A sandwich enzyme‐linked immunosorbent assay (ELISA) for detection of Pseudomcnas fluorescens and related psychrotrophic bacteria in refrigerated milk

Abstract

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 psychrotropic micro-organisms.

Culturability and Expression of Outer Membrane Proteins during Carbon, Nitrogen, or Phosphorus Starvation of Pseudomonas fluorescens DF57 and Pseudomonas putida DF14

“…A limited number of stable starvation markers were found, including representatives of both specific and general starvation proteins. The outer membrane starvation proteins in P. fluorescens DF57 and P. putida DF14 do not seem closely related in spite of the conservation of major outer membrane proteins within the fluorescent pseudomonads (9,19,24). Analogous to our comparison of Pseudomonas strains, Albertson et al (2) found major differences in the number of starvation-induced outer membrane proteins appearing in two Vibrio strains.…”

Outer Membrane Protein Heterogeneity within Pseudomonas fluorescens and P. putida and Use of an OprF Antibody as a Probe for rRNA Homology Group I Pseudomonads

“…However, no individual monoclonal antibody recognizes all fluorescent pseudomonads. Finally, González et al (9) reported only weak reactions of a polyclonal antibody produced against OprF from a psychrophilic P. fluorescens strain with other P. fluorescens strains, P. aeruginosa, and Pseudomonas fragi.…”

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

“…Previous work carried out on the detection of P. jluorescens in refrigerated milk revealed that anti-PF antibodies of P.jluorescens AH-70 recognized strongly the presence of this strain in milk samples, whereas their capacity to recognize other P. jluorescens strains was reduced (13). However, analysis on meat surfaces have shown (Fig.…”

“…It is known that protein F from the outer membrane of Pseudomonas aeruginosa is conserved and antigenic ally related in all tested strains of P. aeruginosa (20). In previous work we demonstrated (13) that protein F from P. fluorescens could be used to generate antibodies useful to detect and quantify the presence of P. fluorescens and related psychrotrophic bacteria in refrigerated raw milk.…”

“…As a result, several rapid, automated methods have been proposed to overcome the disadvantages inherent in traditional plate count methods for estimating psychrotrophic bacteria. These include fluorescence microscopy (26), adenosine triphosphate (ATP) photometry (5), the aminopeptidase method (25), the Limu-Ius test (17) and antibody based analytical techniques (13,15). Among the different methods, immunological techniques are promising because of their sensitivity and rapidity, although they need to select for appropriate antigens and antibodies.…”