User Manual for Human Vascular Endothelial Growth Factor A (VEGF-A) ELISA Kit Experiment

BS-9809 Human Vascular Endothelial Growth Factor A (VEGF-A) ELISA Kit

1、 Product Overview

This kit uses a double antibody sandwich enzyme-linked immunosorbent assay (ELISA), specifically designed to detect the concentration of VEGF-A in samples such as human serum, plasma, tissue homogenates, and cell culture supernatants. For scientific research purposes only.

2、 Composition of reagent kit

Pre coated microplate: 12 wells x 8 strips (96 well specification) or 12 wells x 4 strips (48 well specification), pre coated with anti-VEGF-A capture antibody.

Standard: Freeze dried powder, which needs to be diluted with standard diluent and diluted in multiple ratios (recommended concentration gradient: 10, 3.33, 1.11, 0.37, 0.12, 0.04, 0 ng/mL).

Detection of antibody HRP: horseradish peroxidase labeled anti-VEGF-A antibody.

Wash buffer: 20 x concentrated solution, diluted with distilled water at a ratio of 1:20.

Substrate solution: TMB color solution A/B, mixed 1:1 before use and used up within 15 minutes.

Termination solution: 2M solution.

Sample diluent: used to dilute high concentration samples.

Sealing film: 2 sheets, used to seal the microporous plate during incubation.

3、 Experimental principle

VEGF-A binds to pre packaged captured antibodies to form an "antibody antigen" complex; After adding HRP labeling to detect antibodies, a "sandwich structure" is formed. After washing to remove unbound substances, TMB substrate was added for color development (blue), and the reaction was terminated with a stop solution before turning yellow. Measure the absorbance (OD value) at a wavelength of 450nm, and the concentration is directly proportional to the OD value.

4、 Experimental steps

Sample preparation

Serum/plasma: Centrifuge to remove particles and avoid hemolysis; Storage at 2-8 ℃ ≤ 48 hours, long-term storage at -20 ℃/-80 ℃ (avoid repeated freezing and thawing).

Cell supernatant: Centrifuge the supernatant to avoid containing high concentrations of protease agents.

Reagent preparation

Washing solution: When the concentrated washing solution crystallizes at room temperature, it is dissolved in a 40 ℃ water bath and diluted at a ratio of 1:20.

Standard product: Dilute multiple times after reconstitution, ready to use.

Substrate: Mix TMB A/B solution in a 1:1 ratio and store in the dark.

Operation process

Sample addition: Add 100 μ L of standard/sample to each well and incubate at 37 ℃ for 90 minutes.

Washing: Shake dry the liquid in the hole, soak it in the washing solution for 1 minute/time, repeat 3 times.

Add detection antibody: Add 100 μ L HRP labeled antibody to each well and incubate at 37 ℃ for 60 minutes.

Washing: Same as step 2.

Color development: Add 90 μ L TMB substrate to each well and incubate at 37 ℃ in the dark for 15-20 minutes.

Termination: Add 100 μ L of termination solution to each well, mix well, and measure the OD value within 30 minutes.

Result calculation

Draw a standard curve with standard concentration (ng/mL) as the horizontal axis and OD value as the vertical axis.

Calculate the concentration by substituting the sample OD value into the curve and multiplying it by the dilution factor to obtain the result.

5、 Precautions

Reagent storage: Unused reagent kits should be stored at 2-8 ℃ in the dark, with a shelf life of 6 months; The reconstitution standard can be packaged and frozen to avoid repeated freeze-thaw cycles.

Sample processing: Avoid using samples containing substances to prevent HRP activity.

Operation standard: When transferring liquid, avoid bubbles; Washing is necessary to prevent cross contamination.

Safety protection: If the termination liquid contains strong acid, gloves should be worn during operation.

6、 Expected results

Sensitivity: ≤ 1.54 pg/mL (actual subject to instructions).

Detection range: The typical range is 3.12-200 pg/mL, with some kits expanding to 62.5-2000 pg/mL.

Repeatability: The intra plate/inter plate coefficients of variation are<10% and<15%, respectively.

7、 Frequently Asked Questions and Answers

Q: Is the color too light or too dark?

A: Adjust the dilution factor or incubation time of the sample; Ensure that the substrate is freshly prepared.

Q: Linear deviation of standard curve?

A: Check the reconstitution operation to avoid bubbles; Replace the pipette tip.

Note: The above is for reference only. This product is for scientific research use only and is not considered as actual data. Experiments must strictly follow the instructions or consult technical teachers.

Tianjin Bensheng has always regarded quality control as the lifeblood of enterprises and pursued continuous improvement of their competitiveness. The company always adheres to the corporate spirit of abiding by laws and regulations, being strict with oneself, being tolerant and willing to take on responsibilities as the standard in its operations. It maintains and expands the market with excellent quality and service, and maximizes the satisfaction of customer needs. Our development goal is to achieve mutual benefit with our customers. This student! A partner you trust. We are willing to cooperate sincerely with you and create a better future together.