user's Guide

【Kit name】

Canine Activated Protein C (APC) Quantitative Detection Kit (ELISA)

【Use of the kit】

Quantitative detection of activated protein C (APC) in dog serum, plasma and related liquid samples.

【Detection principle】

This kit uses double-antibody two-step sandwich enzyme-linked immunosorbent assay (ELISA). Add the standard and the test sample to the pre-coated canine activated protein C (APC) monoclonal antibody transparent enzyme label coated plate. After incubation for a sufficient time, wash to remove unbound components, and then add the enzyme label working solution. After incubation for a sufficient time, washing to remove unbound components. Substrates A and B are added in sequence. The substrate (TMB) is converted into a blue product catalyzed by horseradish peroxidase (HRP) and turns yellow under the action of an acid. (APC) The concentration is positively correlated. The OD value is measured at a wavelength of 450 nm. Based on the OD value of the standard and the sample, the canine activated protein C (APC) content in the sample is calculated.

【Composition of the kit】

Enzyme label coating plate 12 well × 8 strips 7
Developer A liquid 6mL

Standard product: 1600pg / mL
Developer B liquid 6mL

20 times concentrated washing liquid 25mL
Stop solution 6mL

Standard dilution 6mL
Instructions 1
Sample diluent 6mL
Sealing film 2 sheets
Enzyme reagent 6mL
1 sealed bag

Remarks: The standard product is diluted with standard product diluent in order: 1600, 800, 400, 200, 100, 50pg / mL

[Reagents and equipment needed but not provided]

1. 37 ℃ thermostat

2. Standard specification microplate reader

3. Precision pipettes and disposable tips

4. Distilled water

5. Disposable test tubes

6. Absorbent paper


1. Preparation: Remove the reagent kit from the refrigerator and re-equilibrate at room temperature for 30 minutes.

2. Mixing solution: dilute the 20-fold concentrated washing solution with distilled water to the original one.

3. Add standard products and samples to be tested: take a sufficient number of enzyme-coated plates and fix them to the frame. Set up standard wells, sample wells to be tested and blank control wells, record the positions of each well in the standard wells. Add 50μL of standard product; first add 10μL of sample to be tested, and then add 40μL of sample diluent (that is, the sample is diluted 5 times); blank control well is not added.

4. Incubation: Incubate in a 37 ° C water bath or thermostat for 30 minutes.

5. Wash the plate: discard the liquid, pat dry on the absorbent paper, fill each hole with the washing solution, let stand for 1min, shake off the washing solution, pat dry on the absorbent paper, repeat the washing 4 times (you can also use the washing machine to press Instructions for washing the board ).

6. Add enzyme-labeled working solution: add 50μL of enzyme-labeled working solution to each well, without adding blank control wells.

7. Incubation: Repeat operation 4.

8. Wash the board: repeat the operation of 5.

9. Color development: Add 50μL of developer A solution to each well, then add 50μL of developer B solution, and develop color at 37 ° C in the dark for 15min.

10. Termination: Remove the enzyme labeling plate and add 50μL of stop solution to each well to stop the reaction (the color changes from blue to yellow)

11. Determination: Zero the blank holes, and within 15 minutes after termination, measure the absorbance (OD value) of each well with a wavelength of 450 nm.

12. Calculation: According to the concentration of the standard product and the corresponding OD value, calculate the linear regression equation of the standard curve, 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 times the dilution factor.

[Sample requirements]

1. The sample cannot contain sodium azide (NaN3) because sodium azide (NaN3) is an inhibitor of horseradish peroxidase (HRP).

2. The specimen should be extracted as soon as possible after collection. The extraction should be carried out according to relevant literature. If the test cannot be performed immediately, the specimen can be stored at -20 ℃, but repeated freezing and thawing should be avoided.

3. The sample should be fully centrifuged, without hemolysis and particles.


1. The experiment is carried out in strict accordance with the instructions, and the result of the experiment must be determined by the reading of the microplate reader.

2. If the enzyme-labeled coated board is not used up after opening, it should be put into a sealed bag and added with desiccant immediately.

3. It is recommended that all standards, samples and blank controls be tested in duplicate, and the average value is taken to reduce the experimental error.

4. Keep in mind that the sample has been diluted 5 times. The calculation result is multiplied by 5 to obtain the actual concentration of the sample.

5. The quantitative range of this kit is 50-1600pg / mL, beyond this range, it is calculated from the extension of the standard curve, not as an accurate quantitative result, please use the standard dilution to determine the accurate result after dilution (50-1600pg / mL range Inside), 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 properly.

7. In order to avoid cross-contamination, the tips, samples and blank controls should be replaced with a new one for each addition; the common components such as enzyme working solution, sample diluent and substrate should be cantilevered, and they should not touch the microwells. ; Do not reuse the sealing film.

8. The kits are used within the warranty period, and different batches of reagents should not be mixed.

9. Substrate B is sensitive to light and avoid prolonged exposure to light.

[Summary of operating procedures]

Prepare reagents, samples and standards

Add prepared samples and standards, and react at 37 ℃ for 30 minutes

Wash the plate 4 times, add the enzyme reagent, and react at 37 ℃ for 30 minutes

Wash the plate 4 times, add color developing solutions A and B, and develop at 37 ℃ for 15 minutes

Add stop solution

Read OD value within 15 minutes


【examination range】

50-1600pg / mL


96 servings / box


Store at 2-8 ℃, protected from light and moisture.


6 months

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