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J. R. Ramos Filho, L. Taniwaki, R. Mendonça, J. A. S. Ribeiro, I. U. Scott, A. S. Cunha-Junior, R. Jorge; Effect of Lyophilization on the in vitro Biological Activity of Bevacizumab. Invest. Ophthalmol. Vis. Sci. 2010;51(13):2806.
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To evaluate the effect of the lyophilization process of bevacizumab solution on the in vitro binding activity of bevacizumab to VEGF165.
The commercial solution of bevacizumab (Avastin®, 100mg/4ml) was frozen at -40ºC for 24 hours and then lyophilized (E-C Modulyo, E-C Apparatus Inc., NY, USA). It was then stored in dry bottles in a low moist environment. VEGF165 (Sigma-Aldrich, St. Louis, MO) was denatured with β-mercaptoethanol at 95oC for 15 minutes and submitted to electrophoresis on 15% SDS polyacrylamide gel. The protein was transferred to nitrocellulose membranes (Transblot 0,45 µM, Bio-RAD Laboratories, Richmond, CA). The blot was blocked by 5% skim milk in Tris-buffered saline with 0.05% Tween (TTBS) for 1 hour at room temperature and incubated overnight at 4oC with primary antibody against VEGF (bevacizumab) at 1:2500 dilution in Tris-buffered saline. After washing with TTBS, blots were incubated with peroxidase-conjugated goat anti-human secondary antibody for 30 minutes at room temperature. After the final wash, the blot was incubated with chemiluminescence reagent (Santa Cruz Biotechnology Inc, Santa Cruz, CA) and the signal was detected with Amersham film.
Sodium dodecyl sulfate polyacrylamide gel electrophoresis confirmed the migration pattern of VEGF165, showing a precise band on the 19-kDa region. Subsequently, Western blot analysis showed that bevacizumab could still bind to VEGF165 molecule in vitro. A positive immunoreaction of bevacizumab with VEGF165 transferred to nitrocellulose membranes was revealed by a chemiluminescent reaction from peroxidase-conjugated goat anti-human secondary antibody.
Bevacizumab still binds VEGF165 after lyophilization and, consequently, it may be incorporated into an intravitreal biodegradable implant for anti-angiogenic activity tests in vivo.
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