Laboratory Research Canine High Sensitive Interleukin 10
Immunoassay Test Kit 2 Hours Assay Length
Cat.No E0006Ca
Assay Principle
This elisa test kit is an Enzyme-Linked Immunosorbent Assay
(ELISA). The plate has been pre-coated with Canine IL-10 antibody.
IL-10 present in the sample is added and binds to antibodies coated
on the wells. And then biotinylated Canine IL-10 Antibody is added
and binds to IL-10 in the sample. Then Streptavidin-HRP is added
and binds to the Biotinylated IL-10 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 IL-10. The reaction is terminated by
addition of acidic stop solution and absorbance is measured at 450
nm.
Intended Use
This elisa test kit is for the accurate quantitative detection of
Canine Interleukin 10 (also known as IL-10) in serum, plasma, cell
culture supernates, cell lysates, tissue homogenates.
*This product is for research use only, not for use in diagnosis
procedures. It’s highly recommend to read this instruction entirely
before use.
Reagent Provided
Components | Quantity |
Standard Solution (800pg/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 IL-10 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 other body fluids 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.
*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 Preparation
All reagents should be brought to room temperature before use.
Standard Reconstitute the 120μl of the standard (800pg/ml) with
120μl of standard diluent to generate a 400pg/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 (400pg/ml)
1:2 with standard diluent to produce 200pg/ml, 100pg/ml, 50pg/ml
and 25pg/ml solutions. Standard diluent serves as the zero
standard(0 pg/ml). Any remaining solution should be frozen at -20°C
and used within one month. Dilution of standard solutions suggested
are as follows:
400pg/ml | Standard No.5 | 120μl Original Standard + 120μl Standard Diluent |
200pg/ml | Standard No.4 | 120μl Standard No.5 + 120μl Standard Diluent |
100pg/ml | Standard No.3 | 120μl Standard No.4 + 120μl Standard Diluent |
50pg/ml | Standard No.2 | 120μl Standard No.3 + 120μl Standard Diluent |
25pg/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 |
800pg/ml | 400pg/ml | 200pg/ml | 100pg/ml | 50pg/ml | 25pg/ml |
Wash Buffer Dilute 20ml of Wash Buffer Concentrate 30x into deionized or
distilled water to yield 600 ml of 1x Wash Buffer. If crystals have
formed in the concentrate, mix gently until the crystals have
completely dissolved.
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.