Abstract: This study established a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) for Salmonella Typhimurium based on nanobodies (Nbs). New Zealand white rabbits were immunized five times with inactivated Salmonella Typhimurium to obtain rabbit anti-Salmonella Typhimurium sera. The titer of rabbit anti-IgG after the fifth immunization was determined to be 1:24 300. Alpacas were immunized five times, and lymphocytes were isolated from the alpacas to extract RNA, which was then reverse-transcribed into cDNA. Nb sequences were amplified by polymerase chain reaction (PCR). The Nb genes and the phagemid vector pComb3XSS were digested with restriction enzymes, ligated, and recovered. The recovered products were electroporated into competent Escherichia coli ER2738 cells to obtain an immune library of nanobodies against Salmonella Typhimurium. The library size was 1.10 × 1010 CFU/mL, with an Nb gene insertion rate of 100%. After rescue with helper phage M13KO7, the library titer reached 1.80 × 1013 PFU/mL. Four rounds of solid-phase panning yielded 16 positive clones using phage-ELISA, and sequencing identified them as the same nanobody, designated Nb-16. The nanobody was expressed in competent E. coli BL21(DE3) PLysS cells, and a double-antibody sandwich ELISA based on Nb-16 was established using rabbit polyclonal antibodies. The half-maximal effective concentration (EC50) of this method was 6.071 × 105 CFU/mL, with 20% effective concentration (EC20) and 80% effective concentration EC80 in the range of 8.944 × 104–4.121 × 106 CFU/mL. The limit of detection (LOD) was 1.3 × 104 CFU/mL, and the limit of quantification (LOQ) was 1.7 × 104 CFU/mL. This method showed no cross-reactivity with other common pathogenic bacteria. The recovery for spiked lettuce samples was approximately 89%. Our method provides a reliable new approach for monitoring Salmonella Typhimurium in vegetables and offers a new pathway for the rapid detection of Salmonella Typhimurium in food and feed.

Key words: Salmonella Typhimurium; nanobody library; phage display technology; nanobodies; double-antibody sandwich enzyme-linked immunosorbent assay