Use of a Light-Addressable Potentiometric Sensor for the Detection ofEscherichia coliO157:H7

Abstract

We describe the development of an immunoligand assay (ILA) in conjunction with a light-addressable potentiometric sensor (LAPS) for the rapid detection ofEscherichia coliO157:H7 cells in buffered saline. The ILA protocol consists of “sandwiching” bacterial analyte between biotinylated and fluoresceinated antibodies, indirect enzyme labeling of the bacteria with urease-labeled anti-fluorescein antibody, and active capture of the immune complex at a biotinylated bovine serum albumin-blocked nitrocellulose filter membrane with streptavidin. Using liveE. coliO157:H7, the efficiency of the ILA was compared using various ratios of the biotinylated and fluoresceinated antibodies. Simultaneous addition of equimolar biotinylated and fluoresceinated antibodies effected optimal urease labeling and subsequent active capture of the bacteria in the ILA. Equimolar concentrations of the antibodies were varied to achieve optimal LAPS detection response for the live bacteria. Using ILA with LAPS, a minimum detectable level of ca. 7.1 × 102cells/ml of heat-killed or ca. 2.5 × 104cells/ml of liveE. coliO157:H7 bacteria was achieved in Tris-buffered saline in an assay time of ca. 45 or ca. 30 min, respectively.