Neutralizing anti-diphtheria toxin scFv produced by phage display

Area of Science:

• Biotechnology
• Immunology
• Molecular Biology

Background:

• Diphtheria remains a significant threat despite vaccination, necessitating effective treatments.
• Current diphtheria antitoxin (DAT) is animal-derived, posing limitations.
• Recombinant human antibody fragments, like single-chain variable fragments (scFv), offer a promising alternative.

Purpose of the Study:

• To construct a human single-chain fragment (HuscFv) library using B cells from immunized volunteers.
• To isolate and characterize HuscFv with specific neutralizing activity against diphtheria toxin.
• To evaluate the potential of HuscFv as a substitute for animal-derived antitoxins.

Main Methods:

• Construction of a large HuscFv library (1.3 × 10^6 members).
• Four rounds of phage bio-panning using Diphtheria toxoid (DTd) for selection.
• Expression and purification of selected scFv fragments, followed by neutralization assays on Vero cells.

Main Results:

• Isolation of 40 DTd-reactive antibody clones from the library.
• Identification of five clones with significant diphtheria toxin neutralization capabilities.
• Characterization of soluble scFv fragments with neutralizing activity (0.6-1.2 µg) and an affinity constant of approximately 10^7 M⁻¹.

Conclusions:

• Successful construction and isolation of HuscFv that specifically neutralize diphtheria toxin.
• The developed HuscFv demonstrates potential as a therapeutic and diagnostic agent for diphtheria.
• This study highlights the viability of using recombinant human antibody fragments to combat infectious diseases.