Sandwich Type High Precision and Specificity Human Eosinophil
Cationic Protein ELISA Test Kit
This ELISA Assay kit is an Enzyme-Linked Immunosorbent Assay
(ELISA). The plate has been pre-coated with Human ECP antibody. ECP
present in the sample is added and binds to antibodies coated on
the wells. And then biotinylated Human ECP Antibody is added and
binds to ECP in the sample. Then Streptavidin-HRP is added and
binds to the Biotinylated ECP antibody. After incubation unbound
Streptavidin-HRP is washed away during a washing step. Substrate
solution is then added and color develops in proportion to the
amount of Human ECP. The reaction is terminated by addition of
acidic stop solution and absorbance is measured at 450 nm.
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
- Prior to use, the ELISA Assay 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.
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
All reagents should be brought to room temperature before use.
Standard Reconstitute the 120μl of the standard (120ng/ml) with 120μl of
standard diluent to generate a 60ng/ml 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 (60ng/ml) 1:2 with standard
diluent to produce 30ng/ml, 15ng/ml, 7.5ng/ml and 3.75ng/ml
solutions. Standard diluent serves as the zero standard(0 ng/ml).
Any remaining solution should be frozen at -20°C and used within
one month. Dilution of standard solutions suggested are as follows:
|60ng/ml||Standard No.5||120μl Original Standard + 120μl Standard Diluent|
|30ng/ml||Standard No.4||120μl Standard No.5 + 120μl Standard Diluent|
|15ng/ml||Standard No.3||120μl Standard No.4 + 120μl Standard Diluent|
|7.5ng/ml||Standard No.2||120μl Standard No.3 + 120μl Standard Diluent|
|3.75ng/ml||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 25x 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-ECP
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.