The immunoreactivity of the anti-p21Ras single-chain fragment variant KGH-R1 and its predicted binding sites to p21Ras

Previously, we constructed a novel anti-p21Ras single-chain antibody fragment, KGH-R1-single-chain fragment variant (ScFv). However, the immunoreactivity of this antibody toward p21Ras is still unclear. Materials &

KGH-R1-ScFv shows specific immunoreactivity toward wild-type and mutant p21Ras as well as the corresponding tumors, which suggests that KGH-R1-ScFv shows potential as a therapeutic antibody for therapy of RAS-driven tumors.

ELISAs, immunohistochemistry, western blotting and immunofluorescence were used to identify the immunoreactivity of KGH-R1-ScFv toward p21Ras. An in silico approach was used to determine the protein structures of KGH-R1-ScFv and p21Ras and then to predict the site involved in the binding of KGH-R1-ScFv to p21Ras.

KGH-R1-ScFv had a specific immune reaction with prokaryotically expressed p21Ras, human tumor cells and tumor tissues with RAS mutations or overexpression of RAS. Molecular docking showed that KGH-R1-ScFv could stably interact with wild-type and mutant p21Ras and the binding sites were in the complementarity-determining regions of KGH-R1-ScFv.