Phage-ELISA for ultrasensitive detection of Salmonella enteritidis†

Abstract

The M13 phage carries approximately 5 copies of the pIII protein, each of which is capable of displaying a single-chain variable fragment (scFv) that targets a specific antigen. This feature enables the M13 phage to be widely employed in the construction of scFv libraries, thereby facilitating the identification of antibodies with high specificity and affinity for target antigens. In this study, mice were immunized three times with Salmonella enteritidis (strain C50041) to induce diverse antibodies. The variable region sequences were subsequently amplified by PCR using genome extracted from the mice's splenic cells and fused to the pIII protein to construct the scFv phage display library (C50041-M13-scFv). Through biopanning with the C50041-M13-scFv library, a phage clone (C50041-scFv-4) exhibiting high affinity for the target bacteria was successfully obtained. Moreover, the scFv antibody (scFv-4) derived from C50041-scFv-4 was expressed in a prokaryotic expression system and validated to possess high specificity and affinity for C50041 through in vitro adsorption assays. Additionally, a phage-ELISA method was established: initially, bacteria were immobilized on the bottom surface of a 96-well plate. Next, the positive clone C50041-scFv-4 was introduced to specifically bind to the host cells. Finally, horseradish peroxidase (HRP)-conjugated anti-pVIII antibodies were used to detect the pVIII proteins of the bound phage clones. Owing to the capacity of multiple C50041-scFv-4 probes to simultaneously bind to a single target Salmonella and each phage clone's ability to accommodate hundreds of HRP-labeled antibodies, the proposed phage-ELISA demonstrated remarkable sensitivity (10⁴ CFU mL⁻¹) for detecting Salmonella enteritidis samples. This sensitivity surpasses that of traditional ELISA by one order of magnitude in this study. Our phage-ELISA technology exhibits broad applicability across various biological species and provides an improved and robust platform for pathogen detection including bacteria and viruses.