Epidermal Growth Factor Receptor (EGFR) Inhibitors Screened from Autodisplayed Fv-Antibody Library.

Inhibitors of the epithermal growth factor receptor (EGFR) were screened from an autodisplayed Fv-antibody library using an anti-EGF antibody. The Fv-antibody library was expressed on the outer membrane of Escherichia coli, which corresponds to the heavy chain VH region of immunoglobulin G. The library was constructed by randomizing the CDR3 region of expressed VH regions (11 amino acid residues) by site-directed mutagenesis. Using an anti-EGF antibody as a screening probe, amino acid sequences (CDR3 region) with antibody binding affinity were screened from the Fv-antibody library. These amino acid sequences were considered to have similar chemical properties to EGF, which can bind to EGFR. Two autodisplayed clones with Fv-antibodies against EGFR were screened from the Fv-antibody library, and the screened Fv-antibodies were expressed as soluble proteins. The binding affinity (KD) was estimated using an SPR biosensor, and the inhibitory activity of expressed Fv-antibodies was observed for PANC-1 pancreatic tumor cells and T98G glioblastoma cells using Western blot analysis of proteins in the EGFR-mediated signaling pathway. The viability of PANC-1 and T98G cells was observed to decrease via the inhibitory activity of expressed Fv-antibodies. Finally, interactions between Fv-antibodies and EGFR were analyzed by using molecular docking simulations.

A one-step immunoassay based on switching peptides for diagnosis of porcine epidemic diarrhea virus (PEDV) using screened Fv-antibodies.

In this study, a one-step immunoassay for porcine epidemic diarrhea virus (PEDV) based on Fv-antibodies and switching peptides was developed, and the assay results of PEDV were obtained by just mixing samples without any further reaction or washing steps. The Fv-antibodies with binding affinity to the spike protein of PEDV were screened from the Fv-antibody library using the receptor-binding domain (RBD) of the spike protein as a screening probe. Screened Fv-antibodies with binding affinities to the RBD antigen were expressed, and the binding constants (KD) were calculated to be 83-142 nM. The one-step immunoassay for the detection of PEDV was configured as a displacement immunoassay using a fluorescence-labeled switching peptide. The one-step immunoassay based on switching peptides was performed using PEDV, and the limit of detection (LOD) values for PEDV detection were estimated to be Ct = 39.7-36.4. Compared with the LOD value for a conventional lateral flow immunoassay (Ct = 33.0), the one-step immunoassay showed a remarkably improved LOD for the detection of PEDV. Finally, the interaction between the screened Fv-antibodies and the PEDV RBD was investigated using docking simulations and compared with the amino acid sequences of the receptors on host cells, such as aminopeptidase N (APN) and angiotensin-converting enzyme-2 (ACE-2).