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.

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 (Cat.# GM3000).

Recombinant VEGF may be used as a positive control ligand in the VEGF Bioassay (Cat.# GA2001, GA2005). Human VEGF-165, amino acids Ala27–Arg191 (Accession# AAM03108), has a predicted molecular mass of 19kDa and is supplied in a solution of 5mM citric acid, 5mM NaHPO₄, 0.15M NaCl (pH 4.0) and at a concentration of 200μg/ml.

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 Bioassay is also available in a Cell Propagation Model (CPM) format, as cryopreserved cells, that can be thawed, propagated and banked for long-term use (Cat.# GA1082).

The Workflow

Traditional Method

Standard bioassay workflow with cultured cells

'Cells as reagents' Method

Improved bioassay workflow with thaw-and-use cells

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: Measure of ability to replicate a reference or "target" value.

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

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

The VEGF Bioassay Shows Precision, Accuracy and Linearity.

Parameter	Results
Accuracy	% Expected Relative Potency	% Recovery
	50	100.1
	75	96.67
	150	102.75
	200	107.25
Repeatability (% CV)	100% (Reference)	8.73
Intermediate Precision (% CV)		9.28
Linearity (r²)		0.9984
Linearity (y = mx + b)		y = 1.1x–7.792
A 50–200% theoretical potency series of bevacizumab (anti-VEGF antibody) was analyzed in triplicate in three independent experiments performed on three days by each of two analysts (for a total of six independent experiments). Bio-Glo™ Reagent was added and luminescence quantified using the GloMax® Discover System. Data were analyzed and relative potencies calculated after parallelism determination using JMP® software. Data were generated using thaw-and-use cells.

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

VEGF Bioassay is available in 1X and 5X sizes.
VEGF Bioassay is also available in CPM format (Cat.# GA1082).
Recombinant VEGF is available separately (Cat.# J2371).

Protocolos

Complete Protocol

VEGF Bioassay Technical ManualPDF (2741 KB) – English

VEGF Bioassay Propagation Model Technical ManualPDF (2994 KB) – English

Contenido

Item	Part #	Presentación
KDR/NFAT-RE HEK293 Cells	GA109A	1 × 0.5ml
Fetal Bovine Serum	J121A	1 × 4ml
DMEM with 4.5g/L Glucose, L-Glutamine, Sodium Pyruvate	J236A	1 × 60ml
Bio-Glo™ Luciferase Assay Buffer	G719A	1 × 10ml
Bio-Glo™ Luciferase Assay Substrate	G720A	1 × 1 vial

SDS

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Certificado de Análisis

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Use Restrictions

Not For Medical Diagnostic Use.

Condiciones de Almacenaje

Please see the Protocol for detailed storage conditions.

Patentes y Exclusiones

NOT FOR MEDICAL DIAGNOSTIC USE. FOR IN VITRO USE ONLY. BY USE OF THIS PRODUCT, RECIPIENT AGREES TO BE BOUND BY THE TERMS OF THIS LIMITED USE STATEMENT. If the recipient is not willing to accept the conditions of this limited use statement, and the product is unused, Promega will accept return of the unused product and provide the recipient with a full refund.

This product may not be further sold or transferred by the recipient and may be used only by the recipient, and then only for (1) research use, (2) discovery, development and monitoring of biologic drugs and vaccines, (3) quality assurance testing of biologic drugs and vaccines, and (4) product release assays for biologic drugs and vaccines. No other commercial use is allowed. "Commercial use” means any and all uses of this product by recipient for monetary or other consideration, including providing a service, information or data to unaffiliated third parties, and resale of this product for any use. Recipient has no right to propagate, modify, derivatize, genetically engineer or otherwise create variations of the cells or genes stably transfected within the cells. In addition, recipient must use Bio-Glo™ Luciferase Assay System purchased from Promega Corporation for all luminescence assays using this product or contact Promega to obtain a license for use of this product with reagents other than Promega’s. PROMEGA MAKES NO REPRESENTATIONS OR WARRANTIES OF ANY KIND, EITHER EXPRESSED OR IMPLIED, INCLUDING AS TO MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE WITH REGARDS TO THIS PRODUCT. The terms of this agreement shall be governed under the laws of the State of Wisconsin, USA.

U.S. Pat. No. 10,077,244 and other patents.

HEK293 cells were obtained under license from AdVec Inc.

Contenido

Item	Part #	Presentación
KDR/NFAT-RE HEK293 Cells	GA109A	5 × 0.5ml
Fetal Bovine Serum	J121A	5 × 4ml
DMEM with 4.5g/L Glucose, L-Glutamine, Sodium Pyruvate	J236A	5 × 60ml
Bio-Glo™ Luciferase Assay Buffer	G719A	5 × 10ml
Bio-Glo™ Luciferase Assay Substrate	G720A	5 × 1 vial

SDS

Search for SDS

Certificado de Análisis

Search by lot number

Use Restrictions

Not For Medical Diagnostic Use.

Condiciones de Almacenaje

Please see the Protocol for detailed storage conditions.

Patentes y Exclusiones

NOT FOR MEDICAL DIAGNOSTIC USE. FOR IN VITRO USE ONLY. BY USE OF THIS PRODUCT, RECIPIENT AGREES TO BE BOUND BY THE TERMS OF THIS LIMITED USE STATEMENT. If the recipient is not willing to accept the conditions of this limited use statement, and the product is unused, Promega will accept return of the unused product and provide the recipient with a full refund.

This product may not be further sold or transferred by the recipient and may be used only by the recipient, and then only for (1) research use, (2) discovery, development and monitoring of biologic drugs and vaccines, (3) quality assurance testing of biologic drugs and vaccines, and (4) product release assays for biologic drugs and vaccines. No other commercial use is allowed. "Commercial use” means any and all uses of this product by recipient for monetary or other consideration, including providing a service, information or data to unaffiliated third parties, and resale of this product for any use. Recipient has no right to propagate, modify, derivatize, genetically engineer or otherwise create variations of the cells or genes stably transfected within the cells. In addition, recipient must use Bio-Glo™ Luciferase Assay System purchased from Promega Corporation for all luminescence assays using this product or contact Promega to obtain a license for use of this product with reagents other than Promega’s. PROMEGA MAKES NO REPRESENTATIONS OR WARRANTIES OF ANY KIND, EITHER EXPRESSED OR IMPLIED, INCLUDING AS TO MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE WITH REGARDS TO THIS PRODUCT. The terms of this agreement shall be governed under the laws of the State of Wisconsin, USA.

U.S. Pat. No. 10,077,244 and other patents.

HEK293 cells were obtained under license from AdVec Inc.

Contenido

Item	Part #	Presentación
KDR/NFAT-RE HEK293 Cells (CPM)	GA108A	2 × 1ml

SDS

Search for SDS

Certificado de Análisis

Search by lot number

Use Restrictions

Not For Medical Diagnostic Use.

Condiciones de Almacenaje

Please see the Protocol for detailed storage conditions.

Patentes y Exclusiones

NOT FOR MEDICAL DIAGNOSTIC USE. FOR IN VITRO USE ONLY. BY USE OF THIS PRODUCT, RECIPIENT AGREES TO BE BOUND BY THE TERMS OF THIS LIMITED USE STATEMENT. If the recipient is not willing to accept the conditions of this limited use statement, and the product is unused, Promega will accept return of the unused product and provide the recipient with a full refund.

This product may not be further sold or transferred by the recipient and may be used only by the recipient, and then only for (1) research use, (2) discovery, development and monitoring of biologic drugs and vaccines, (3) quality assurance testing of biologic drugs and vaccines, and (4) product release assays for biologic drugs and vaccines. No other commercial use is allowed. "Commercial use" means any and all uses of this product by recipient for monetary or other consideration, including providing a service, information or data to unaffiliated third parties, and resale of this product for any use. Recipient has no right to modify, derivatize, genetically engineer or otherwise create variations of the cells or genes stably transfected within the cells except that recipient may propagate and store the cells for long-term use. In addition, recipient must use Bio-Glo™ Luciferase Assay System purchased from Promega Corporation for all luminescence assays using this product or contact Promega to obtain a license for use of this product with reagents other than Promega's. PROMEGA MAKES NO REPRESENTATIONS OR WARRANTIES OF ANY KIND, EITHER EXPRESSED OR IMPLIED, INCLUDING AS TO MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE WITH REGARDS TO THIS PRODUCT. The terms of this agreement shall be governed under the laws of the State of Wisconsin, USA.

HEK293 cells were obtained under license from AdVec Inc.

Contenido

Item	Part #	Presentación	Concentración
Recombinant VEGF	J237A	1 × 10μg	200μg/ml

SDS

Search for SDS

Certificado de Análisis

Search by lot number

Use Restrictions

Not For Medical Diagnostic Use.

Condiciones de Almacenaje

Please see the Protocol for detailed storage conditions.

Recursos

Posters

Novel MOA-Based Bioluminescent Bioassay Quantitation Anti-VEGF Antibody Potency and Stability Poster

Otros

Immune Checkpoint Bioassays Power Combination Immunotherapy

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VEGF Bioassay

A Simpler, More Consistent VEGF Bioassay

Simple VEGF Bioassay, Consistent Results

Assay Components

The Workflow

The Assay Principle

Assay Performance Data

The VEGF Bioassay response to recombinant human VEGF.

VEGF Bioassay used to measure anti-VEGF antibody activity.

Product Kit Formats and Related Products

Protocolos

Complete Protocol

Especificaciones

Contenido

SDS

Certificado de Análisis

Use Restrictions

Condiciones de Almacenaje

Patentes y Exclusiones

Especificaciones

Contenido

SDS

Certificado de Análisis

Use Restrictions

Condiciones de Almacenaje

Patentes y Exclusiones

Especificaciones

Contenido

SDS

Certificado de Análisis

Use Restrictions

Condiciones de Almacenaje

Patentes y Exclusiones

Especificaciones

Contenido

SDS

Certificado de Análisis

Use Restrictions

Condiciones de Almacenaje

Recursos

Posters

Otros

Productos Similares

T Cell Activation Bioassay

PD-1/PD-L1 Blockade Bioassay

ADCC Reporter Bioassay, V Variant

Membrane VEGF Target Cells

Usado con frecuencia junto con

Bio-Glo™ Luciferase Assay System

GloMax® Discover System

RealTime-Glo™ MT Cell Viability Assay

CellTiter-Glo® 2.0 Cell Viability Assay

pHAb Reactive Dyes

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