Rat vascular endothelial growth factor receptor-2 (VEGF R2) ELISA kit instruction manual

Rat vascular endothelial growth factor receptor -2 (VEGF R2) ELISA kit

  ( used in serum, plasma, cell culture supernatants and other biological fluids )

principle

This experiment used double antibody sandwich ABC-ELISA. The anti-rat VEGF R2 monoclonal antibody was coated on the microtiter plate, the VEGF R2 in the standard and the sample was combined with the monoclonal antibody, and the biotinylated anti-rat VEGF R2 was added to form an immune complex attached to the plate, which was spicy. The root peroxidase-labeled Streptavidin is combined with biotin, and the substrate working solution is blue. Finally, the stop solution sulfuric acid is added, and the OD value is measured at 450 nm. The concentration of VEGF R2 is proportional to the OD value, which can be obtained by drawing a standard curve. The concentration of VEGF R2 in the specimen was obtained.

Kit composition ( 2-8 ° C preservation)

Prepare reagents and collect blood samples

1. Collect specimens: serum, plasma (EDTA), cell culture supernatant, tissue homogenate, etc., as soon as possible, store at 2-8 ° C for 48 hours; longer time must be frozen (-20 °C or -70 °C) Save to avoid repeated freezing and thawing.

2. Standard solution preparation: Add 1ml of distilled water before use and mix well to prepare a solution of 16000pg/ml. Set 8 tubes of standard tubes, and add 300 ul of standard dilution solution to each tube. Add 1 6,000 pg/ml of the standard solution to the first tube and mix 300 ul, then aspirate 300 ul with the sampler and transfer to the second tube. Repeat the dilution in this way, and remove 300 ul from the seventh tube and discard it. The eighth tube is a blank control.

3. The 10× specimen dilution was diluted 1:10 with distilled water (example: 1 ml concentrated dilution + 9 ml distilled water).

4. Washing solution: diluted 1:20 with distilled water (example: 1 ml concentrated washing solution added to 19 ml of distilled water)

Test procedure

1. Loading: Add 100 ul of standard or sample to be tested in each well. Mix the reaction plate thoroughly and let it stand at 37 °C for 120 minutes.

2. Wash the plate: Wash the plate thoroughly with washing solution 4-6 times, and dry it on the filter paper.

3. Add 100 ul of the first antibody working solution to each well. The reaction plate was thoroughly mixed and placed at 37 ° C for 60 minutes.

4. Wash the board: the same as before.

5. Add 100 ul of enzyme-labeled antibody working solution per well. The reaction plate was placed at 37 ° C for 30 minutes.

6. Wash the board: same as before.

7. Add 100 ul of substrate working solution per well, set 37 The reaction was carried out in the dark at °C for 15 minutes.

8. Add 100 ul of stop solution to each well and mix.

9. Measure the absorbance at 450 nm using a microplate reader within 30 minutes.

Result calculation and judgment

1. All OD values ​​should be subtracted from the blank value before calculation.

2. Take the standard products 8000, 4000, 2000, 1000, 500, 250, 125, 0 pg/ml as the abscissa and OD as the ordinate. Draw on the coordinate paper and draw the standard curve.

3. Find the corresponding VEGF R2 content on the graph based on the OD value of the sample.

Kit performance

1. Sensitivity: The minimum VEGF R2 detection concentration is less than 62pg/ml.

2. Specificity: Recombinant or natural rat VEGF R2 can be detected simultaneously. Does not cross-react with other cytokines in rats.

3. Repeatability: The coefficient of variation in the plate and plate is less than 7.9%.

Precautions

1. It is recommended to make double holes for the above standard holes and samples to be tested. The standard curve should be made at the same time for each measurement.

2. The washing process is critical. Insufficient washing will result in an accuracy error and an erroneous rise in the OD value.

3. After the slats are opened, the remaining slats should be sealed again to keep the slats dry .

4. This kit should be stored in a 4 ^o C refrigerator.

5. This kit is for scientific research only and cannot be used for clinical diagnosis!