Canine tartaric acid phosphatase 5b (TRACP5b)
ELISA
Kit
Experimental operation guide
This kit is for research use only.
content.
Experimental principle
Canine tartaric acid phosphatase 5b (TRACP5b)
ELISA
Kit
The level of dog-resistant tartrate acid phosphatase 5b (TRACP5b) in the specimen was determined by double antibody sandwich method. The microplate was coated with purified dog anti-tartaric acid phosphatase 5b (TRACP5b) antibody to prepare a solid phase antibody, and tartrate-resistant acid phosphatase 5b (TRACP5b) was sequentially added to the microcapsules of the coated monoclonal antibody, and then labeled with HRP. The anti-tartaric acid phosphatase 5b (TRACP5b) antibody binds to form an antibody-antigen-enzyme-labeled antibody complex, which is thoroughly washed and then added to the substrate TMB for color development. TMB is converted to blue under the catalysis of HRP enzyme and converted to the final yellow color by the action of an acid. The color depth is positively correlated with the tartrate-resistant acid phosphatase 5b (TRACP5b) in the sample. The absorbance (OD value) was measured by a microplate reader at a wavelength of 450 nm, and the concentration of canine acid tartrate acid phosphatase 5b (TRACP5b) in the sample was calculated from a standard curve.
Canine tartaric acid phosphatase 5b (TRACP5b)
ELISA
Kit
composition
130 times concentrated washing solution 20ml × 1 bottle 7 stop solution 6ml × 1 bottle
2 enzyme standard reagent 6ml × 1 bottle 8 standard (480ng / L) 0.5ml × 1 bottle
3 enzyme label coating plate 12 holes × 8 9 standard dilutions 1.5ml × 1 bottle
4 sample dilution 6ml × 1 bottle 10 instructions 1 copy
5 color developing agent A liquid 6ml × 1 bottle 11 sealing film 2 sheets
6 color developer B liquid 6ml × 1 bottle 12 sealed bag 1
Specimen requirements
1. The specimens should be extracted as soon as possible after collection, and the extraction should be carried out according to the relevant literature. The experiment should be carried out as soon as possible after extraction. If the test cannot be performed immediately, the specimen can be stored at -20 °C, but repeated freezing and thawing should be avoided.
2. Samples containing NaN3 could not be detected because NaN3 inhibited horseradish peroxidase (HRP) activity.
Canine tartaric acid phosphatase 5b (TRACP5b)
ELISA
Kit
Steps
1. Dilution of standard: This kit provides one original standard, which can be diluted in a small tube according to the following chart.
Add 150 μl of standard dilution to the original standard of 150 μl of 240 ng/L No. 5 standard
Add 150 μl of standard dilution to 120 ng/L No. 4 standard 150 μl of No. 5 standard
Add 60 μl of standard dilution to the standard No. 4 of 60 ng/L No. 3 standard 150 μl
Add 30 μl of standard dilution to 150 μl of Standard No. 3 of 30 ng/L No. 2 Standard
Add 150 μl of standard dilution to 15 ng/L No. 1 standard 150 μl of No. 2 standard
2. Loading: Set blank holes separately (the blank control wells are not added with the sample and the enzyme standard reagent, the other steps are the same), the standard holes, and the sample holes to be tested. Accurately load 50 μl of the standard on the enzyme-labeled plate, add 40 μl of the sample dilution to the well to be tested, and then add 10 μl of the sample to be tested (the final dilution of the sample is 5 times). Add the sample to the bottom of the well of the microplate, try not to touch the wall of the well, and shake gently to mix.
3. Incubation: The plate was sealed with a sealing film and incubated at 37 ° C for 30 minutes.
4. Dosing: 30 times concentrated washing solution diluted with distilled water 30 times and used
5. Washing: Carefully remove the sealing film, discard the liquid, dry it, fill each well with the washing liquid, let stand for 30 seconds, then discard it, repeat 5 times, and pat dry.
6. Add enzyme: 50 μl of enzyme labeling reagent was added to each well, except for blank wells.
7. Incubation: The operation is the same as 3.
8. Washing: The operation is the same as 5.
9. Color development: Add 50 μl of the developer to each well, then add 50 μl of the developer, gently shake and mix, and develop at 37 ° C for 10 minutes.
10. Termination: 50 μl of stop solution was added to each well to terminate the reaction (when the blue color turned yellow).
11. Measurement: The absorbance (OD value) of each well was measured sequentially with a blank air conditioner of zero and a wavelength of 450 nm. The measurement should be carried out within 15 minutes after the addition of the stop solution.
Calculation
Taking the concentration of the standard as the abscissa and the OD as the ordinate, draw a standard curve on the coordinate paper, and find the corresponding concentration from the standard curve according to the OD value of the sample; multiply by the dilution factor; or use the concentration of the standard Calculate the linear regression equation of the standard curve with the OD value, substitute the OD value of the sample into the equation, calculate the sample concentration, and multiply by the dilution factor, which is the actual concentration of the sample.
Canine tartaric acid phosphatase 5b (TRACP5b)
ELISA
Kit
Precautions
1. The kit should be taken out from the refrigerated environment and should be used at room temperature for 15-30 minutes before use. If the enzyme label is unsealed after opening, the slats should be stored in a sealed bag.
2. Concentrated washing liquid may crystallize out. When diluted, it can be heated and dissolved in a water bath. The washing will not affect the result.
3. The sampler should be used for each step, and the accuracy should be corrected frequently to avoid test errors. It is best to control the loading time within 5 minutes. If the number of specimens is large, it is recommended to use a gun.
4. Please make a standard curve at the same time of each measurement, it is best to make a double hole. If the content of the substance to be tested in the specimen is too high (the OD value of the sample is larger than the OD value of the first hole of the standard well), please first dilute the sample dilution with a certain multiple (n times) and then measure it. When calculating, multiply the total dilution by the total dilution. Multiple (×n×5).
5. The sealing film is intended for single use only to avoid cross-contamination.
6. Please keep the substrate away from light.
7. Strictly follow the instructions of the manual, the test results must be based on the microplate reader reading.
8. All samples, washings and various wastes should be treated as infectious materials.
9. The different batch components of this reagent must not be mixed.
Canine tartaric acid phosphatase 5b (TRACP5b)
ELISA
Kit
Storage conditions and expiration date
1. Kit storage: 2-8 ° C.
2. Validity: 6 months
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