The antibody conjugation was also quantified using an enzyme-linked immunosorbent assay (ELISA) kit to detect bevacizumab specifically. Following conjugation, PLGA NPs, NHS-PLGA NPs and Bev-PLGA NPs were diluted in a ratio of 1:50, and 100μL was added to each well and incubated for an hour at room temperature (RT). Each sample’s concentration was also measured in duplicates to measure sample variation as instructed by the manufacturer. After incubation, the wells were washed four times with the wash solution provided. 100μL Biotin Anti-Bevacizumab Antibody was added to each well to detect the antibody, incubated for 30 minutes at RT, and then washed again four times. 100μL of the enzyme conjugate, Streptavidin-horseradish peroxidase (HRP), was added to each well, incubated for 10 minutes and rewashed four times. 100μL of the substrate for the enzyme, TMB (3,3',5,5'-Tetramethylbenzidine), was then added to react with the previously used enzyme for 15 minutes in the dark at RT. Finally, 50μL of the stop solution was added to halt the substrate-enzyme reaction, and the OD was measured on a SpectraMax absorbance plate reader at the wavelength of 450nm.The bevacizumab concentration from the adsorbed NPs Bev-PLGA NPs (Ads) and Bev-PLGA (Cd) NPs was measured using the ELISA kit. The dataset above shows the samples’ bevacizumab amount which was determined from a calibration curve created using bevacizumab standards.