Vol. 69, Nr. 6, 2004—JOURNAL OF FOOD SCIENCE M151 Published on Web 7/27/2004 © 2004 Institute of Food Technologists Further reproduction without permission is prohibited M: Food Microbiology & Safety JFS M: Food Microbiology and Safety Immunoliposomes Sandwich Fluorometric Assay (ILSF) for Detection of Escherichia coli O157:H7 SUNGSU PARK, SEJONG OH, AND RICHARD A. DURST ABSTRACT: We report the development of automated flourometric immunoassay for the detection of Escherichia coli O157:H7, using antibody-directed liposomes (immunoliposomes) encapsulating fluorophore as an ana- lytical reagent. Thiolated antibodies (anti–E. coli O157:H7) were coupled to malemide-tagged liposomes encap- sulating dye. To automate the assay, a fluorescence plate reader was included in the assay system to detect fluorophore released from lysed liposomes in a microplate. The detection limit of the current assay with pure cultures of the serotype was about 10 4 colony-forming units (CFU)/mL. The assay can detect E. coli O157 in ground beef samples inoculated with as few as 0.8 CFU/mL after a 12-h enrichment. These results demonstrate the feasibility of using fluorophore-encapsulated immunoliposomes in a microtiter plate for the rapid and automated detection of molecules with multivalent antigenic sites. Keywords: liposome, Escherichia coli O157:H7, detection, fluorometric assay, multivalent antigen Introduction T he need for rapid and automated analytical methods for the detection of foodborne pathogens has become more urgent in recent years (Fung 1995). Conventional culture methods are gen- erally labor intensive and usually take several days to weeks to iso- late suspected pathogens (Feng 1998). Because of this lengthy pro- cedure of the cultural methods, the food industry has often delayed the release of a particular food commodity until the com- modity has been certified to be microbiologically safe. Therefore, it is not surprising that many practicing microbiologists would like to have inexpensive and accurate systems, which could provide rapid and automated detection of foodborne pathogens (Fung and oth- ers 1989). Immunoassays, which offer high selectivity, sensitivity, speed, and simplicity of operation, are potentially useful for routine micro- biological analyses of foods (Helfle 1995). For the detection of an- tigens containing at least 2 independent epitopes, such as bacte- rial cells, the antigens can be sandwiched between 2 different but specific antibodies. This sandwich format is widely adapted in en- zyme-linked immunosorbent assay (ELISA) methods, in which the detection antibody is enzyme-labeled (Helfle 1995). The signal generated by enzyme-substrate reactions is measured in a micro- titer plate (96-wells) by a ultraviolet (UV)-visible detector at appro- priate wavelengths. Microtiter plates lead the popularity of the ELISAs because of the ease of separating immunoreactants from unbound molecules by simple washing and the capacity for test- ing many samples at a time. Most importantly, the plates have a potential for automation. Liposomes are closed spherical vesicles consisting of 1 or more phospholipid bilayers surrounding an aqueous cavity that can encapsulate a variety of marker molecules, such as enzymes and fluorophores (Umeda and others 1986; Rongen and others 1994; Singh and others 1995; Lee and others 1997). The amount of encap- sulated marker molecules needs to be related to the concentration of the analyte in the sample to measure the analyte. For this pur- pose, liposomes have to carry either antigens (or hapten) or anti- bodies on their outer surface. Immunoassays using antigen (or hapten) sensitized liposomes are mainly used in competitive as- says for detecting antigens (or haptens) with small molecular size (Lee and others 1997). Immunoassays using antibody-coupled liposomes (immunoli- posomes) have been performed in both homogeneous and heter- ogeneous formats mainly for detecting antigens with multiple binding sites (Umeda and others 1986; Singh and others 1995). In many of these homogeneous immunoassays, immunoliposomes are designed to lyse when exposed to targeted antigens that bind to antibodies on the surface of the liposomes (Singh and others 1995). On the other hand, most of heterogeneous immunoassays using immunoliposomes have been using a sandwich format (Singh and others 1995). Singh and others (1995) used bifunctional vesicles tagged with enzymes and antibodies for detecting d-dimer in human plasma and reported a detection limit 9 times lower than what was achieved using conventional ELISAs. However, this meth- od still relies on prolonged incubation for enzyme-substrate reac- tions to amplify signals, thereby not being applicable in routine automated assays. Rongen and others used biotinylated liposomes encapsulating carboxyfluorescein (CF) with a microtiter plate in a sandwich format and reported a comparable sensitivity to the best colorimetric immunoassay for detecting human interferon-g. They also automated this assay by placing microtiter plates in a flow in- jection system consisting of a pump and autosampler. Most recent- ly, Emanuel and others (1995) suggested that the steric barrier of polyethylene glycols (PEGs) on the liposomal surface improves the specific binding of immunoliposomes to targeted cells by prevent- ing nonspecific binding of the liposomes to the cell surfaces, and immunodiagnostic assays using these liposomes are ready to be developed for a broad spectrum of analytes. Here we demonstrate the use of immunoliposomes encapsulat- MS 20030699 Submitted 12/11/03, Revised 1/20/04, Accepted 3/23/04. Author Park is with the Div. of Nano Science, Ewha Womans Univ., Seoul 120-750, South Korea. Author Oh is with the Dept. of Animal Science, Chonnam Natl. Univ., Gwangju 500-600, South Korea. Author Durst is with the Cornell Analytical Chemistry Laboratories, Cornell Univ., Geneva, N.Y. Direct in- quiries to author Oh (E-mail: soh@chonnam.ac.kr).