Development of a flow injection analysis (FIA) immunosensor for the detection of Escherichia coli.
A flow injection immunoanalysis (FIA) system has been developed for the detection of Escherichia coli in artificially contaminated food samples. Anti-E. coli antibodies were covalently immobilized onto porous aminopropyl glass beads via glutaraldehyde activation to form an immunoreactor. After adsorption of the cells onto anti-E. coli antibody bound glass beads, 4-methylumbelliferyl-beta-D-glucuronide was injected into the system which was then hydrolyzed by the adsorbed E. coli cells containing beta-D-glucuronidase, an enzyme which is very specific to E. coli and to a few other strains of Shigella. Fluorescent 4-methylumbelliferone released from the enzymatic reaction was then detected by a fluorometer. Owing to the specificity of the antibody towards E. coli, the FIA system was very selective for detection of E. coli whereas Shigella boydii, another GUD-positive bacterium, did not give any response. The FIA system was successfully used for detecting as low as 5 x 10(7) CFU/ml E. coli in less than 30 min and was reusable for at least 300 repeated assays. The immunoreactor yielded reproducible results during 3 months of experimentation if stored overnight at 4 degrees C in carrier buffer containing 0.05 to 0.25% Tween 20.
Bouvrette
P
P
Biotechnology Research Institute, National Research Council Canada, Montreal, Quebec, Canada.
Luong
J H
JH
D004926:Escherichia coli / Q000201:enzymology / Q000276:immunology / Q000302:isolation & purification*; D017022:Flow Injection Analysis / Q000295:instrumentation*; D005516:Food Microbiology; D014453:Glucuronosyltransferase / Q000378:metabolism; D015203:Reproducibility of Results
C021788:4-methylumbelliferone glucuronyltransferase; D014453:Glucuronosyltransferase