Development of a novel FRET method for detection of Listeria or Salmonella

Abstract

Listeria and Salmonella contamination in foods results in not only foodborne outbreaks, but also a large economical burden for the industry due to product recalls. As a result, a fluorescence resonance energy transfer (FRET)-based method was developed to detect Listeria and Salmonella. Sensors utilizing FRET switch their fluorescence wavelength between donor and acceptor fluorophores as the distance between the two fluorophores change. This change in distance is a result of the conformational change in the 3D structure of the antibody as it binds to the target antigen. Listeria or Salmonella antibodies were labeled with FRET donor fluorophores (Alexa Fluor 546 (AF546)) while protein G or A was labeled with the acceptor fluorophores (Alexa Fluor 594 (AF594)). The labeled antibody–protein G or A complex was formed via incubation of the labeled antibody with protein G/A which specifically attaches to the Fc fragment of antibodies. The labeled antibody–protein G or A complex was tested in solution and specific and nonspecific antigens were exposed to the in solution complex. Changes in fluorescence were monitored by a spectrofluorometer. For “in-solution” tests, the optimal acceptor/donor fluorophore (A/D) ratios were 0.2 and 1.0 for Listeria and Salmonella, respectively, and both of Listeria and Salmonella antigen detection limits were 2.0 μg/ml.