Figure 3From: Retinoblastoma-independent antiproliferative activity of novel intracellular antibodies against the E7 oncoprotein in HPV 16-positive cells Mapping of the scFv 43M2 and 51 epitopes on E7 protein by immunoassays. Western Blotting and ELISA were performed using as antigens two 16E7 deletion mutants respectively representing NH2-term and COOH-term of the 16E7 protein. Panel A: schematic representation, with functional domains, of full-length 16E7 and 16E7 deletion mutants produced as GST-fusion proteins and used for the mapping of the scFvs. Panel B: Western blotting. Each antigen-containing strip was incubated with the purified scFvs 43M2 (M2), 51 and a mouse anti-Flag mAb. The control stripes were incubated with commercial anti-16E7 (M) and anti-GST monoclonal antibodies (mAb) in mouse, an in-house anti-16E7 polyclonal antibody (P) or no antibody (CTR). An anti-mouse peroxidase-conjugated mAb was used as a secondary antibody in all cases. The immunocomplexes were visualised by chemioluminescence. Panel C: ELISA. The coating was performed with the same proteins utilised for WB, and the incubation performed with the same primary and secondary antibodies, as indicated. The immune-complexes were revealed following incubation with the peroxidase substrate by reading the OD at 450 nm (OD450). The dashed lines represent the cut-off values for each experiment, corresponding to the OD450 values obtained using the anti-GST mAb.Back to article page