Vascular Endothelial Growth Factor (VEGF) ELISA Kit

(For serum, plasma, tissue homogenate, cell culture supernatant, and other biological fluids)

(Product Code: F03060)

Principle

This experiment uses the double antibody sandwich ABC-ELISA method. Anti-human VEGF monoclonal antibody is coated on the enzyme-labeled plate. VEGF in the standard and sample binds to the monoclonal antibody. Biotinylated anti-human VEGF is added, forming an immune complex attached to the plate. Horseradish peroxidase-labeled Streptavidin binds to the biotin. Substrate working solution is added to develop a blue color, followed by the stop solution. The OD value is measured at 450 nm. The VEGF concentration is proportional to the OD value and can be determined by plotting a standard curve.

Kit Components (Store at 2-8°C)

Reagent Preparation and Blood Sample Collection

1. Dilute the 10× Sample Buffer 1:10 with distilled water (e.g., 1 ml concentrated diluent + 9 ml distilled water).

2. Collect specimens: serum, plasma (EDTA, citrate, heparin anticoagulated), cell culture supernatant, tissue homogenate, etc. Test as soon as possible; store at 2-8°C for 48 hours. For longer storage, freeze (-20°C or -70°C) and avoid repeated freeze-thaw cycles.

3. Standard solution preparation: Take 8 1.5 ml centrifuge tubes. Add 900 μl of sample buffer to the first tube and 500 μl to tubes 2 to 8. Add 100 μl of 40 ng/ml standard solution to the first tube, mix thoroughly on a vortex mixer, then pipette 500 μl and transfer to the second tube. Repeat this serial dilution, discarding 500 μl from the seventh tube. The eighth tube serves as a blank control.

4. Wash Buffer: Dilute 1:20 with distilled water (e.g., 1 ml concentrated wash buffer + 19 ml distilled water).

Assay Procedure

1. Sample addition: Add 100 μl of standard or test sample to each well. Mix the plate thoroughly and incubate at 37°C for 40 minutes.

2. Washing: Wash the plate thoroughly with wash buffer 4-6 times, and blot dry on filter paper.

3. Add 50 μl of distilled water and 50 μl of Biotinylated Antibody to each well (except blank). Mix thoroughly and incubate at 37°C for 20 minutes.

4. Washing: As before.

5. Add 100 μl of Enzyme Conjugate to each well. Incubate the plate at 37°C for 10 minutes.

6. Washing: As before.

7. Add 100 μl of TMB Solution to each well. Incubate in the dark at 37°C for 15 minutes.

8. Add 100 μl 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 Interpretation

1. It is recommended to subtract the blank OD value from all OD values before calculation. If the blank OD is below 0.1, direct calculation may be used.

2. Using the standard concentrations of 4000, 2000, 1000, 500, 250, 125, 62.5, and 0 pg/ml as the x-axis and OD values as the y-axis, plot the standard curve using software.

3. Calculate the corresponding VEGF concentration based on the sample OD value. Software can be requested from the company via email.

Kit Performance

1. Sensitivity: The minimum detectable concentration of VEGF is less than 30 pg/ml.

2. Specificity: Can detect both recombinant and natural human VEGF. No cross-reactivity with other human cytokines.

3. Reproducibility: Intra- and inter-assay coefficients of variation are both less than 10%.

Precautions

1. It is recommended to run duplicates for both standard and test samples. A standard curve should be included in each assay.

2. The washing process is critical. Inadequate washing may lead to precision errors and falsely elevated OD values.

3. All reagents should be brought to room temperature before use. After opening, reseal the remaining strips to keep them dry.

4. This kit uses a highly sensitive TMB solution. If color development is too intense, precipitation may occur, which is normal and does not affect result interpretation.

5. The kit components listed are for a 96-well plate. For a 48-well plate, halve the amounts.

6. Store the kit at 4°C. For research use only. Not for clinical diagnosis.

The above instructions are for reference only! Please refer to the instructions inside the kit for actual use!