Construction of multiform scFv antibodies using linker peptide

Area of Science:

• Biotechnology
• Molecular Biology
• Immunotechnology

Background:

• Single-chain variable fragments (scFvs) are crucial for developing targeted diagnostic and therapeutic agents.
• Optimizing linker length in scFvs is essential for maintaining or enhancing antigen-binding affinity.
• Bispecific scFvs offer a promising platform for simultaneous detection of multiple analytes.

Purpose of the Study:

• To investigate the impact of linker length on the affinity of anti-deoxynivalenol (anti-DON) scFvs.
• To construct and characterize a bispecific scFv capable of binding both DON and zearalenone (ZEN).
• To evaluate the applicability of the developed bispecific scFv for detecting DON and ZEN in contaminated wheat.

Main Methods:

• Construction and affinity analysis of anti-DON scFvs with varying linker lengths (0-15 amino acids).
• Genetic fusion of anti-DON scFv and anti-zearalenone (anti-ZEN) scFv genes to create a bispecific scFv.
• Affinity constant determination for parental and bispecific scFvs.
• Competitive direct enzyme-linked immunosorbent assay (ELISA) for detecting DON and ZEN in wheat samples.

Main Results:

• scFv affinity increased with linker lengths up to 12 amino acids, plateauing thereafter.
• The constructed bispecific scFv retained the individual affinities of its parental anti-DON and anti-ZEN scFvs.
• The bispecific scFv demonstrated successful application in detecting DON and ZEN in naturally contaminated wheat samples.

Conclusions:

• Optimal linker length is critical for maximizing scFv affinity.
• Bispecific scFvs can be successfully engineered to maintain dual antigen-binding capabilities.
• This bispecific scFv holds significant potential for rapid and simultaneous detection of mycotoxins like DON and ZEN, aiding in food safety and agricultural diagnostics.