| Products/Services Used | Details | Operation |
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| Recombinant Proteins | For the determination of the affinity and cross-reactivity of GLAF-2 to VEGF from different species, an ELISA was performed. For this purpose recombinant feline (5844-CV-010, R&D Systems, Minneapolis, MN, USA), murine (V4512, Sigma-Aldrich, St. Louis, MO, USA) and human (V7259, Sigma-Aldrich) VEGF proteins were pre-coated at a concentration of 100 ng/well in 96-well ELISA plates and incubated overnight at 4°C. The wells were washed once with bidest. water and twice with PBS/0.05% Tween (PBST) and blocked with 100 μl 1% w/v Blocker Casein in PBS (Pierce, 37528) for 2 h at 37°C. After washing the wells four times with PBST, wells were incubated with seven two-fold dilution series of GLAF-2 (2000 to 31.25 ng/ml, GenScript,?E.coli?expressed and purified tag-free) for 1 h at room temperature (RT). PBS was used as a negative control. The wells were washed again and incubated with a polyclonal rabbit anti-GLAF-2 antibody (1∶1000, GenScript) for 1 h at RT. After washing the wells were incubated with a HRP-conjugated polyclonal goat anti-rabbit IgG (H+L) (170-6515, Biorad, 1∶5000) for 1 h at RT. The plate was washed and staining was developed using TMB (T0440, Sigma-Aldrich) and stopped with 2 N HCl. The ELISA was read at a wavelength of 450 nm. | Get A Quote |
Virotherapy on the basis of oncolytic vaccinia virus (VACV) infection is a promising approach for cancer therapy. In this study we describe the establishment of a new preclinical model of feline mammary carcinoma (FMC) using a recently established cancer cell line, DT09/06. In addition, we evaluated a recombinant vaccinia virus strain, GLV-5b451, expressing the anti-vascular endothelial growth factor (VEGF) single-chain antibody (scAb) GLAF-2 as an oncolytic agent against FMC. Cell culture data demonstrate that GLV-5b451 virus efficiently infected, replicated in and destroyed DT09/06 cancer cells. In the selected xenografts of FMC, a single systemic administration of GLV-5b451 led to significant inhibition of t... More
