3β-hydroxysteroid dehydrogenase I (3βHSDI) enzyme-linked immunoassay kit instruction manual

3 β-hydroxysteroid dehydrogenase I (3βHSDI) enzyme-linked immunosorbent assay

Kit instruction manual

This reagent is for research use only. Purpose: This kit is used to determine the content of 3β-hydroxysteroid dehydrogenase I (3βHSDI) in rat serum, plasma, cell supernatant and related liquid samples.

Experimental principle :

The kit uses a double antibody sandwich assay to determine the level of rat 3β-hydroxysteroid dehydrogenase I (3βHSDI) in the specimen. The microplate was coated with purified rat 3β-hydroxysteroid dehydrogenase I (3βHSDI) antibody to prepare a solid phase antibody, and 3β-hydroxysteroid dehydrogenase I (3βHSDI) was sequentially added to the microcapsules of the coated monoclonal antibody. Then, it is combined with an HRP-labeled 3β-hydroxysteroid dehydrogenase I (3βHSDI) antibody to form an antibody-antigen-enzyme-labeled antibody complex, which is thoroughly washed and then added with a 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 3β-hydroxysteroid dehydrogenase I (3βHSDI) in the sample. The absorbance (OD value) was measured by a microplate reader at a wavelength of 450 nm, and the concentration of rat 3β-hydroxysteroid dehydrogenase I (3βHSDI) in the sample was calculated by a standard curve.

Kit composition :

Sample processing and requirements :

1. Serum: The blood is naturally coagulated for 10-20 minutes at room temperature and centrifuged for about 20 minutes (2000-3000 rpm). The supernatant is carefully collected, and if precipitation occurs during storage, it should be centrifuged again.

2. Plasma: EDTA or sodium citrate should be selected as an anticoagulant according to the requirements of the specimen. After mixing for 10-20 minutes, centrifuge for about 20 minutes (2000-3000 rpm). The supernatant is carefully collected, and if a precipitate forms during storage, it should be centrifuged again.

3. Urine: Collect with a sterile tube and centrifuge for about 20 minutes (2000-3000 rpm). The supernatant is carefully collected, and if a precipitate forms during storage, it should be centrifuged again. The chest and ascites and cerebrospinal fluid are referred to.

4. Cell culture supernatant: When detecting secreted components, collect them in a sterile tube. Centrifuge for about 20 minutes (2000-3000 rpm). Collect the supernatant carefully. When the components in the cells were detected, the cell suspension was diluted with PBS (pH 7.2-7.4), and the cell concentration reached about 1 million/ml. By repeated freezing and thawing, the cells are destroyed and the intracellular components are released. Centrifuge for about 20 minutes (2000-3000 rpm). Collect the supernatant carefully. If a precipitate forms during storage, it should be centrifuged again.

5. Tissue specimen: After cutting the specimen, weigh the weight. Add a certain amount of PBS, pH 7.4. It was quickly frozen and stored in liquid nitrogen for use. The specimen still maintains a temperature of 2-8 ° C after melting. A certain amount of PBS (pH 7.4) was added, and the specimen was homogenized by hand or homogenizer. Centrifuge for about 20 minutes (2000-3000 rpm). Collect the supernatant carefully. One part of the package is to be tested, and the rest is frozen for use.

6. 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.

7. Samples containing NaN3 cannot be detected because NaN3 inhibits horseradish peroxidase (HRP) activity.

Steps:

1. Dilution and loading of standard products: 10 holes of standard wells are placed on the enzyme labeling plate, 100 μl of standard is added to the first and second holes, and then standard is added to the first and second holes. 50 μl of the dilution solution, and mix; then, 100 μl of each of the first hole and the second hole are respectively added to the third hole and the fourth hole, and then 50 μl of the standard dilution solution is added to the third and fourth holes, respectively, and mixed; Then, 50 μl of each of the third hole and the fourth hole is discarded, and then 50 μl of each is added to the fifth and sixth holes, respectively, and 50 ul of the standard dilution solution is added to the fifth and sixth holes, respectively, and mixed; After mixing, 50 μl of each of the fifth and sixth wells is separately added to the seventh and eighth wells, and then 50 μl of the standard dilution solution is added to the seventh and eighth wells, and the mixture is mixed from the seventh and the seventh. 50 μl of each of the eight wells 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 after dilution was 50 μl, and the concentrations were 120 U/L, 80 U/L, 40 U/L, 20 U/L, 10 U/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. Solution: 30 (48 times of 48T) concentrated washing solution was diluted with distilled water 30 (20 times of 48T) 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.

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.

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.

Kit performance:

1. The linear regression coefficient of the sample and the expected concentration correlation coefficient R value is 0.990 or more.

2. Within and within the batch should be less than 9% and 11% respectively

Storage conditions and expiration date:

1. Kit storage: 2-8 ° C.

2. Validity: 6 months