Abstract
Purpose: With the growing research interest in therapeutic monoclonal antibodies (mAbs) and their broad range of applications, the development of reliable analytical approaches for their quantification has become increasingly important. Since, microbead (MB)-based flow cytometric immunoassays have emerged as highly sensitive analytical methods, the aim of this study was to develop and evaluate a flow cytometry-based method using poly(ε-caprolactone) (PCL) MBs to quantify bevacizumab (BVZ) concentration.
Methods: PCL MBs were synthesized through the emulsion/solvent evaporation method and modified by reaction with 1,2-ethylenediamine (EDA) to enable covalent binding of biomolecules. Based on the optimal biomolecule immobilization conditions determined using a model protein, purified recombinant vascular endothelial growth factor (VEGF) protein was immobilized on the surface of amine-functionalized MBs (A-MBs(45)) to capture BVZ, which was then detected using fluorescein isothiocyanate (FITC)-conjugated anti-human IgG antibody in flow cytometry.
Results: The synthesized PCL MBs, with an average diameter of 6.62 ± 0.17 µm, were within the suitable particle size range for immunoassay applications. The optimal aminolysis time was found to be 45 min at 37 °C, and the maximum protein coupling was achieved 5 h post-incubation at pH 8.0. The developed MB-based flow cytometric immunoassay demonstrated the potential to quantify BVZ with a limit of detection (LOD) and limit of quantification (LOQ) of about 6.71 and 22.73 ng mL-1, respectively.
Conclusion: Considering the ease of fabrication and functionalization of PCL MBs under the obtained optimal conditions, along with their capability for analyte detection, these cost-effective analytical platforms may serve as promising candidates for BVZ quantification.