Specificity Canine Sandwich ELISA Kit , 96 Wells Cortisol COR ELISA Kit
Brand Name:BT Lab
Certification:CE, ISO9001:2005, MSDS
Model Number:Cat.No E0077Ca
Minimum Order Quantity:Negotiation
Delivery Time:1-3 business days, bulk order within one week
Payment Terms:T/T, Western Union
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Product Details
Specificity and sensitivity Canine Cortisol COR ELISA Kit 96 wells
Cat.No E0077Ca
Standard Curve Range: 2ng/ml - 600ng/ml
Sensitivity: 1.04ng/ml
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
before use.
Intended Use
This sandwich kit is for the accurate quantitative detection of
Canine Cortisol (also known as COR) in serum, plasma, cell culture
supernates, cell lysates, tissue homogenates.
Assay Principle
This kit is an Enzyme-Linked Immunosorbent Assay (ELISA). The plate
has been pre-coated with Canine COR antibody. COR present in the
sample is added and binds to antibodies coated on the wells. And
then biotinylated Canine COR Antibody is added and binds to COR in
the sample. Then Streptavidin-HRP is added and binds to the
Biotinylated COR 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 Canine COR. The reaction is terminated by addition of
acidic stop solution and absorbance is measured at 450 nm.
Reagent Provided
| Components | Quantity |
| Standard Solution (800ng/ml) | 0.5ml x1 |
| Pre-coated ELISA Plate | 12 * 8 well strips x1 |
| Standard Diluent | 3ml x1 |
| Streptavidin-HRP | 6ml x1 |
| Stop Solution | 6ml x1 |
| Substrate Solution A | 6ml x1 |
| Substrate Solution B | 6ml x1 |
| Wash Buffer Concentrate (30x) | 20ml x1 |
| Biotinylated Canine COR Antibody | 1ml x1 |
| User Instruction | 1 |
| Plate Sealer | 2 pics |
| Zipper bag | 1 pic |
Specimen Collection
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
above-mentioned.
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
minutes.
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
minutes.
Reagent Preparation
All reagents should be brought to room temperature before use.
Standard Reconstitute the 120μl of the standard (800ng/ml) with 120μl of
standard diluent to generate a 400ng/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 (400ng/ml) 1:2 with standard
diluent to produce 200ng/ml, 100ng/ml, 50ng/ml and 25ng/ml
solutions. Standard diluent serves as the zero standard(0 ng/ml).
Any remaining solution should be frozen at -20℃ and used within one
month. Dilution of standard solutions suggested are as follows:
| 400ng/ml | Standard No.5 | 120μl Original Standard + 120μl Standard Diluent |
| 200ng/ml | Standard No.4 | 120μl Standard No.5 + 120μl Standard Diluent |
| 100ng/ml | Standard No.3 | 120μl Standard No.4 + 120μl Standard Diluent |
| 50ng/ml | Standard No.2 | 120μl Standard No.3 + 120μl Standard Diluent |
| 25ng/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 |
| 800ng/ml | 400ng/ml | 200ng/ml | 100ng/ml | 50ng/ml | 25ng/ml |
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
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-COR 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.
Specificity Canine Sandwich ELISA Kit , 96 Wells Cortisol COR ELISA Kit
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