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).

Monoamine Oxidase-A (MAO-A) Inhibitors Screened from the Autodisplayed Fv-Antibody Library.

Serotonin-like mimotopes were screened from the Fv-antibody library to be used as inhibitors against monoamine oxidase A (MAO-A). The Fv-antibody [corresponding to the VH region of immunoglobulin G (IgG)] consists of three complementarity-determining regions and four frame regions. The Fv-antibody library was prepared by site-directed mutagenesis of CDR3, which consists of 11 amino acid residues. Three target clones were screened from the Fv-antibody library, and the binding affinity of the screened clones to the monoclonal anti-serotonin antibody was analyzed using fluorescence-activated cell sorting. The screened Fv-antibodies were expressed as soluble proteins fused with green fluorescence protein. Additionally, the screened CDR3 regions (11 residues) of the selected Fv-antibodies were synthesized as peptides with linking amino acid residues. The binding constants (KD) of the three serotonin-like mimotopes (Fv-antibodies and peptides) were estimated using a surface plasmon resonance biosensor. The inhibitory activity (IC50) of the serotonin-like mimotopes (Fv-antibodies and peptides) was estimated separately for MAO-A and MAO-B enzymes and compared with that of conventional inhibitors. Finally, the screened serotonin-like mimotopes were used to treat a cell line (SH-SY5Y, ATCC code: CRL-2266) expressing serotonin receptors. This was done to confirm the following two aspects: (1) the binding of mimotopes to the serotonin receptors on the cell surface and (2) the inhibitory activity of mimotopes against MAO-A enzymes in the cell lysates.

Phage-targeting bimetallic nanoplasmonic biochip functionalized with bacterial outer membranes as a biorecognition element.

The use of phages-a natural predator of bacteria-has emerged as a therapeutic strategy for treating multidrug-resistant bacterial infections; thus, the isolation and detection of phages from the environment is crucial for advancing phage therapy. Herein, for the first time, we propose a nanoplasmonic-based biodetection platform for phages that utilizes bacterial outer membranes (OMs) as a biorecognition element. Conventional biosensors based on phage-bacteria interactions encounter multiple challenges due to the bacteriolytic phages and potentially toxic bacteria, resulting in instability and risk in the measurement. Therefore, instead of whole living bacteria, we employ a safe biochemical OMs fraction presenting phage-specific receptors, allowing the robust and reliable phage detection. In addition, the biochip is constructed on bimetallic nanoplasmonic islands through solid-state dewetting for synergy between Au and Ag, whereby sensitive detection of phage-OMs interactions is achieved by monitoring the absorption peak shift. For high detection performance, the nanoplasmonic chip is optimized by systematically investigating the morphological features, e.g., size and packing density of the nanoislands. Using our optimized device, phages are detected with high sensitivity (≥∼104 plaques), specificity (little cross-reactivity), and affinity (stronger binding to the host OMs than anti-bacterial antibodies), further exhibiting the cell-killing activities.