2.5.1. Integrin Human ITG αvβ3 dan VEGF

This test was carried out using sandwich ELISA for both human integrin αvβ3 and VEGF (Fine Test, Wuhan, China). The procedure was performed according to the manual in the kit (Fine Test, Wuhan, China). The plate was washed twice before adding the sample in the form of lysate, along with the negative control, to a 96-well plate. A total of 100 mL of sample and standard was added to each well and incubated for 90 min at 37 ◦C. The plate then washed for 2 times. A volume of 100 uL of biotin-labeled antibody was added to each well and incubated for 60 min at 37 ◦C. The plate then washed 3 times. Working solution of 100 mL HRP–streptavidin conjugate (SABC) was added to each well and incubated for 30 min at 37 ◦C, and the plate was then washed for 5 times. TMB substrate (90 mL) was added and incubated for 15–30 min at 37 ◦C. A 50 mL stop solution was added, and the well plates were immediately read at a wavelength of 450 nm (BioRad, Hercules, CA, USA).
