Filtration capture immunoassay for bacteria: optimization and potential for urinalysis

A novel assay utilizing immune-labeling, filtration, and electrochemistry for the rapid detection of bacteria has been optimized for the detection of Escherichia coli O157:H7. Bacteria were specifically labeled with alkaline phosphatase conjugated polyclonal antibodies and captured on a polycarbonate track-etched membrane filter (0.2 mu m pore size). The filter was then placed directly against a glassy carbon electrode, incubated with enzyme substrate, and the product detected by square wave voltammetry. The high speed and capture efficiency of membrane filtration and inherent sensitivity of electrochemical detection produced a 25-min assay with a detection limit of 5 X 10(3) E. coli O157:H7 per ml using a filtration volume of 100 mu l (i.e. 500 cells filtered). The labeling, filtration, and electrochemical steps were optimized, and the assay performance using electrochemical and colorimetric detection methods was compared. The assay was used to detect E. coli O157:H7 that was spiked into filter-sterilized urine at clinically relevant concentrations. (C) 1999 Elsevier Science B.V. All rights reserved.