Streptavidin hrp conjugated antibody

Competition inhibition ELISA was designed to determine that VEGF-specific NBs are able to detect VEGF in the solution phase and inhibit its binding to immobilized VEGF-antibodies. ELISA assay was set using the avidin-biotin-complex (ABC). For competition inhibition analysis, a microtiter well was coated with 1 µg/ml of anti-hVEGF or goat anti-mVEGF antibody. The wells were blocked with BSA 2% and incubated at RT for 1 hr. Fifty µl of hVEGF (500 ng/ml) or mVEGF (500 ng/ml) were mixed with 50 µl of anti-VEGF NB (0 and 10 µg/ml), bevacizumab (0 and 10 µg/ml), or the anti-mVEGF antibody (0 and 10 µg/ml) and added to the wells coated with anti-hVEGF or the anti-mVEGF antibody, respectively and incubated for 1 hr at 37°C. The wells were emptied and washed 4 times with PBST. One hundred µl of biotinylated polyclonal anti-VEGF (R&D) (1:100) were added to each well and the plate was incubated for 1 hr at 37 °C. After washing, wells were incubated with 100 µl of streptavidin HRP conjugated antibody (R&D, Minneapolis) (1:5000) for 1 hr at 37 °C. The peroxide activity was detected using TMB and subsequently, absorption was measured at a wavelength of 450 nm. Maximal signal value refers to 0% inhibition and minimal signal refers to 100 inhibitions.