Quantitative detection of Escherichia coli O157 in surface waters by usingimmunomagnetic electrochemiluminescence

A protocol for the quantitative detection of Escherichia coli O157 in raw a nd concentrated surface waters using immunomagnetic electrochemiluminescenc e (IM-ECL) was developed and optimized. Three antibody sandwich formats wer e tested: commercial anti-O157:H7 IR I beads, IR I beads made in-house with a polyclonal anti-O157:H7 immunoglobulin G (IgG), or IM beads made in-hous e with a monoclonal anti-O157:H7 IgG coupled with a polyclonal anti-O157:H7 Ige to which an electrochemiluminescent label (TAG) was attached. The mono clonal IM bead-polyclonal TAG format was chosen for optimization because it gave Io,ver background levels and linear regression slopes of ca, 1.0, ind icative of a constant ECL signal per cell, The dynamic range was ca, 10(1) to 10(5) cells ml(-1) in phosphate-buffered saline and in raw water samples . The monoclonal IM beads selectively captured E, coli O157 cells in the pr esence of ca, 10(8) cells of a non-O157 strain of E. coli ml(-1). Backgroun d ECL signals from concentrated (100-fold) water samples were substantially higher and more variable than ra,v water samples, The background signal wa s partially eliminated by the addition of polyvinylpolypyrrolidone. Success ive cell capture incubations, termed sequential bead capture (SBC), were op timized for establishing baseline ECL values for individual water samples, The linear dynamic range with SEC was ca, 10(2) to 10(5) E. coli O157 cells ml of concentrated water(-1). To validate the protocol, 10-liter surface m ater samples were spiked with ca, 5,000 E. coli O157 (Odwalla) cells and co ncentrated by vortex filtration, and 1- or 3-ml aliquots were analyzed by I M-ECL. Differential ECL signals (SBC) from 1- and 3-ml samples were statist ically significant and were generally consistent with standard curves for t hese cell concentrations. Enrichments were conducted with aliquots of spike d raw water and concentrated water using EC broth and minimal lactose broth (MLB). All tubes with concentrated water became turbid and gave a positive ECL response for E, coli O157 (> 10,000 ECL units); MLB gave a somewhat hi gher detection rate with spiked ram water. The potential sensitivity of the IM-ECL assay is ca, 25 E. coli O157 cells mi of raw water(-1), 25 cells 10 0 mi of 100-fold concentrated water(-1), or 1 to 2 viable cells liter(-1) w ith concentration and enrichment, The IM-ECL assay appears suitable for rou tine analysis and screening of water samples.