Application of Recombinant Human scFv Antibody as a Powerful Tool to Monitor Nitrogen Fixing Biofertilizer in Rice and Legume

Human scFv antibody generated from phage display technology was successfully used for the generation of specific recombinant antibodies: yiN92-1e10 and yiDOA9-162 for the detection of Bradyrhizobium strains SUTN9-2 and DOA9, respectively.

Characterization of an In Vivo Neutralizing Anti-Vaccinia Virus D8 Single-Chain Fragment Variable (scFv) from a Human Anti-Vaccinia Virus-Specific Recombinant Library

A panel of potent neutralizing antibodies are protective against orthopoxvirus (OPXV) infections. For the development of OPXV-specific recombinant human single-chain antibodies (scFvs), the IgG repertoire of four vaccinated donors was amplified from peripheral B-lymphocytes. The resulting library consisted of ≥4 × 108 independent colonies. The immuno-screening against vaccinia virus (VACV) Elstree revealed a predominant selection of scFv clones specifically binding to the D8 protein. The scFv-1.2.2.H9 was engineered into larger human scFv-Fc-1.2.2.H9 and IgG1-1.2.2.H9 formats to improve the binding affinity and to add effector functions within the human immune response. Similar binding kinetics were calculated for scFv-1.2.2.H9 and scFv-Fc-1.2.2.H9 (1.61 nM and 7.685 nM, respectively), whereas, for IgG1-1.2.2.H9, the Michaelis-Menten kinetics revealed an increased affinity of 43.8 pM. None of the purified recombinant 1.2.2.H9 formats were able to neutralize VACV Elstree in vitro. After addition of 1% human complement, the neutralization of ≥50% of VACV Elstree was achieved with 0.0776 µM scFv-Fc-1.2.2.H9 and 0.01324 µM IgG1-1.2.2.H9, respectively. In an in vivo passive immunization NMRI mouse model, 100 µg purified scFv-1.2.2.H9 and the IgG1-1.2.2.H9 partially protected against the challenge with 4 LD50 VACV Munich 1, as 3/6 mice survived. In contrast, in the scFv-Fc-1.2.2.H9 group, only one mouse survived the challenge.

A Nano-Emulsion Platform Functionalized with a Fully Human scFv-Fc Antibody for Atheroma Targeting: Towards a Theranostic Approach to Atherosclerosis

Atherosclerosis is at the onset of the cardiovascular diseases that are among the leading causes of death worldwide. Currently, high-risk plaques, also called vulnerable atheromatous plaques, remain often undiagnosed until the occurrence of severe complications, such as stroke or myocardial infarction. Molecular imaging agents that target high-risk atheromatous lesions could greatly improve the diagnosis of atherosclerosis by identifying sites of high disease activity. Moreover, a “theranostic approach” that combines molecular imaging agents (for diagnosis) and therapeutic molecules would be of great value for the local management of atheromatous plaques. The aim of this study was to develop and characterize an innovative theranostic tool for atherosclerosis. We engineered oil-in-water nano-emulsions (NEs) loaded with superparamagnetic iron oxide (SPIO) nanoparticles for magnetic resonance imaging (MRI) purposes. Dynamic MRI showed that NE-SPIO nanoparticles decorated with a polyethylene glycol (PEG) layer reduced their liver uptake and extended their half-life. Next, the NE-SPIO-PEG formulation was functionalized with a fully human scFv-Fc antibody (P3) recognizing galectin 3, an atherosclerosis biomarker. The P3-functionalized formulation targeted atheromatous plaques, as demonstrated in an immunohistochemistry analyses of mouse aorta and human artery sections and in an Apoe−/− mouse model of atherosclerosis. Moreover, the formulation was loaded with SPIO nanoparticles and/or alpha-tocopherol to be used as a theranostic tool for atherosclerosis imaging (SPIO) and for delivery of drugs that reduce oxidation (here, alpha-tocopherol) in atheromatous plaques. This study paves the way to non-invasive targeted imaging of atherosclerosis and synergistic therapeutic applications.

Isolation and characterizations of a novel recombinant scFv antibody against exotoxin A of Pseudomonas aeruginosa

Background Pseudomonas aeruginosa is the leading cause of nosocomial infections, especially in people with a compromised immune system. Targeting virulence factors by neutralizing antibodies is a novel paradigm for the treatment of antibiotic-resistant pseudomonas infections. In this respect, exotoxin A is one of the most potent virulence factors in P. aeruginosa. The present study was carried out to identify a novel human scFv antibody against the P. aeruginosa exotoxin A domain I (ExoA-DI) from a human scFv phage library. Methods The recombinant ExoA-DI of P. aeruginosa was expressed in E. coli, purified by Ni-NTA column, and used for screening of human antibody phage library. A novel screening procedure was conducted to prevent the elimination of rare specific clones. The phage clone with high reactivity was evaluated by ELISA and western blot. Results Based on the results of polyclonal phage ELISA, the fifth round of biopanning leads to the isolation of several ExoA-DI reactive clones. One positive clone with high affinity was selected by monoclonal phage ELISA and used for antibody expression. The purified scFv showed high reactivity with the recombinant domain I and full-length native exotoxin A. Conclusions The purified anti-exotoxin A scFv displayed high specificity against exotoxin A. The human scFv identified in this study could be the groundwork for developing a novel therapeutic agent to control P. aeruginosa infections.

Isolation and Characterizations of A Novel Recombinant scFv Antibody Against Exotoxin-A of Pseudomonas Aeruginosa

Background: Pseudomonas aeruginosa is known as the leading cause of nosocomial infections especially in people with a compromised immune system. Targeting virulence factors by neutralizing antibodies is a novel paradigm for the treatment of antibiotic-resistant pseudomonas infections.Methods: In this respect, exotoxin A is one of the most potent virulence factors in P. aeruginosa. The present study was conducted to identify a novel human scFv antibody against domain I of P. aeruginosa exotoxin A from a human scFv phage library. For this, the recombinant domain I of exotoxin A was expressed in E. coli and purified by Ni-NTA column. A novel screening procedure was conducted to prevent the elimination of rare specific clones. Based on polyclonal phage ELISA results, the fifth round of biopanning was selected to identify specific phage clones.Results: Two positive clones were found by monoclonal phage. The phage clone with high reactivity was evaluated by ELISA and western blot. The purified scFv also showed high reactivity with full length exotoxin.Conclusions: In conclusion, the purified anti-exotoxin A scFv displayed high specificity against exotoxin A. The human scFv identified can be the groundwork for development of a novel therapeutic agent for control of P. aeruginosa infections.

A Novel Fully Human Recombinant Antibody Neutralizing the α-hemolysin of Staphylococcus Aureus

BackgroundMethicillin-resistant Staphylococcus aureus (MRSA) is resistant to almost all β-lactam antibiotics. Hence, new ways to control MRSA infection, such as antibacterial antibodies, need to be explored. α-Hemolysin is the most important virulence factor widely expressed in S. aureus. This study aimed to develop a new fully human antibody against α-hemolysin of S. aureus and explore its neutralizing effect. ResultsThe single-chain antibody fragments（scFvs）against S. aureus were screened from a fully human scFv library using phage display technology with purified α-hemolysin protein. The selected scFvs had good binding affinities to α-hemolysin and were highly specific to S. aureus. The IgG-like scFv-Fc inserted into the pcDNA3.1 or pMH3 vector was expressed in HEK293F suspension cells to extend the antibody's in vivo half-life and restore Fc function. The size of purified scFv-Fc was about 55 kDa. The functions of expressed scFv-Fcs against α-hemolysin were validated. The results of A549 cytotoxicity assays showed that scFv10-Fc and scFv555-Fc had better protective effects than other scFv-Fcs on A549 cells. The results of anti-rabbit erythrocyte lysis assay confirmed that scFv555-Fc had a significant neutralizing effect on toxins. The scFv555-Fc was used to construct the docking model of antigen–antibody complexes using Discovery Studio software. It predicted that the key binding sites of α-hemolysin were TYR28, LYS37, PHE39, ARG56, and LYS58, which might be the key toxic sites of α-hemolysin.ConclusionsA novel fully human scFv-Fc antibody neutralizing the α-hemolysin toxin of S. aureus was successfully developed. The findings might provide a new theoretical basis and treatment method for preventing MRSA infection.