Customized 96 Well Plate Human ELISA Kit Angiopoietin 4 2 - 8°C Storage OEM
Brand Name:BT Lab
Certification:CE, ISO9001:2005, MSDS
Model Number:Cat.No E1263Hu
Minimum Order Quantity:Negotiation
Delivery Time:1-3 business days, bulk order within one week
Place of Origin:Shanghai, China
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Product Details
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Product Details
Human Strong Sensitivity and Specificity Angiopoietin 4 Sandwich
ELISA Kit Customized 96 Well Plate
Cat.No E1263Hu
Standard Curve Range: 15ng/L - 3000ng/L
Sensitivity: 7.73ng/L
Size: 96 wells
Assay Principle
This elisa test kit is an Enzyme-Linked Immunosorbent Assay
(ELISA). The plate has been pre-coated with human ANG-4 antibody.
ANG-4 present in the sample is added and binds to antibodies coated
on the wells. And then biotinylated human ANG-4 Antibody is added
and binds to ANG-4 in the sample. Then Streptavidin-HRP is added
and binds to the Biotinylated ANG-4 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 ANG-4. The reaction is terminated by
addition of acidic stop solution and absorbance is measured at 450
nm.
*This product is for research use only, not for use in diagnosis
procedures. It’s highly recommend to read this instruction entirely
before use.
Precautions
- Prior to use, the elisa test 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 elisa test kit should not be used beyond the expiration date.
Reagent Preparation
All reagents should be brought to room temperature before use.
Standard Reconstitute the 120μl of the standard (3200ng/L) with 120μl of
standard diluent to generate a 1600ng/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 (1600ng/L) 1:2 with standard
diluent to produce 800ng/L, 400ng/L, 200ng/L and 100ng/L solutions.
Standard diluent serves as the zero standard(0 ng/L). Any remaining
solution should be frozen at -20°C and used within one month.
Dilution of standard solutions suggested are as follows:
| 1600ng/L | Standard No.5 | 120μl Original Standard + 120μl Standard Diluent |
| 800ng/L | Standard No.4 | 120μl Standard No.5 + 120μl Standard Diluent |
| 400ng/L | Standard No.3 | 120μl Standard No.4 + 120μl Standard Diluent |
| 200ng/L | Standard No.2 | 120μl Standard No.3 + 120μl Standard Diluent |
| 100ng/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 |
| 3200ng/L | 1600ng/L | 800ng/L | 400ng/L | 200ng/L | 100ng/L |
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
completely dissolved.
Assay Procedure
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-ANG-4
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.
Summary
1. Prepare all reagents, samples and standards.
2. Add sample and ELISA reagent into each well. Incubate for 1 hour
at 37°C.
3. Wash the plate 5 times.
4. Add substrate solution A and B. Incubate for 10 minutes at 37°C.
5. Add stop solution and color develops.
6. Read the OD value within 10 minutes.
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.
Customized 96 Well Plate Human ELISA Kit Angiopoietin 4 2 - 8°C Storage OEM
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