Development of an Immunomagnetic Bead-Immunoliposome Fluorescence Assay for Rapid Detection of Escherichia coli O157:H7 in Aqueous Samples and Comparison of the Assay with a Standard Microbiological Method

Abstract

The objective of this study was to develop and optimize a protocol for the rapid detection of Escherichia coli O157:H7 in aqueous samples by a combined immunomagnetic bead-immunoliposome (IMB/IL) fluorescence assay. The protocol consisted of the filtration or centrifugation of 30-to 100-ml samples followed by incubation of the filter membranes or pellet with anti-E. coli O157:H7 immunomagnetic beads in growth medium specific for E. coli O157:H7. The resulting E. coli O157:H7-immunomagnetic bead complexes were isolated by magnetic separation, washed, and incubated with sulforhodamine B-containing immunoliposomes specific for E. coli O157:H7; the final immunomagnetic bead-E. coli O157:H7-immunoliposome complexes were again isolated by magnetic separation, washed, and lysed with a n-octyl-␤-D-glucopyranoside to release sulforhodamine B. The final protocol took less than 8 h to complete and had a detection limit of less than 1 CFU of E. coli O157:H7 per ml in various aqueous matrices, including apple juice and cider. To validate the protocol at an independent facility, 100-ml samples of groundwater with and without E. coli O157:H7 (15 CFU) were analyzed by a public health laboratory using the optimized protocol and a standard microbiological method. While the IMB/IL fluorescence assay was able to identify E. coli O157:H7-containing samples with 100% accuracy, the standard microbiological method was unable to distinguish E. coli O157:H7-spiked samples from negative controls without further extensive workup. These results demonstrate the feasibility of using immunomagnetic beads in combination with sulforhodamine B-encapsulating immunoliposomes for the rapid detection of E. coli O157:H7 in aqueous samples.

Polymeric micelle-based bioassay with femtomolar sensitivity

“…This creates a highly sensitive bioassay due to the signal amplification process, in which one binding event translates into the fluorescence signal of hundred thousands of dye molecules released from a single polymeric micelle to the solution. While linking an antibody molecule with many dye molecules has been an effective way to increase an assay's sensitivity, it has typically been achieved by attaching the antibody to dendrimers or particles [8,9,10], on which the dye molecules were attached on the surface. In our case, the dye molecules are encapsulated inside the micelles.…”

Methods of Rapid Microbiological Assay and Their Application to Pharmaceutical and Medical Device Fabrication

“…However, they are not always suited to pharmaceutical and medical devices due to the large difference in the number of contaminating microorganisms. Their procedures of detection may be direct, in which individual microorganisms or populations of organisms are directly observed, or indirect, whereby microbial metabolism, metabolites, or components can be Pettipher, 1983;Hutcheson et al, 1988;Rodrigues and Kroll, 1988;Rodrigues and Kroll, 1990;Diaper and Edwards, 1994;Nebe-von Caron et al, 1998;Newby, 2000;Van Poucke and Nelis, 2000;DeCory et al, 2005, electrical resistance Baynes et al, 1983Owens and WacherViveros, 1986;Silley and Forsythe, 1996;Newby, 2000 , enzyme monitoring Kroll andRodrigues, 1986;Watling and Leech, 1996;Newby, 2000 , Limulus amoebocyte lysate Jorgensen and Alexander, 1981;Bussey and Tsuji, 1984;Baines, 2000, nucleic acid probes Bauters et al, 1999Jordan, 2000;Newby, 2000;Dunsmoor et al, 2001;Jimenez et al, 2001;Serin et al, 2005;Chaieb et al, 2007;Zuluaga et al, 2009 , phage-interaction technology Wolber andGreen, 1990;Turpin et al, 1993;Stewart et al, 1996;Stewart et al, 1998;Mole et al, 1999;Wu et al, 2001 , andcarbon dioxide radiometry Cutler et al, 1989 . In addition to the methods summarized in Table 2, other techniques have been inve...…”

Rapid colorimetric detection of proteins and bacteria using silver reduction/precipitation catalyzed by gold nanoparticles

“…Detection assays play a very important role in medical diagnoses, quality control, biosafety, and research and development applications, especially in the fields of pharmaceuticals, medicine [1,2], food safety [3,4], environmental monitoring [5] and biosciences [6]. Rapid detection of target substances in an economical way is desirable for a variety of practical and potential applications.…”

“…Rapid detection of target substances in an economical way is desirable for a variety of practical and potential applications. In conjunction with commonly used detection schemes such as fluorometric [3,7], spectrophotometric, electrochemical [8], and optical [9], among others, immunoassays are becoming more prominent due to their specificity and reliability. Immunoassays are based on the biospecific molecular recognition interaction of antibodies with the target substances (antigens) to be detected and have been widely used to measure low concentrations of antigens in clinical specimens such as blood and other body fluids [1,10] and the detection of trace amounts of drugs, chemicals (e.g., pesticides), bacteria and viruses in biological and environmental samples [11].…”