NK natural killer cell ELISA kit

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We can tailor testing kits according to your application needs, such as special requirements for detection range, species, and sensitivity, to effectively control the cost of testing reagents. And free proxy testing can be provided.

Kit composition and reagent preparation:

1. Enzyme linked assay plate: one piece (96 well).

2. Standard: 2 bottles (freeze-dried).

3. Sample Diluent: 1 × 20ml/bottle.

4. Labeling antibody diluent: 1 × 10ml/bottle.

5. HRP avidin Diluent: 1 × 10ml/bottle.

6. Biotin antibody labeling: 1 × 120 μ l/bottle (1:100)

7. Horseradish peroxidase labeled avidin (HRP avidin): 1 × 120 μ l/bottle (1:100)

8. TMB Substrate: 1 × 10ml/bottle.

9. Wash Buffer: 1 × 20ml/bottle, diluted 25 times with distilled water per bottle during use.

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

Sample preparation:

1) Serum - Avoid any cellular stimulation during operation. Use test tubes without pyrogen and endotoxin. After collecting blood, centrifuge at 1000 × g for 10 minutes to quickly and carefully separate serum and red blood cells.

2) Plasma - EDTA, citrate, heparin plasma can be used for detection. Centrifuge at 1000 × g for 30 minutes to remove particles.

3) Cell supernatant - Centrifuge at 1000 × g for 10 minutes to remove particles and polymers.

4) Tissue homogenate - add an appropriate amount of physiological saline and crush the tissue. Centrifuge at 1000 × g for 10 minutes and collect the supernatant

5) Storage - If the sample is not used immediately, it should be divided into small parts and stored at -70 ℃ to avoid repeated freezing. Try not to use hemolytic or hyperlipidemic blood as much as possible. If there are a large number of particles in the serum, centrifuge or filter before detection. Do not thaw by heating at 37 ℃ or higher. Thaw at room temperature and ensure that the sample is evenly thawed.

Usage:

The sensitivity of the assay comes from the enzyme used as the report. Enzymes are organic catalysts, and a small amount of enzyme can induce a large number of catalytic reactions, resulting in observable color reactions. Therefore, this system is often referred to as an enzyme amplification system.

1. Dilution of standard samples: This kit provides one original standard sample, and users can dilute it in a small test tube according to the following chart. Add 150 μ l of standard dilution to 150 μ l of the original standard of standard 5 at a concentration of 24 μ g/ml

Add 150 μ l of standard dilution solution to 150 μ l of standard 5, with a concentration of 12 μ g/ml

Add 150 μ l of standard dilution to 150 μ l of standard dilution solution for standard 4, with a concentration of 6 μ g/ml

Add 150 μ l of standard dilution solution to 150 μ l of standard 2 at a concentration of 3 μ g/ml

Add 150 μ l of standard dilution to 150 μ l of standard dilution solution for standard 1 at a concentration of 1.5 μ g/ml

2. Sample addition: Set up blank holes, standard holes, and sample holes for testing. Accurately add 50 μ l of the standard sample on the enzyme-linked immunosorbent assay (ELISA) coated plate, first add 40 μ l of sample diluent to the well of the test sample, and then add 10 μ l of the test sample. Add the sample to the bottom of the enzyme-linked immunosorbent assay plate well, avoiding touching the well wall as much as possible, and gently shake and mix well. |

3. Incubation: Cover the plate with a sealing film and incubate at 37 ℃ for 30 minutes.

4. Solution preparation: Dilute 30 times the concentrated washing solution with distilled water and set aside for later use

5. Washing: Carefully remove the sealing film, discard the liquid, shake dry, fill each hole with washing solution, let it stand for 30 seconds, then discard. Repeat this process 5 times and pat dry.

6. Enzyme addition: Add 50 μ l of enzyme labeled reagent to each well, except for blank wells.

7. Incubation: Follow the same procedure as in 3.

8. Washing: Follow the same procedure as in 5.

9. Color development: Add 50 μ l of color developing agent A50 to each well, then add 50 μ l of color developing agent B50, gently shake and mix well, and develop color at 37 ℃ in the dark for 15 minutes

10. Termination: Add 50 μ l of termination solution to each well to terminate the reaction (at this point, blue will turn yellow).

11. Measurement: Measure the absorbance (OD value) of each well in sequence using blank air conditioner zero and 450nm wavelength. The measurement should be conducted within 15 minutes after adding the termination solution.

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Notes:

1. The kit should be balanced at room temperature for 15-30 minutes before use when taken out from the cold storage environment. If the enzyme coated plate is not used up after opening, the Flat noodles should be stored in a sealed bag.

2. There may be crystal precipitation in concentrated washing solution. When diluting, it can be dissolved by heating in a water bath, and washing does not affect the results.

3. Sample dispensers should be used for each step of sample addition, and their accuracy should be regularly checked to avoid experimental errors. The sample addition time should be controlled within 5 minutes. If there are a large number of specimens, it is recommended to use a sampling gun for sample addition.

4. If the content of the substance to be tested in the specimen is too high (the OD value of the sample is greater than the OD value of the standard well * well), please dilute the sample by a certain multiple (n times) before measuring. When calculating, please multiply it by the dilution multiple (x 5 x n).

5. The sealing film is only for one-time use to avoid cross contamination.

6. Components from different batch numbers of this reagent must not be mixed. Please store color developer B away from light.

7. Strictly follow the instructions for operation, and the judgment of the test results must be based on the reading of the enzyme-linked immunosorbent assay reader

8. All samples, detergents, and various waste materials should be treated as infectious agents.

If there is any discrepancy with the English manual, the English manual shall prevail.