An Ultrasensitive Sandwich Chemiluminescent Enzyme Immunoassay Based on Phage-Mediated Double-Nanobody for Detection of Salmonella Typhimurium in Food

ABSTRACT In this study, we demonstrated the first report of a nanobody-based sandwich chemiluminescent ELISA (P-CLISA) for Salmonella Typhimurium (S. Typhimurium) determination. Epitope mapping was first conducted for pairing nanobodies, and then soluble and phage-displayed nanobodies were used as detection antibodies in Nb-ELISA and P-ELISA, respectively. Compared with Nb-ELISA, P-ELISA has a 100-fold improvement in sensitivity given the signal amplification mediated by phage. Moreover, a chemiluminescence reaction was applied to replace the traditional chromogenic reaction in the assay, which achieved the detection of S. Typhimurium with an LOD of 3.63 × 103 CFU/mL in a linear range from 5.1 × 103 to 1.2 × 106 CFU/mL. The phage-mediated double-nanobody sandwich chemiluminescent enzyme immunoassay (P-CLISA) was successfully applied in actual sample analysis and able to detect fewer than 10 S. Typhimurium cells within 6~8 h of incubation. This study provides guidelines to develop reliable and sensitive double nanobody sandwich immunoassays, which also have great potential for other large molecule monitoring in food samples.