Development of an Immunoassay for Chloramphenicol Based on the Preparation of a Specific Single-Chain Variable...

Area of Science:

• Immunotechnology
• Analytical Chemistry
• Food Safety

Background:

• Specific antibodies are crucial for detecting small molecule contaminants like chloramphenicol (CAP).
• Hybridoma technology produces monoclonal antibodies (mAbs) for targeted contaminant detection.

Purpose of the Study:

• To clone and sequence immunoglobulin genes (V(H) and V(L)) from a CAP-specific mAb.
• To construct and express a single-chain variable fragment (scFv) for improved CAP detection.
• To evaluate the performance of the engineered scFv in a direct competitive ELISA.

Main Methods:

• Cloning and sequencing of immunoglobulin heavy and light variable regions (V(H) and V(L)).
• Separation and analysis of antibody chains using SDS-PAGE and Orbitrap mass spectrometry.
• Recombinant expression of a soluble scFv-alkaline phosphatase fusion protein.
• Development of a direct competitive ELISA for CAP detection.

Main Results:

• The V(H) and V(L) chains specific for CAP were identified and sequenced.
• A soluble scFv-alkaline phosphatase fusion protein was successfully expressed.
• The developed scFv demonstrated lower sensitivity but enhanced food matrix resistance compared to the parent mAb.
• The scFv-based ELISA is a viable method for CAP detection in food samples.

Conclusions:

• Engineered antibodies, specifically scFv fragments, offer a promising tool for detecting small molecule contaminants like CAP.
• The developed scFv-ELISA provides a robust method for CAP detection with improved performance in complex food matrices.
• This study underscores the potential of antibody engineering in advancing food safety analytical methods.