Affinity maturation of single-chain variable fragment specific for aflatoxin B(1) using yeast surface display

Area of Science:

• Immunology
• Biotechnology
• Analytical Chemistry

Background:

• Aflatoxin B1 is a highly toxic mycotoxin requiring accurate detection and quantification.
• Immunological methods are crucial for detecting and quantifying aflatoxin B1.
• Enhancing the binding affinity of single-chain variable fragments (scFvs) is key to improving these methods.

Purpose of the Study:

• To improve the aflatoxin B1-binding affinity of a single-chain variable fragment (scFv).
• To develop a more sensitive and specific immunological detection method for aflatoxin B1.

Main Methods:

• Yeast surface display technique was employed to create a mutated scFv library.
• Fluorescence-activated cell sorting (FACS) was used for 4 rounds to select high-affinity variants.
• The binding affinity and specificity of isolated scFv variants were characterized.

Main Results:

• Five scFv variants with improved aflatoxin B1-binding affinity were isolated.
• The best variant, scFv-M37, showed a 9-fold increase in binding affinity compared to the wild type.
• scFv-M37 demonstrated specificity comparable to a monoclonal antibody.
• Mutations in scFv-M37 were primarily outside antigen-contact loops, suggesting an allosteric binding enhancement.

Conclusions:

• Yeast surface display and FACS effectively enhance scFv binding affinity for aflatoxin B1.
• scFv-M37 represents a significant advancement for sensitive and specific aflatoxin B1 detection.
• The identified mutations suggest allosteric mechanisms contribute to improved mycotoxin binding.