Structural Analyses of An Epidermal Growth Factor Receptor-Specific Single-Chain Fragment Variable via An In Silico Approach.

Single-chain fragment variable (scFv) antibodies were previously constructed of variable light and heavy chains joined by a (Gly4-Ser) 3 linker. The linker was created using molecular modeling software as a loop structure. Here, we introduce a protocol forin silico analysis of a complete scFv antibody that interacts with the epidermal growth factor receptor (EGFR). The homology modeling, with Pyrx of protein-protein docking and molecular dynamic simulation of the interacting scFv antibody and EGFR First, the authors used a protein structure modeling program and Python for homology modeling, and the antibody scFv structure was modeled for homology. The investigators downloaded Pyrx software as a platform in the docking study. The Molecular dynamic simulation was run using modeling software. Results show that when the MD simulation was subjected to energy minimization, the protein model had the lowest binding energy (-5.4 kcal/M). In addition, the MD simulation in this study showed that the docked EGFR-scFv antibody was stable for 20-75 ns when the movement of the structure increased sharply to 7.2 Å. In conclusion, in silicoanalysiswas performed, and the molecular docking and molecular dynamics simulations of the scFv antibody proved the effectiveness of the designed immune-therapeutic drug scFv as a specific drug therapy for EGFR.

Construction, expression and characterisation of a single chain variable fragment in the Escherichia coli periplasmic that recognise MCF-7 breast cancer cell line.

BACKGROUND: A functional  single-chain fragment variable (scFv) recognizing the  MCF-7 breast cancer carcinoma cell line was constructed from the C3A8 hybridoma using phage display technology. AIM OF STUDY: This study was conducted to evaluate the binding activity of scFv antibody recognise MCF-7 breast cancer cells carcinoma, the scfv antibody constructed and expressed in Escherichia coli periplasmic. MATERIALS AND METHODS: The scFv coding sequence was cloned in frame with the pIII phage coat protein. The signal sequence included in the C terminus directed the expression of the scFv in the Escherichia coli periplasm. Following several rounds of biopanning, colonies that expressed a scFv that recognized MCF-7 cells in Western blots, ELISAs, and flow cytometry test were isolated. RESULTS: A 750-bp scFv gene was successfully isolated. Cloning and two rounds of biopanning isolated the candidate with the highest activity (clone B7), as screened by ELISA. Following poly-acrylamide gel electrophoresis (SDS-PAGE) of the purified product, a 32-kDa band was observed. A similar-sized band was observed following Western blot analysis with an E tag-specific antibody. Binding reactivity of scFv antibody with MCF cells was determined using indirect ELISA and compared with monoclonal antibodies' reactivity. Also, flow cytometry was useful in further characterization to the binding reactivity of scFv antibody with MCF-7 cells. CONCLUSIONS: The recombinant antibody technology used in this study is a rapid and effective approach that will aid in the development of the next generation of immunodiagnostic reagents.