Detection of Pseudomonas fluorescens and Related Psychrotrophic Bacteria in Refrigerated Meat by a Sandwich ELISA

Abstract

A sandwich enzyme-linked immunosorbent assay (ELISA) was developed for detection of Pseudomonas fluorescens and related psychrotrophic bacteria in refrigerated meat. Polyclonal antibodies were raised in rabbits against protein F from the cell envelope of P. fluorescens AH-70. The ELISA involved capturing antigens from the microorganisms present on meat samples with the anti-protein F antibodies immovilized on 96-well plates, and detecting bound antigens with the same anti-PF antibodies conjugated to biotin. Commercial ExtrAvidin-peroxidase conjugate was used to detect the biotinylated antibodies bound to their specific antigens. Subsequent enzymatic conversion of substrate gave distinct absorbance differences when assaying meat samples containing P. fluorescens strains of different origin as well as related psychrotrophic microorganisms. The detection threshold for the ELISA assay developed in this work is 105 CFU/cm2.

Rapid Detection of Pseudomonas in Seafoods Using Protease Indicator

“…The activity and stability of protease at various temperatures needs further study. ELISA using protein-F, toxins, or whole cells as antigens are used for detection of Pseudomonas species in milk, dairy products and meat (Azcona et al, 1989;Gonzalez et al, 1994;Eriksson et al, 1995). In our work, protease enzyme produced by Pseudomonas species was used as an antigen for the development of a wholly indige-nous ELISA system and applied to seafoods for the first time.…”