Pentameric nanobodies serve as a capture agent and RANbodies function as immunoprobes for the sensitive detection of Salmonella typhimurium in immunoassays.

A simpler, more convenient, and faster diagnostic method for detection of Salmonella typhimurium (S. typhimurium) is urgently needed. In this study, the pentameric nanobodies (pST-Nb4) and HRP fusion nanobodies (ST-Nb1-vHRP) were produced in vitro, and pST-Nb4 displayed a binding affinity to S. typhimurium that was 61.8 times higher than that of the monomeric ST-Nb4. Ultimately, the sandwich ELISA based on pST-Nb4 as a capture agent and ST-Nb1-vHRP as an immunoprobe was developed for rapid detection of S. typhimurium. The assay was validated to have good specificity with a lower detection limit of 2.37 × 104 CFU/mL and a recovery rate of 95.00 to 108.66% for detection of S. typhimurium from contaminated milk and egg samples. After bacterial enrichment, the developed immunoassay was able to detect less than 10 CFU of S. typhimurium in milk or egg samples, even in mouse tissues infected with S. typhimurium.