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大鼠肾上腺素(EPI)ELISA试剂盒使用说明
Rat Epinephrine/Adrenaline (EPI) ELISA Kit
For the quantitative determination of endogenic rat
Epinephrine/Adrenaline (EPI) concentrations in serum, plasma, tissue
homogenates.
This package insert must be read in its entirety before using this product.
In order to obtain higher efficiency service, please ready to supply the lot number
of the kit to us (found on the outside of the box).2
PRINCIPLE OF THE ASSAY
This assay employs the competitive inhibition enzyme immunoassay technique.
The microtiter plate provided in this kit 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 EPI and Horseradish Peroxidase (HRP)
conjugated EPI. The competitive inhibition reaction is launched between with
HRP labeled EPI and unlabeled EPI with the antibody. A substrate solution is
added to the wells and the color develops in opposite to the amount of EPI in the
sample. The color development is stopped and the intensity of the color is
measured.
DETECTION RANGE
1 pg/ml-200 pg/ml.
SENSITIVITY
The minimum detectable dose of rat EPI is typically less than 0.5 pg/ml.
The sensitivity of this assay, or Lower Limit of Detection (LLD) was defined as
the lowest rat EPI concentration that could be differentiated from zero. It was
determined the mean O.D value of 20 replicates of the zero standard added by
their three standard deviations.
SPECIFICITY
This assay has high sensitivity and excellent specificity for detection of rat EPI.
No significant cross-reactivity or interference between rat EPI and analogues
was observed.
Note: Limited by current skills and knowledge, it is impossible for us to complete
the cross-reactivity detection between rat EPI and all the analogues, therefore,
cross reaction may still exist.3
PRECISION
Intra-assay Precision (Precision within an assay): CV%<15%
Three samples of known concentration were tested twenty times on one plate to
assess.
Inter-assay Precision (Precision between assays): CV%<15%
Three samples of known concentration were tested in twenty assays to assess.
LIMITATIONS OF THE PROCEDURE
? FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC
PROCEDURES.
? 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 and repeat the assay.
? 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 Immunoassay, the possibility of
interference cannot be excluded.4
MATERIALS PROVIDED
Reagents Quantity
Assay plate 1(96 wells)
Standard 6 x 0.5 ml
Antibody 1 x 6 ml
HRP-conjugate 1 x 6 ml
Wash Buffer (20 x concentrate) 1 x 15 ml
Substrate A 1 x 7 ml
Substrate B 1 x 7 ml
Stop Solution 1 x 7 ml
Adhesive Strip (For 96 wells) 4
Instruction manual 1
STANDARD CONCENTRATION
Standard S0 S1 S2 S3 S4 S5
Concentration
(pg/ml)
0 1 4 16 50 200
STORAGE
Unopened kit Store at 2 - 8°C. Do not use the kit beyond the expiration date.
Opened kit May be stored for up to one month at 2 - 8° C.
*Provided this is within the expiration date of the kit.
OTHER SUPPLIES REQUIRED
? Microplate reader capable of measuring absorbance at 450 nm, with the
correction wavelength set at 600 nm - 630 nm.
? An incubator which can provide stable incubation conditions up to
37°C±0.5°C.
? Squirt bottle, manifold dispenser, or automated microplate washer.
? Absorbent paper for blotting the microtiter plate.5
? 100 mL and 500 mL graduated cylinders.
? Deionized or distilled water.
? Pipettes and pipette tips.
? Test tubes for dilution.
PRECAUTIONS
The Stop Solution provided with this kit is an acid solution. Wear eye, hand, face,
and clothing protection when using this material.
SAMPLE COLLECTION AND STORAGE
? Serum Use a serum separator tube (SST) and allow samples to clot for
two hours at room temperature or overnight at 4°C before centrifugation
for 15 minutes at 1000 ×g. Remove serum and assay immediately or
aliquot and store samples at -20°C or -80°C. Avoid repeated freeze-thaw
cycles.
? Plasma Collect plasma using EDTA, or heparin as an anticoagulant.
Centrifuge for 15 minutes at 1000 ×g at 2-8°C within 30 minutes of
collection. Assay immediately or aliquot and store samples at -20°C or
-80°C. Avoid repeated freeze-thaw cycles.
? Tissue Homogenates 100mg tissue was rinsed with 1X PBS,
homogenized in 1 ml of 1X PBS and stored overnight at -20°C. After two
freeze-thaw cycles were performed to break the cell membranes, the
homogenates were centrifuged for 5 minutes at 5000 x g, 2 - 8°C. The
supernate was removed and assayed immediately. Alternatively, aliquot
and store samples at -20°C or -80°C. Centrifuge the sample again after
thawing before the assay. Avoid repeated freeze-thaw cycles.6
Note:
1. CUSABIO is only responsible for the kit itself, but not for the samples
consumed during the assay. The user should calculate the possible
amount of the samples used in the whole test. Please reserve sufficient
samples in advance.
2. Samples to be used within 5 days may be stored at 2-8°C, otherwise
samples must be stored at -20°C (≤1month) or -80°C (≤2month) to avoid
loss of bioactivity and contamination.
3. Grossly hemolyzed samples are not suitable for use in this assay.
4. If the samples are not indicated in the manual, a preliminary experiment to
determine the validity of the kit is necessary.
5. 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.
6. Tissue or cell extraction samples prepared by chemical lysis buffer may
cause unexpected ELISA results due to the impacts of certain chemicals.
7. Owing to the possibility of mismatching between antigen from other
resource and antibody used in our kits (e.g., antibody targets
conformational epitope rather than linear epitope), some native or
recombinant proteins from other manufacturers may not be recognized by
our products.
8. Influenced by the factors including cell viability, cell number and also
sampling time, samples from cell culture supernatant may not be detected
by the kit.
9. Fresh samples without long time storage are recommended for the test.
Otherwise, protein degradation and denaturalization may occur in those
samples and finally lead to wrong results.7
REAGENT PREPARATION
Note:
? Kindly use graduated containers to prepare the reagent.
? Bring all reagents to room temperature (18-25°C) before use for 30min.
? Distilled water is recommended to be used to make the preparation for
reagents or samples. Contaminated water or container for reagent
preparation will influence the detection result.
? Wash Buffer(1x)- If crystals have formed in the concentrate, warm up to
room temperature and mix gently until the crystals have completely
dissolved. Dilute 15 ml of Wash Buffer Concentrate (20 x) into deionized
or distilled water to prepare 300 ml of Wash Buffer (1 x).8
ASSAY PROCEDURE
Bring all reagents and samples to room temperature before use. Centrifuge
the sample again after thawing before the assay. It is recommended that all
samples and standards be assayed in duplicate.
1. Prepare all reagents and samples as directed in the previous sections.
2. Determine the number of wells to be used and put any remaining wells
and the desiccant back into the pouch and seal the ziploc, store unused
wells at 4°C.
3. Set a Blank well without any solution.
4. Add 50μl of Standard or Sample per well. Standard need test in duplicate.
5. Add 50μl of HRP-conjugate to each well (not to Blank well), then 50μl
Antibody to each well. Mix well and then incubate for 1 hour at 37°C.
6. Aspirate each well and wash, repeating the process two times for a total of
three washes. Wash by filling each well with Wash Buffer (200μl) using a
squirt bottle, multi-channel pipette, manifold dispenser, or autowasher,
and let it stand for 10 seconds, complete removal of liquid at each step is
essential to good performance. After the last wash, remove any remaining
Wash Buffer by aspirating ordecanting. Invert the plate and blot it against
clean paper towels.
7. Add 50μl of Substrate A and 50μl of 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.
8. Add 50μl of Stop Solution to each well, gently tap the plate to ensure
thorough mixing.
9. Determine the optical density of each well within 10 minutes, using a
microplate reader set to 450 nm.9
Note:
1. The final experimental results will be closely related to validity of the
products, operation skills of the end users and the experimental
environments.
2. Samples or reagents addition: Please carefully add samples to wells and
mix gently to avoid foaming. Do not touch the well wall as possible. For
each step in the procedure, total dispensing time for addition of reagents
or samples to the assay plate should not exceed 10 minutes. This will
ensure equal elapsed time for each pipetting step, without interruption.
Duplication of all standards and specimens, although not required, is
recommended. 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.
3. Incubation: 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. Incubation time and temperature must be observed.
4. Washing: The wash procedure is critical. Complete removal of liquid at
each step is essential to good performance. After the last wash, remove
any remaining Wash Solution by aspirating or decanting and remove any
drop of water and fingerprint on the bottom of the plate. Insufficient
washing will result in poor precision and falsely elevated absorbance
reading. When using an automated plate washer, adding a 10 second
soak period following the addition of wash buffer, and/or rotating the plate
180 degrees between wash steps may improve assay precision.
5. Controlling of reaction time: Observe the change of color after adding
Substrates (e.g. observation once every 10 minutes). Substrates should
change from colorless or light blue to gradations of blue. If the color is too
deep, add Stop Solution in advance to avoid excessively strong reaction
which will result in inaccurate absorbance reading.
6. Substrates are easily contaminated. Substrates should remain colorless or
light blue until added to the plate. Please protect it from light.
7. Stop Solution should be added to the plate in the same order as the
Substrates. 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 Substrates.10
ASSAY PROCEDURE SUMMARY11
CALCULATION OF RESULTS
Using the professional soft "Curve Expert 1.3" to make a standard curve is
recommended, which can be downloaded from our web.
Average the duplicate readings for each standard and sample and subtract the
average optical density of Blank.
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 EPI 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.
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