Synthesis and application of firefly luciferase antibody conjugates in a bioluminescent immunoassay of Salmonella cells

Abstract

A method has been developed for synthesizing stable and highly active conjugates of firefly luciferase with antibodies using the heterobifunctional coupling agent N-succinimidyl 3-(2-pyridyldithio) propionate linking NH2 groups of antibodies with free SH groups of thermostable Luciola mingrelica firefly luciferase (Luc). A novel specific matrix for sorption of Salmonella cells has been designed that includes monodisperse polystyrene microparticles (φ = 240 nm) coated with Pluronic F108-PDS, which are then covalently linked to monoclonal antibodies against Salmonella cells (Sal). The Luc-Sal conjugates are used as detecting agents for bioluminescent immunoassay of Salmonella cells. The use of the novel matrix and highly active conjugates allow for a ∼100-fold increase in the method’s sensitivity compared with the standard sandwich assay.