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[The principle of detection of mouse interleukin 10 receptor (IL-10R) ELISA kit]
The kit uses a two-antibody two-step sandwich enzyme-linked immunosorbent assay (ELISA). The standard and the sample to be tested are added to the pre-coated chemotactic factor CC-primary ligand 3 (CCL3) transparent enzyme-labeled plate, and after incubation for a sufficient time, the unbound component is washed and removed, and the enzyme label is added. The working fluid, after incubation for a sufficient period of time, is washed to remove unbound components. Substrate A and B were sequentially added, and the substrate (TMB) was converted to a blue product under the catalysis of horseradish peroxidase (HRP), which turned yellow under the action of acid, the color depth and the chemotactic factor CC in the sample. - The concentration of the ligand 3 (CCL3) was positively correlated, and the OD value was measured at a wavelength of 450 nm. The content of the chemotactic factor CC-primary ligand 3 (CCL3) in the sample was calculated according to the OD value of the standard and the sample.
Remarks: Standards are diluted with standard dilutions to: 200, 100, 50, 25, 12.5, 6.25 ng/ml
[Mouse Interleukin 10 Receptor (IL-10R) ELISA Kit Procedure]
1. Preparation: Remove the kit from the refrigerator and re-balance at room temperature for 30 minutes.
2. Dosing: Dilute the 20 times concentrated washing solution into the original washing solution with distilled water.
3. Add standard and sample to be tested: Take a sufficient number of enzyme label coated plates, fix them on the frame, set standard wells, sample holes to be tested and blank control holes, record the position of each hole, in the standard hole Add 50 μL of the standard sample; add 10 μL of the sample to be tested first, and then add 40 μL of the sample dilution (ie, the sample is diluted 5 times); the blank control well is not added.
4. Incubation: Incubate for 30 min in a 37 °C water bath or incubator.
5. Wash the board: discard the liquid, pat dry on the absorbent paper, fill each well with the washing liquid, let stand for 1 min, remove the washing liquid, pat dry on the absorbent paper, and repeat the washing 4 times (can also be used by the washing machine) Instructions for washing the board).
6. Add the enzyme standard working solution: add 50 μL of the enzyme standard working solution to each well, and the blank control well is not added.
7. Incubation: Repeat 4 operations.
8. Wash the plate: Repeat the operation of 5.
9. Color development: 50 μL of the developer A solution was added to each well, then 50 μL of the developer B solution was added, and the color was developed at 37 ° C for 15 minutes.
10. Termination: The enzyme plate was taken out, and 50 μL of the stop solution was added to each well to terminate the reaction (the color was changed from blue to yellow).
11. Measurement: Zeroing was performed with a blank hole, and the absorbance (OD value) of each well was measured with a wavelength of 450 nm within 15 minutes after termination.
12. Calculation: Calculate the linear regression equation of the standard curve according to the concentration of the standard product and the corresponding OD value, and then calculate the corresponding sample concentration on the regression equation according to the OD value of the sample. You can also use various application software to Calculation. The final concentration is the actual measured concentration multiplied by the dilution factor.
[Notes on Mouse Interleukin 10 Receptor (IL-10R) ELISA Kit]
1. The experiment is carried out in strict accordance with the operation of the manual. The determination of the experimental results must be based on the reading of the microplate reader.
2. If the enzyme label is not used up after being opened, it should be immediately placed in a sealed bag and desiccant.
3. It is recommended that all standards, samples and blanks be double-tested and averaged to reduce experimental error.
4. Keep in mind that the sample has been diluted 5 times and the calculated result is multiplied by 5 to determine the actual concentration of the sample.
5. The quantitative range of this kit is 0.1-200ng/ml. If it exceeds this range, it is calculated by the extension of the standard curve. It is not used as an accurate quantitative result. Please dilute with special dilution solution to determine the accurate result (0.1-200ng/ml). ), multiplied by the total dilution factor is the final concentration of the sample.
6. If the color is too light, the substrate incubation time can be extended appropriately.
7. In order to avoid cross-contamination, the standard, product and blank control should be replaced once for each additional one; the common components such as enzyme standard working solution, sample diluent and substrate should be cantilevered and should not touch micropores. The sealing film must not be reused.
8. The kit is used during the shelf life. The reagents of different batches must not be mixed.
9. Substrate B is sensitive to light and avoids prolonged exposure to light.
[Mice Interleukin-10 Receptor (IL-10R) ELISA Kit Operating Procedure Summary]
- Prepare reagents, samples and standards
- Add prepared samples and standards and react at 37 ° C for 30 minutes
- Wash the plate 4 times, add the enzyme standard reagent, and react at 37 ° C for 30 minutes.
- Wash the plate 4 times, add the coloring solution A, B, and develop color at 37 ° C for 15 minutes.
- Add stop solution
- Read OD value within 15 minutes
- Calculation
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Mouse Interleukin 10 Receptor (IL-10R) ELISA Kit Procedure
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