Quantifying CD13 and αvβ3 Integrin Binding

HUVECs expressing CD13 and α_vβ₃ integrin receptors [2 (link), 8 (link)] were cultured overnight in 96-well microplates in RPMI 1640 supplemented with 10% FBS at 37 °C, 5% CO₂. After washing the wells with PBS, tCoa-NGR or tCoa was added to the wells in sequential nanomolar dilutions of 0, 0.25, 0.05, 0.1, 0.2, 0.4, and 0.8. In reference to our previous work [14 (link)], the absorbance for reaction was developed using an HRP-labeled anti-His tag antibody (Sigma).
For competitive binding, ELISA was conducted by addition of either non His-tagged tCoa-NGR fusion protein, as integrin α_vβ₃ and CD13 antagonist, and non His-tagged tCoa-RGD fusion protein [14 (link)], as α_vβ₃ integrin receptor antagonist only.