Rat α-smooth muscle actin-linked immunosorbent assay kit instruction manual

This kit is for research use only.

Detection range: 96T

2.5ng/ml-80ng/ml

purpose of usage:

This kit is used to determine the content of α-smooth muscle actin (α-SMA) in rat serum , plasma and related fluid samples.

Experimental principle

The kit measures the level of rat α-smooth muscle actin (α-SMA) in the specimen by double antibody sandwich method. A solid phase antibody was prepared by coating a microplate with purified rat human α-smooth muscle actin (α-SMA) antibody, and α-smooth muscle actin (α-SMA) was sequentially added to the microcapsule of the coated monoclonal antibody. The antibody is then combined with an HRP-labeled α-smooth muscle actin (α-SMA) antibody 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 α-smooth muscle actin (α-SMA) in the sample. The absorbance (OD value) was measured by a microplate reader at a wavelength of 450 nm, and the concentration of rat α-smooth muscle actin (α-SMA) in the sample was calculated from a standard curve.

Kit composition

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.

Steps

1. Dilution of standard: This kit provides one original standard, which can be diluted in a small tube according to the following chart.

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), 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. Liquor: 20 times concentrated washing solution diluted with distilled water 20 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.

Summary of operating procedures:

1. Prepare reagents, samples and standards

2. Add prepared samples and standards and react at 37 ° C for 30 minutes.

3. Wash the plate 5 times, add the enzyme standard reagent, and react at 37 ° C for 30 minutes.

4. Wash the plate 5 times, add the coloring solution A, B, and react at 37 ° C for 10 minutes.

5. Add stop solution

OD value within 6.15 minutes

7. Calculation

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.

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.

10. In the case of an English manual, the English manual shall prevail.

Storage conditions and expiration date

1. The kit is stored at: 2-8 °C.

2. Validity: 6 months