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Background
Vascular Endothelial Growth Factor (VEGF) A plays a central role in tumor angiogenesis by activating VEGFR1-3 on endothelial cells, promoting their growth, survival, and increasing vessel permeability to meet the tumor’s metabolic needs. Bevacizumab is a humanized monoclonal IgG1 antibody that binds to the receptor-binding site of VEGF-A, blocking its interaction with VEGFRs. This prevents VEGFR-2 dimerization and activation of intracellular tyrosine kinase domains, thereby disrupting downstream signaling. By inhibiting both the formation and function of tumor blood vessels, bevacizumab reduces angiogenesis and suppresses tumor growth.
PathHunter Bevacizumab Bioassay Kits model this molecular MOA in a simple, homogenous format, suitable for implementation in characterization, comparability studies, potency testing in QC lot release, stability program, and detection of neutralizing antibodies.
Product Highlights
- Cell-based functional assay for anti-VEGF drugs, Bevacizumab or Biosimilar based on their mechanism of action
- Simple, homogenous protocol with results in less than 24 hours
- Significantly better assay performance compared to difficult HUVEC assay
- Suitable for rank ordering, comparability studies, potency measurement, stability studies and neutralizing antibody detection
- Applicable across various stages from characterization and QC lot-release
Assay Principle
The PathHunter Bevacizumab bioassay utilizes EFC-based platform and is designed to detect VEGF-A-induced functional homodimerization of the kinase insert domain (KDR), also known as the VEGFR2 receptor. Activation of the KDR receptor through VEGF-A leads to receptor dimerization, which is an essential event in the receptor's signaling cascade. A. The PathHunter KDR/KDR bioassay cells have been engineered to co-express KDR fused to PK, and another KDR construct fused to EA. B. Upon activation by VEGF-A, the VEGFR2 receptors naturally dimerize forcing the two fragments to complement and create an active β-gal enzyme. Active β-gal hydrolyzes its substrate and produces a chemiluminescent signal, indicating receptor activation C. Anti-VEGF antibodies like bevacizumab, inhibit VEGF-A’s ability to activate the receptors resulting in a dose-dependent reduction of the chemiluminescent signal.
