96 Wells Mouse High Precision Endothelin 1 ET 1 Sandwich ELISA Kit
Standard Curve Range: 1ng/L - 400ng/L
Size: 96 wells
Storage: Store the reagents at 2-8°C. For over 6-month storage refer to
the expiration date keep it at -20°C. Avoid repeated thaw cycles.
If individual reagents are opened it is recommended that the kit be
used within 1 month.
*This product is for research use only, not for use in diagnosis
procedures. It’s highly recommend to read this instruction entirely
Intra-Assay Precision (Precision within an assay) Three samples of
known concentration were tested on one plate to assess intra-assay
Inter-Assay Precision (Precision between assays) Three samples of
known concentration were tested in separate assays to assess
CV(%) = SD/mean x 100
This sandwich kit is for the accurate quantitative detection of
Mouse Endothelin 1 (also known as ET-1) in serum, plasma, cell
culture supernates, cell lysates, tissue homogenates.
|Standard Solution (480ng/L)||0.5ml x1|
|Pre-coated ELISA Plate||12 * 8 well strips x1|
|Standard Diluent||3ml x1|
|Stop Solution||6ml x1|
|Substrate Solution A||6ml x1|
|Substrate Solution B||6ml x1|
|Wash Buffer Concentrate (30x)||20ml x1|
|Biotinylated Mouse ET-1 Antibody||1ml x1|
|Plate Sealer||2 pics|
|Zipper bag||1 pic|
Material Required But Not Supplied
- 37°C±0.5°C incubator
- Absorbent paper
- Precision pipettes and disposable pipette tips
- Clean tubes
- Deionized or distilled water
- Microplate reader with 450 ± 10nm wavelength filter
- Prior to use, the kit and sample should be warmed naturally to room
temperature 30 minutes.
- This instruction must be strictly followed in the experiment.
- Once the desired number of strips has been removed, immediately
reseal the bag to protect the remain from deterioration. Cover all
reagents when not in use.
- Make sure pipetting order and rate of addition from well-to-well
when pipetting reagents.
- Pipette tips and plate sealer in hand should be clean and
disposable to avoid cross-contamination.
- Avoid using the reagents from different batches together.
- Substrate solution B is sensitive to light, don’t expose substrate
solution B to light for a long time.
- Stop solution contains acid. Please wear eye, hand and skin
protection when using this material. Avoid contact of skin or
mucous membranes with kit reagent.
- The kit should not be used beyond the expiration date.
Serum Allow serum to clot for 10-20 minutes at room temperature.
Centrifuge at 2000-3000 RPM for 20 minutes.
Plasma Collect plasma using EDTA or heparin as an anticoagulant.
Centrifuge samples for 15 minutes at 2000-3000 RPM at 2 - 8°C
within 30 minutes of collection.
Urine Collect by sterile tube. Centrifuge at 2000-3000 RPM for
approximately 20 minutes. When collecting pleuroperitoneal fluid
and cerebrospinal fluid, please follow the procedures
Cell Culture Supernatant Collect by sterile tubes when examining secrete components.
Centrifuge at 2000-3000 RPM for approximately 20 minutes. Collect
the supernatants carefully. When examining the components within
the cell, use PBS (pH 7.2-7.4) to dilute cell suspension to the
cell concentration of approximately 1 million/ml. Damage cells
through repeated freeze-thaw cycles to let out the inside
components. Centrifuge at 2000-3000 RPM for approximately 20
Tissue Rinse tissues in PBS (pH 7.4) to remove excess blood thoroughly
and weigh before homogenization. Mince tissues and homogenize them
in PBS (pH7.4) with a glass homogenizer on ice. Thaw at 2-8°C or
freeze at -20°C. Centrifuge at 2000-3000 RPM for approximately 20
- Sample concentrations should be predicted before being used in the
assay. If the sample concentration is not within the range of the
standard curve, users must contact us to determine the optimal sample for their particular experiments.
- Samples to be used within 5 days should be stored at 2-8°C. Samples
should be aliquoted or must be stored at -20°C within 1 month or
-80°C within 6 months. Avoid repeated freeze thaw cycles.
- Samples should be brought to room temperature before starting the
- Centrifuge to collect sample before use.
- Samples containing NaN3 can’t be tested as it inhibits the activity
of Horse Radish Peroxidase (HRP).
- Collect the supernatants carefully. When sediments occurred during
storage, centrifugation should be performed again.
- Hemolysis can greatly impact the validity of test results. Take
care to minimize hemolysis.
*Sample can't be diluted with this kit. Owing to the the material we use to prepare the kit, the sample
matrix interference may falsely depress the specificity and
accuracy of the assay.
Reagent PreparationAll reagents should be brought to room temperature before use.Standard Reconstitute the 120μl of the standard (480ng/L) with 120μl of
standard diluent to generate a 240ng/L standard stock solution.
Allow the standard to sit for 15 mins with gentle agitation prior
to making dilutions. Prepare duplicate standard points by serially
diluting the standard stock solution (240ng/L) 1:2 with standard
diluent to produce 120ng/L, 60ng/L, 30ng/L and 15ng/L solutions.
Standard diluent serves as the zero standard(0 ng/L). Any remaining
solution should be frozen at -20℃ and used within one month.
Dilution of standard solutions suggested are as follows:
|240ng/L||Standard No.5||120μl Original Standard + 120μl Standard Diluent|
|120ng/L||Standard No.4||120μl Standard No.5 + 120μl Standard Diluent|
|60ng/L||Standard No.3||120μl Standard No.4 + 120μl Standard Diluent|
|30ng/L||Standard No.2||120μl Standard No.3 + 120μl Standard Diluent|
|15ng/L||Standard No.1||120μl Standard No.2 + 120μl Standard Diluent|
|Standard Concentration||Standard No.5||Standard No.4||Standard No.3||Standard No.2||Standard No.1|
Wash Buffer Dilute 20ml of Wash Buffer Concentrate 30x into deionized or
distilled water to yield 500 ml of 1x Wash Buffer. If crystals have
formed in the concentrate, mix gently until the crystals have
1. Prepare all reagents, standard solutions and samples as
instructed. Bring all reagents to room temperature before use. The
assay is performed at room temperature.
2. Determine the number of strips required for the assay. Insert
the strips in the frames for use. The unused strips should be
stored at 2-8°C.
3. Add 50μl standard to standard well. Note: Don’t add antibody to standard well because the standard solution
contains biotinylated antibody.
4. Add 40μl sample to sample wells and then add 10μl
anti-CAMP/LL-37 antibody to sample wells, then add 50μl
streptavidin-HRP to sample wells and standard wells ( Not blank
control well ). Mix well. Cover the plate with a sealer. Incubate
60 minutes at 37°C.
5. Remove the sealer and wash the plate 5 times with wash buffer.
Soak wells with at least 0.35 ml wash buffer for 30 seconds to 1
minute for each wash. For automated washing, aspirate all wells and
wash 5 times with wash buffer, overfilling wells with wash buffer.
Blot the plate onto paper towels or other absorbent material.
6. Add 50μl substrate solution A to each well and then add 50μl
substrate solution B to each well. Incubate plate covered with a
new sealer for 10 minutes at 37°C in the dark.
7. Add 50μl Stop Solution to each well, the blue color will change
into yellow immediately.
8. Determine the optical density (OD value) of each well
immediately using a microplate reader set to 450 nm within 10
minuets after adding the stop solution.
Calculation of Result
Construct a standard curve by plotting the average OD for each
standard on the vertical (Y) axis against the concentration on the
horizontal (X) axis and draw a best fit curve through the points on
the graph. These calculations can be best performed with
computer-based curve-fitting software and the best fit line can be
determined by regression analysis.