Branched Polyethylenimine as a Carrier for Significantly Improving the Biopanning Efficiency of Phages Specific to Hapten

Unlike macromolecule-specific phage biopanning, traditional phage biopanning specific to small molecules (hapten) often faces the dilemma of low efficiency due to the low hapten density and non-target-specific binding attributed to the protein carriers. In this study, an enrofloxacin (ENR)-immunized library with a capacity of 1.75 × 107 CFU/mL was constructed. ENR was covalently coupled with polyethylenimine (PEI) by the carbodiimide method with a high coupling ratio of 46:1 (ENR:PEI), and the polymeric carrier for hapten was first used as a coating antigen in the biopanning of phages. After optimization in the blocking and washing procedure, the biopanning method based on the ENR–PEI conjugate is established with only three rounds, and the tedious incubation of the protein carrier in biopanning for up to 1 h in every biopanning round was omitted. Compared with the traditional biopanning method using ENR–BSA, the developed approach increased the number of positive clones by approximately 5.67 times in the verification of monoclonal phage ELISA, significantly improving the biopanning efficiency for phages specific to hapten. Verification ELISA, gene sequencing, and BLI experiments showed that the positive clones screened by ENR–PEI have a similar binding ability compared to those screened by ENR–BSA. This study provides a simple and efficient method for phage biopanning in the preparation of single-domain antibodies against haptens.