A SANDWICH ENZYME-LINKED-IMMUNOSORBENT-ASSAY (ELISA) FOR DETECTION OF PSEUDOMONAS-FLUORESCENS AND RELATED PSYCHROTROPHIC BACTERIA IN REFRIGERATED MILK

A sandwich ELISA (enzyme-linked immunosorbent assay) was developed for detection of Pseudomonas fluorescens and related psychrotrophic bacteria in refrigerated milk. Polyclonal antibodies were raised in rabbits against protein F from the cell envelope of Pseudomonas fluorescens AH-70. The anti-protein F antibodies (anti-PF) bound to the wells of a microtitre plate were used to capture this protein from the micro-organisms on milk samples. Further immunorecognition of the captured antigen was attained with the same anti-PF antibodies conjugated to biotin. ExtrAvidin-peroxidase was used to detect the biotinylated antibodies bound to their specific antigens. Subsequent enzymic conversion of substrate gave clear absorbance differences when assaying milk samples containing Ps.fluorescens strains of different origin as well as related psychrotrophic micro-organisms.