Structural Changes in Stx1 Engineering Monoclonal Antibody Improves Its Functionality as Diagnostic Tool for a Rapid...

Area of Science:

• Microbiology
• Immunotechnology
• Molecular Biology

Background:

• Shiga toxin-producing Escherichia coli (STEC) causes foodborne illness.
• Stx1 is a key toxin produced by STEC.
• Recombinant antibody fragments like scFv offer diagnostic potential but face production challenges.

Purpose of the Study:

• To develop a stable and functional single-chain variable fragment (scFv) targeting Stx1 toxin.
• To optimize scFv production in bacteria for improved solubility and antigen affinity.
• To evaluate the utility of the engineered scFv for STEC detection.

Main Methods:

• Obtained scFv-Stx1 from hybridoma-produced IgG.
• Engineered structural modifications including protein orientation, vector, and linker.
• Assessed scFv solubility, affinity (K_D), and antigen recognition via ELISA and latex agglutination test.

Main Results:

• Achieved stable and functional bacterial expression of scFv-Stx1.
• Increased scFv solubility and antigen affinity, with a K_D of 2.26 × 10^-7 M.
• Demonstrated recognition of purified Stx1, cross-reactivity with Stx2, and detection of 88% of Stx1-producing strains.

Conclusions:

• The engineered scFv-Stx1 is a stable, bacteria-produced tool.
• This scFv fragment shows promise for rapid STEC diagnosis.
• Provides an alternative diagnostic approach for STEC infections.