Construction of a human scFv antibody library with VH regions randomized and its application

Abstract 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.

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Construction of immune human library and screening of neutralizing antibodies specific to glycoprotein of severe fever with thrombocytopenia syndrome virus

10.21203/rs.2.23499/v1 ◽

2020 ◽

Author(s):

wenshuai zhang ◽

Xiling Guo ◽

Ying Chi ◽

Yongjun Jiao

Keyword(s):

Neutralizing Antibodies ◽

High Capacity ◽

Binding Activity ◽

Immunofluorescence Assay ◽

Antibody Library ◽

Phage Display Technology ◽

Glycoprotein C ◽

Human Scfv ◽

Display Technology ◽

Severe Fever

Abstract Background Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease circulated in eastern Asian countries including China, Japan and South Korea. Currently, there are no effective prophylactic or therapeutic measurements available in the clinical settings. Antibody-mediated prevention and treatment can be an effective complement to the clinical supporting strategies. Glycoprotein N (Gn) and Glycoprotein C (Gc) are two of the highly antigenic envelope proteins on the SFTS virus (SFTSV) surface and contain neutralizing epitopes that can induce neutralizing antibodies. Methods To obtain the neutralizing antibodies specific to glycoprotein of SFTSV, we used phage display technology to generate a human phage antibody library with ScFv format from the peripheral lymphocytes of 8 patients who had recovered from SFTS. The library was bio-panned against recombinant Gn and Gc proteins for four rounds to find their specific antibody clones. Finally, the selected clones were characterized by binding activity test, virus neutralization and Competitive ELISA.Results An immune human ScFv antibody library against SFTSV with high capacity and diversity was constructed. After 4 rounds of panning, 6 distinct clones were found. Of them, 5 were specific to Gn, whereas only 1 was specific to Gc. The immunofluorescence assay showed only three clones with Gn specificity called MAb 4A6, MAb 2B6 and MAb 1F2, respectively, could bind nature virion. All these clones showed broad neutralization activity against the SFTSV, and had different antigenic epitopes from MAb 4-5, a previous identified antibody clone. Conclusions Three new monoclonal antibodies described in our study could be used as potential agents in immunotherapy against SFTSV infection.

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