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VEGF Bioassay

A Simpler, More Consistent VEGF Bioassay

  • Prequalified according to ICH guidelines
  • Mechanism-of-action-based assay
  • No primary cell culture required

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This product is discontinued
VEGF Bioassay

Simple VEGF Bioassay, Consistent Results

Vascular endothelial growth factor (VEGF) is an important signaling protein that is secreted from epithelial cells, tumor cells and macrophages. It has many functions, including stimulation of angiogenesis, increase of vascular permeability, enhancement of tumor invasion and survival, and inhibition of antitumor response in Treg cells. There are several VEGF receptor subtypes—VEGFR1, VEGFR2 and VEGFR3. VEGFR2 (also known as KDR) mediates almost all of the known receptor cellular responses to VEGF. VEGF occurs in four isoforms, including VEGF-121, VEGF-165, VEGF-189 and VEGF-206, of which VEGF-121 and -165 are diffusible forms. VEGF-165 is the predominant isoform in the body. 

All members of the VEGF family stimulate cellular response by binding to receptors of the receptor tyrosine kinase, namely VEGFR-1 (Flt-1) and VEGFR-2 (Flk-1/KDR). When VEGF binds to KDR, the receptor dimerizes and becomes activated through transphosphorylation.

The VEGF Bioassay is a bioluminescent cell-based assay that measures VEGF stimulation and inhibition of KDR (VEGFR-2) using luciferase as a readout. This assay overcomes many of the limitations of current endothelial cell proliferation assays, and can be used for discovery and development of novel biologic therapies aimed at either inducing or inhibiting the VEGF response. 

Assay Components

The VEGF Responsive Cells included are provided in a thaw-and-use format as cryopreserved cells that can be thawed, plated and used in an assay without the need for cell propagation. 

The VEGF Responsive Cells have been engineered to express the response element (RE) upstream of luc2P, as well as exogenous VEGF receptor. When VEGF binds to VEGF Responsive Cells, the receptor transduces intracellular signals resulting in luminescence.The bioluminescent signal is detected and quantified using Bio-Glo™ Luciferase Assay System (Cat.# G7940) and a standard luminometer such as the GloMax® Discover System.

The Workflow

Traditional Method

12052MA-W-a

'Cells as reagents' Method

12052MA-W-b

Standardized Reagents Improve Results

  • Thaw-and-use cell format provides significant time and labor savings over traditional methods while reducing variability.
  • Kit includes all of the required reagents in standardized formats.
  • Easily amenable to high-throughput workflows.

The Assay Principle

 
15062ma-w
Representation of the VEGF Bioassay. The VEGF Bioassay consists of a genetically engineered cell line, VEGF Responsive Cells. When VEGF binds, receptor-mediated signaling induces luminescence that can be detected by adding Bio-Glo™ Reagent and quantitated with  a luminometer. Inhibition of VEGF binding to KDR by either anti-VEGF or anti-KDR antibodies results in a decrease in luminescence.

Assay Performance Data

Accuracy and Precision are critical to bioassays and the development of biologics. The VEGF Bioassay is both accurate and precise. Accuracy is a measure of how close results are to a target or reference value. Precision is a measure of repeatability. This assay was developed and prequalified according to ICH guidelines for accuracy and precision giving it the performance characteristics required for many applications of a bioassay used for antibody drug discovery research and development.

Accuracy for Assay Performance Data
Accurate, but not precise

Accuracy
Measure of ability to replicated a reference or "target" value.
IntraLab Precision
Precise, but not accurate

IntraLab Precision

Measure of repeatability from multiple assays of the same homogeneous sample under the same operating conditions over a short time (same analyst; same lab; same day).

Intermediate Precision Data Target 3
Accurate and precise

Intermediate Precision

Measure of repeatability of multiple assays of the same homogeneous sample with different analysts or equipment or on different days.

32454752-data-linear
Linear

Linearity

Ability of your assay to provide measured values that are proportional to the concentration of the sample.

Accuracy

Expected Relative Potency Recovery
50% 100.1%
75% 96.67%
150% 102.75%
200% 107.25%

Repeatability (100% reference, %CV)

8.73%

Intermediate Precision (%CV)

9.28%

Linearity

r 2 = 0.9984 (50–200% range)
y = 1.1x–7.792


The VEGF Bioassay response to recombinant human VEGF.

VEGF Bioassay used to measure anti-VEGF antibody activity.

Graph showing VEGF Bioassay response to recombinant human VEGF.
KDR/NFAT-RE HEK293 Cells were incubated with serial dilutions of recombinant human VEGF-165. After a 6-hour incubation, Bio-Glo™ Reagent was added and luminescence was quantified using the GloMax® Multi+ Detection System.
15111ma-w Graph showing that VEGF Bioassay used to measure activity of anti-VEGF antibodies.
KDR/NFAT-RE HEK293 Cells were incubated with serial dilutions of antibodies to VEGF in the presence of an EC80 concentration of recombinant human VEGF-165. After a 6-hour incubation, Bio-Glo™ Reagent was added and luminescence was quantified using the GloMax® Multi+ Detection System.

Product Kit Formats and Related Products

  • Kits are available in 1X and 5X sizes.
  • Recombinant VEGF is included in the kits and also available separately.
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