Human Activated Protein C (APC) ELISA Kit

Instruction manual of human activated protein C (APC) ELISA kit

Human Activated Protein C (APC) ELISA Kit is for research use only

Detection range: 156 pg / ml -10,00 pg / ml

Intended application: ELISA method for quantitative determination of activated protein C (APC) content in human serum, plasma or other related fluids.

Experimental principle: This kit uses double antibody sandwich enzyme immunoassay to determine the level of activated protein C in the specimen. The microtiter plate is coated with purified antibody to make a solid-phase antibody. Activated protein C, biotinylated anti-human activated protein C antibody, and HRP-labeled avidin are added to the antibody-coated microwells in sequence. After washing, color was developed with the substrate TMB. TMB is converted into blue under the catalysis of peroxidase, and into the final yellow under the action of acid. The color depth is positively correlated with activated protein C in the sample. The absorbance (OD value) was measured with a microplate reader at a wavelength of 450 nm, and the sample concentration was calculated.

Human activated protein C (APC) ELISA kit composition and reagent preparation:

1. Enzyme-linked plate: one piece (96 wells)

2. Standard product (lyophilized product): 2 bottles, each bottle is diluted to 1ml with the sample diluent before use, and it is left to stand for more than 10 minutes after being capped, and then inverted / rubbed repeatedly to help dissolve, its concentration is 10,000 pg / ml, after serial dilution, dilute to 10,000 pg / mL, 5,000 pg / mL, 2,500 pg / mL, 1,250 pg / mL, 625 pg / mL, 312 pg / mL, 156 pg / mL As the highest standard concentration directly, the sample dilution is directly used as the standard concentration of 0 pg / mL, prepared within 15 minutes before use.

For example, to prepare a 5,000 pg / mL standard: Take 0.5ml 10,000 pg / mL of the above standard into an Eppendorf tube containing 0.5ml of sample diluent, mix well, and the rest of the concentration can be deduced by analogy.

3. Sample diluent: 1 × 20ml / bottle.

4. Test dilution A: 1 × 10ml / bottle.

5. Test diluent B: 1 × 10ml / bottle.

6. Detection solution A: 1 × 120ul / bottle (1: 100) is diluted with detection diluent A 1: 100 before use, prepared according to the pre-calculated total amount required for each experiment before dilution (100ul per well) , The actual preparation should be more 0.1-0.2ml. For example, 1ul detection solution A plus 99ul detection dilution A is prepared in proportion, mix gently and prepare within one hour before use.

7. Detection solution B: 1 × 120ul / bottle (1: 100) diluted with detection diluent B1: 100 before use. The dilution method is the same as that of Test Solution A.

8. Substrate solution: 1 × 10ml / bottle.

9. Concentrated washing solution: 1 × 30ml / bottle, each bottle is diluted 25 times with distilled water.

10. Stop solution: 1 × 10ml / bottle (2N H2SO4).

Collection and preservation of specimens:

1. Plasma: EDTA or heparin can be used as an anticoagulant. Centrifuge the sample at 2-8 ° C 1000 xg for 15 minutes within 30 minutes after collection, or store the specimen at -20 ° C, but avoid repeated freezing and thawing.

2. Serum: Please leave the specimen at room temperature for 2 hours or 4 ° C overnight and centrifuge at 1000 xg for 20 minutes. Take the supernatant for testing, or store the specimen at -20 ° C, but avoid repeated freezing and thawing.

Note: Hemolysis of specimens will affect the final test results, so hemolysis specimens should not be tested.

Steps:

Before starting the experiment, please configure all reagents in advance. When the reagents or samples are diluted, they should be mixed well. Try to avoid foaming when mixing. A standard curve should be made for each test. If the sample concentration is too high, dilute with sample diluent to make the sample meet the detection range of the kit.

1. Add sample: set blank hole, standard hole and sample hole to be tested respectively. Add 100ul of sample diluent to the blank well, and 100ul of the standard or the sample to be tested in the remaining well. Be careful not to have air bubbles. Add the sample to the bottom of the well of the microtiter plate. Do not touch the wall of the well. The target plate is covered with a cover or film and reacted at 37 ° C for 120 minutes.

To ensure the validity of the experimental results, please use a new standard solution for each experiment.

2. Discard the liquid and spin dry without washing. Add 100ul of detection solution A working solution to each well (take 1ul of detection solution A plus 99ul of detection dilution A to prepare, mix gently and prepare within one hour before use), 37 ℃, 60 minutes.

3. After incubating for 60 minutes, discard the liquid in the well, spin dry, wash the plate 3 times, soak for 1-2 minutes each time, 350ul / per well, spin dry.

4. Add 100ul of detection solution B working solution (same as the detection A working solution) to each well at 37 ℃ for 60 minutes.

5. After incubating for 60 minutes, discard the liquid in the wells, spin dry, wash the plate 5 times, soak for 1-2 minutes each time, 350ul / per well, spin dry.

6. Add 90ul of substrate solution to each well in sequence, and develop color at 37 ° C in the dark (within 30 minutes, at this time, the first 3-4 wells of the standard product have a visible blue gradient, and the back 3-4 wells are not obvious. , You can terminate).

7. Add 50ul of stop solution to each well in sequence to stop the reaction (at this time, the blue color turns to yellow). The order of adding the stop solution should be the same as that of the substrate solution. In order to ensure the accuracy of the experimental results, the termination solution should be added as soon as possible after the substrate reaction time expires.

8. Measure the optical density (OD value) of each well in sequence using an enzyme-linked instrument at a wavelength of 450 nm. Test within 15 minutes after adding stop solution.

Note:

1. Leave one hole for each experiment as a blank zero-adjusting hole. No reagent is added to this hole, but the substrate solution and 2NH2SO4 are added at the end. Use this hole to adjust the OD value to zero when measuring.

2. To prevent the sample from evaporating, place the reaction plate in a closed box covered with a damp cloth during the test, and add a cover or film to the enzyme label plate.

3. Store unused microplates or reagents at 2-8 ° C. Standard products, working solution A, and working solution B should be configured and used according to the required amount. Do not reuse the diluted standard, test solution A working solution or test solution B working solution.

4. It is recommended to set a double-hole test when testing samples to ensure the accuracy of the test results.

Washing method:

1. Automatic plate washing: If there is an automatic plate washing machine, it should be used in the formal experiment process after being used skillfully.

2. Manual plate washing method: suck up (do not touch the wall) or shake off the liquid in the enzyme plate; place a few layers of absorbent paper on the experimental table, and force the enzyme plate down to pat several times; the recommended washing buffer Inject at least 0.3ml into the hole, soak for 1-2 minutes, and repeat this process several times as needed.

Specificity: This kit can detect recombinant or natural human activated protein C at the same time, and does not cross-react with other related proteins.

Calculation: Taking the concentration of the standard as the abscissa (logarithmic coordinate) and the OD value as the ordinate (ordinary coordinate), draw a standard curve on semi-logarithmic coordinate paper, and find the corresponding curve from the standard curve according to the OD value of the sample Concentration; multiply by the dilution factor; or calculate the linear regression equation of the standard curve using the concentration of the standard and 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 sample concentration.

Precautions:

1. The washing process is very important, inadequate washing is easy to cause false positives.

2. It is best to control the sampling time within 5 minutes. If there are many specimens, it is recommended to use a volley gun to add samples.

3. Please make a standard curve at the same time of each measurement, it is best to make a complex hole.

4. If the content of the substance to be tested in the specimen is too high, please dilute it and then determine it. When calculating, please multiply by the dilution factor.

5. When preparing standard products and testing solution working fluid, please prepare with corresponding diluent, not to be confused.

6. Please keep the substrate away from light.

Explanation:

1. Avoid exposing the reagent to strong light during storage and incubation. All reagent bottle caps must be tightly closed to prevent evaporation and contamination. Reagents should be protected from microbial contamination, because the interference of proteolytic enzymes will lead to erroneous results.

2. Aspirate the reagents carefully and strictly observe the given incubation time and temperature. Please note that when drawing samples / standards, enzyme conjugates or substrates, if the time interval between the first well and the last well is too large, it will result in different "pre-incubation" time, which obviously Affect the accuracy and repeatability of the measured value. Moreover, insufficient washing will affect the test results.

3. Preservation of the kit: some reagents are stored at -20 ℃, and some reagents are stored at 2-8 ℃, depending on the label.

4. Salt will be precipitated from the concentrated washing liquid, which can be heated and dissolved in the water bath when diluted.

5. The well of the enzyme-linked plate just opened may contain a little water-like substance. This is a normal phenomenon and will not have any impact on the experimental results.

6. There may be inconsistencies between the Chinese and English instructions. Please refer to the English instructions.

7. All samples should be managed, and the samples and testing devices should be processed according to the prescribed procedures.

8. Validity: 6 months