Single‐chain fragment variable produced by phage display technology: Construction, selection, mutation, expression, and recent applications in food safety

Immunoassays are reliable, efficient, and accurate methods for the analysis of small‐molecule harmful substances (such as pesticides, veterinary drugs, and biological toxins) that may be present in food. However, traditional polyclonal and monoclonal antibodies are limited by animal hosts and hinder further development of immunoassays. With the gradual application of phage display technology as an efficient in vitro selection technology, the single‐chain fragment variable (scFv) now provides an exciting alternative to traditional antibodies. Efficiently constructed scFv source libraries and specifically designed biopanning schemes can now yield scFvs possessing specific recognition capabilities. A rational mutation strategy further enhances the affinity of scFv, and allows it to reach a level that cannot be achieved by immunization. Finally, appropriate prokaryotic expression measures ensure stable and efficient production of scFv. Therefore, when developing excellent scFvs, it is necessary to focus on three key aspects of this process that include screening, mutation, and expression. In this review, we analyze in detail the preparation and affinity improvement process for scFv and provide insights into the research progress and development trend of scFv‐based immunoassay methods for monitoring small‐molecule harmful substances.