Canine C-reactive protein (CRP) enzyme-linked immunosorbent assay

Drug Name:
Generic Name: Canine C-Reactive Protein (CRP) Enzyme-Linked Immunoassay Kit Uses:
This kit is used to determine the content of canine C-reactive protein (CRP) in canine serum, plasma and related fluid samples.
Experimental principle This kit uses the double antibody sandwich method to determine the level of canine C-reactive protein (CRP) in the specimen. The microplate was coated with purified canine C-reactive protein (CRP) antibody to prepare a solid phase antibody, and C-reactive protein (CRP) was sequentially added to the microcapsule of the coated monoclonal antibody, followed by HRP-labeled C-reactive protein (CRP). The 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 of zui under the action of acid. The color depth is positively correlated with C-reactive protein (CRP) in the sample. The absorbance (OD value) was measured with a microplate reader at a wavelength of 450 nm, and the concentration of canine C-reactive protein (CRP) in the sample was calculated from a standard curve.
Kit composition
1 20 times concentrated washing solution 30ml × 1 bottle 7 Stop solution 6ml × 1 bottle
2 enzyme standard reagent 6ml × 1 bottle 8 standard (2700μg / L) 0.5ml × 1 bottle
3 Enzyme label coating plate 12 holes × 8 strips 9 standard dilutions 1.5ml × 1 bottle
4 sample dilution 6ml × 1 bottle 10 instructions 1
5 developer A liquid 6ml × 1 bottle 11 sealing film 2 sheets
6 developer B liquid 6ml × 1 bottle 12 sealed bag 1 specimen request
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.
Steps
1. Dilution and loading of standard products: 10 wells of standard wells on the enzyme labeling plate, 100 μl of standard in * and 2, respectively, and then standard dilutions in * and 2 holes 50μl, mix; then add 100μl from the * hole and the second hole to the third hole and the fourth hole respectively, then add 50μl of the standard dilution in the third and fourth holes, and mix; Take 50 μl of each of the three wells and the fourth well, and then add 50 μl to each of the fifth and sixth wells, and then add 50 μl of the standard dilution solution to the fifth and sixth wells, and mix; 50μl from each of the fifth and sixth holes are respectively added to the seventh and eighth holes, and then 50 μl of the standard dilution solution is added to the seventh and eighth holes, respectively, and mixed from the seventh and eighth holes. 50 μl of each was added to the ninth and tenth holes, and 50 μl of the standard dilution was added to the ninth and tenth holes, respectively, and 50 μl of each of the ninth and tenth holes was discarded. (The amount of each well was 50 μl after dilution, and the concentrations were 1800 μg/L, 1200 μg/L, 600 μg/L, 300 μg/L, 150 μg/L, respectively).
2. Adding samples: 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), and the sample holes to be tested. Add 40 μl of the sample dilution to the sample well to be tested on the enzyme-labeled plate, 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. Liquor: 20 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 solution, let stand for 30 seconds, then discard it, repeat 5 times, and pat dry.
6. Add enzyme: Add 50 μl of enzyme labeling reagent to each well, except for blank wells.
7. Incubation: The operation is the same as 3.
8. Wash: Operate the same as 5.
9. Color development: add 50 μl of color developer A, and then add 50 μl of color developer B, gently shake and mix, and color for 15 minutes at 37 °C.
10. Termination: 50 μl of stop solution was added to each well to stop the reaction (the blue color turned yellow).
11. Measurement: The absorbance (OD value) of each well was measured in sequence 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.

Calculate 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 standard Calculate the linear regression equation of the standard curve by the concentration and 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.
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. Once the sample loading time is good, it is controlled 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 for each measurement, and 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 standard pore* hole), please first dilute the sample dilution with a certain multiple (n times) before measuring. Please calculate the total dilution after Zui. 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.
10. In the case of an English manual, the English manual shall prevail.
examination range:
100μg/L -2000μg/L
specification:
96 person/box storage conditions and expiration date
1. The kit is stored at: 2-8 °C.
2. Validity: 6 months

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