Development of a streptavidin-bridged enhanced sandwich ELISA based on self-paired nanobodies for monitoring multiplex Salmonella serogroups

Abstract

Salmonella are major pathogens that cause foodborne diseases. In this work, a broad-spectrum Salmonella nanobody-01 (Nb-01) was isolated and applied in the development of a streptavidin-bridged sandwich ELISA (SAB-ELISA) for simultaneously identifying five Salmonella serovars, including Salmonella Enteritidis (S. Enteritidis), Salmonella Typhimurium (S. Typhimurium), Salmonella London (S. London), Salmonella Paratyphi B (S. Paratyphi B) and Salmonella Hadar (S. Hadar). In this work, streptavidin (SA) was utilized as a scaffold to directionally immobilize biotinylated nanobody (BiNb) and Salmonella was detected by phage-displayed nanobodies. The SAB-ELISA can be accomplished within 180 min with a limit of detection (LOD) of 6.31 × 10³ colony forming units (CFU) mL^-1, 9.15 × 10³ CFU mL^-1, 4.23 × 10³ CFU mL^-1, 7.31 × 10³ CFU mL^-1 and 7.25 × 10³ CFU mL^-1 towards S. Typhimurium, S. Enteritidis, S. London, S. Paratyphi B and S. Hadar, respectively. In comparison of sandwich ELISA by passive immobilization of Nb-01 on polystyrene plate, the sensitivity was increased by around 6-fold, which confirmed the enhanced immobilization efficacy of SAB-ELISA. Furthermore, the feasibility of the assay for S. Typhimurium determination in actual samples was evaluated, showing excellent recovery, inter-day and intra-day precision.

Keywords: Multiplex analysis; Nanobody; Oriented immobilization; Salmonella; Streptavidin-biotin system.