5 ml of Sample Diluent. This reconstitution
produces a stock solution of 400 pg/mL. Allow the standard to sit for about 10 minutes with
gentle agitation prior to making serial dilutions (Making serial dilution in the wells directly is
not permitted). The undiluted standard serves as the highest standard (400 pg/mL). The
Sample Diluent serves as the zero standard ( 0 pg/mL). pg/mL 400 200 100 50 25 12.5 6.25 0 Detection Reagent A and B - Dilute to the working concentration using Assay Diluent A or B (1:100), respectively. Assay procedure
Allow all reagents to reach room temperature (Please do not dissolve the reagents at 37
directly.). All the reagents should be mixed thoroughly by gently swirling before pipetting. Avoid foaming. Keep appropriate numbers of strips for 1 experiment and
remove extra strips from microtiter plate. Removed strips should be resealed and stored
at 4 until the kits expiry date. Prepare all reagents, working standards and samples as
directed in the previous sections. Please predict the concentration before assaying. If
values for these are not within the range of the standard curve, users must determine the
optimal sample dilutions for their particular experiments.
of Standard, Blank, or Sample per well. Cover with the Plate sealer.
2. Remove the liquid of each well, don’t wash.
3. Add 100 μl of Detection Reagent A working solution to each well. Cover with the
Plate sealer. Incubate for 1 hour at 37 . Detection Reagent A working solution may
appear cloudy. Warm to room temperature and mix gently until solution appears
4. Aspirate each well and wash, repeating the process three times for a total of three
washes. Wash by filling each well with Wash Buffer (approximately 400 μl) 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
5. Add 100 μl of Detection Reagent B working solution to each well. Cover with a new
Plate sealer. Incubate for one hour at 37 .
6. Repeat the aspiration/wash process for five times as conducted in step 4.
7. Add 90 μl of Substrate Solution to each well. Cover with a new Plate sealer.
Incubate for 15 - 30 minutes at 37 . Protect from light.
8. Add 50 μl of Stop Solution to each well. If color change does not appear uniform,
gently tap the plate to ensure thorough mixing.
9. Determine the optical density of each well at once, using a microplate reader set to
Important Note:
1. Absorbance is a function of the incubation time. Therefore, prior to starting the assay
it is recommended that all reagents should be freshly prepared prior to use and all
required strip-wells are secured in the microtiter frame. This will ensure equal elapsed
time for each pipetting step, without interruption.
2. Please carefully reconstitute Standards or working Detection Reagent A and B
according to the instruction, and avoid foaming and mix gently until the crystals have
completely dissolved. The reconstituted Standards can be used only once. This
assay requires pipetting of small volumes. To minimize imprecision caused by
pipetting, ensure that pipettors are calibrated. It is recommended to suck more than
3. To ensure accurate results, proper adhesion of plate sealers during incubation steps
is necessary. Do not allow wells to sit uncovered for extended periods between
incubation steps. Once reagents have been added to the well strips, DO NOT let the
strips DRY at any time during the assay.
4. For each step in the procedure, total dispensing time for addition of reagents to the
assay plate should not exceed 10 minutes.
5. To avoid cross-contamination, change pipette tips between additions of each
standard level, between sample additions, and between reagent additions. Also, use
6. The wash procedure is critical. Insufficient washing will result in poor precision and
7. Duplication of all standards and specimens, although not required, is recommended.
8. Substrate Solution is easily contaminated. Please protect it from light.
Specificity
This assay recognizes recombinant and natural rat E2. No significant cross-reactivity or
Sensitivity
The minimum detectable dose of rat E2 is typically less than 3.12 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.
Detection Range
6.25-400 pg/mL. The standard curve concentrations used for the ELISA’s were 400 pg/mL,
200 pg/mL, 100 pg/mL, 50 pg/mL, 25 pg/mL, 12.5 pg/mL, 6.25 pg/mL.
Calculation of results
Average the duplicate readings for each standard, control, and samples and subtract the
average zero standard optical density. Create a standard curve by reducing the data using
computer software 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 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 log of the E2
concentrations versus the log of the O.D. and the best fit line can be determined by
regression analysis. It is recommended to use some related software to do this calculation,
such as curve expert 13.0. 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.
Storage of test kits and instrumentation
1. The Standard, Detection Reagent A and Detection Reagent B should be stored at
-20 upon being received. Other reagents are kept according to the labels on vials.
But for long term storage, please keep the whole kit at -20 . The unused strips
should be kept in a sealed bag with the desiccant provided to minimize exposure to
damp air. The test kit may be used throughout the expiration date of the kit (six
months from the date of manufacture). Opened test kits will remain stable until the
expiring date shown, provided it is stored as prescribed above.
2. There may be some foggy substance in the wells when the plate is opened at the first
time. It will not have any effect on the final assay results.
3. Do not remove microtiter plate from the storage bag until needed.
4. A microtiter plate reader with a bandwidth of 10nm or less and an optical density range
of 0-3 OD or greater at 450nm wavelength is acceptable for use in absorbance
5. Use fresh disposable pipette tips for each transfer to avoid contamination.
6. Do not substitute reagents from one kit lot to another. Use only the reagents supplied
7. The final experimental results will be closely related to validity of the products,
operation skills of the end users and the experimental environments. Please make
sure that sufficient samples are available.
Precaution
The Stop Solution suggested for use with this kit is an acid solution. Wear eye, hand, face,
and clothing protection when using this material.
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