Anal. Chem. 2003, 75, 4330-4334
Procedures for Preparing Escherichia coli O157:H7 Immunoliposome and Its Application in Liposome Immunoassay Ja-an Annie Ho* and Hsiu-Wen Hsu
Department of Applied Chemistry, National Chi-Nan University, Puli, Nantou, 545 Taiwan
Although Escherichia coli serotype O157:H7 was identified as a human pathogen in the ninth decade of the twentieth century, it has become recognized as a major foodborne pathogen. In the United States, the severity of E. coli O157:H7 infection in the young and the elderly has had a tremendous impact on human health, the food industry, and federal regulations regarding food safety. In laboratory diagnosis, most microbiologic assays rely on a single phenotype to selectively isolate this pathogen. However, the process is labor- and time-consuming. It is important eventually to develop new assay procedures to detect them. Immunoliposomes, anti-E. coli O157:H7 antibody-tagged liposomes, encapsulating a visible dye, sulforhodamine B, were used in the present study for the development of a field-portable colorimetric immunoassay to detect E. coli O157:H7. The N-succinimidyl-S-acetylthioacetate derivative of the antibodies (anti-E. coli O157: H7) was first conjugated through the reactive N-(kmaleimidoundecanoyloxy) sulfosuccinimide ester derivative of dipalmitoylphosphatidylethanolamine and subsequently incorported into liposomes to form the immunoliposomes. A plastic-backed nitrocellulose strip with two immobilized zones is the basis for a sandwich assay to detect E. coli O157:H7. The first zone is the antigen capture zone (AC zone), which is used in a sandwich (noncompetitive) assay format; the other is the biotin capture zone (BC zone), which is used as a positive control for the strip. During the capillary migration of the wicking reagent containing 50 µL of immunoliposomes and 90 µL of the test sample, E. coli O157:H7 with surface-bound immunoliposomes is captured at the AC zone, while the unbound immunoliposomes migrate and bind to the antibiotin antibodies coated on BC zone. The color density of the AC zone were directly proportional to the amount of E. coli O157:H7 in the test sample. The detection limit of the current assay with heat-killed E. coli O157:H7 was ∼2500 cells. The selectivity of the newly developed biosensor system was investigated, and pathogens, including Salmonella typhimurium and Listeria genus specific, were proven to have no interference with the detection of E. coli O157:H7.
* Corresponding author. E-mail:
[email protected]. Fax: +886-49-2917956.
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In the past decade, outbreaks of human illness associated with the consumption of raw or unpasteurized products have increased in the United States. Pathogens such as Listeria monocytogenes, Clostridium botulinum, and Bacillus cereus are naturally present in soil, so that their presence on fresh produce is not surprising. Salmonella, Escherichia coli O157:H7, Campylobacter jejuni, Vibrio cholerae, parasites, and viruses are more likely to contaminate fresh produce through vehicles such as raw or improperly composted manure, irrigation water containing untreated sewage, or contaminated wash water. E. coli O157:H7 was first recognized as a cause of illness in 1982 during an outbreak of severe bloody diarrhea.1 The outbreak was traced to contaminated hamburgers. An estimated 73 000 cases of infection and 61 deaths occur in the United States each year. Most illness has been associated with eating undercooked, contaminated ground beef. Person-to-person contact in families and child care centers is also an important mode of transmission. Infection can also occur after drinking raw milk and after swimming in or drinking sewage-contaminated water.2-5 E. coli O157:H7 is one of hundreds of strains of the bacterium E. coli, which is the dominant species found in feces while culturing under aerobic conditions. Normally E. coli serves a useful function in the body by suppressing the growth of harmful bacterial species and by synthesizing appreciable amounts of vitamins. Although most strains are harmless and live in the intestines of healthy humans and animals, E. coli serotype O157:H7 is a rare variety of E. coli that produces large quantities of one or more related, potent toxins that cause severe damage to the lining of the intestine. These toxins are referred to as verotoxins or shiga-like toxins, which closely resemble the toxin produced by Shigella dysenteriae type 1.6 Currently, there are four recognized classes of enterovirulent E. coli (collectively referred to as the EEC group) that cause gastroenteritis in humans. Among these is the enterohemorrhagic strain designated E. coli O157:H7. Hemorrhagic colitis is the name of the acute disease caused by E. coli O157:H7. The illness is (1) Feng, P. Emerging Infect. Dis. 1995, 1 (2), 47-52. (2) Griffin, P. M.; Tauxe, R. V. Epidemiol. Rev. 1991, 13, 60-98. (3) Karmali, M. A.; Arbus, C. S.; Ish Shalom, N.; Fleming, P. C.; Malkin, D.; Petric, M.; Cheung, R.; Louie, S.; Humphreys, G. R.; Strachan, M. Pediatr. Nephrol. 1998, 2, 409-414. (4) Carter, A. O.; Borczyk, A. A.; Carlson, J. A.; Harvey, B.; Hockin, J. C.; Karmali, M. A.; Krishnan, C.; Korn, D. A.; Lior, H. N. Engl. J. Med. 1987, 317, 1496-1500. (5) Belongia, E. A.; Osterholm, M. T.; Soler, J. T.; Ammend, D. A.; Braunm J. E.; MacDonald, K. L. J. Am. Med. Assoc. 1993, 269, 883-888. (6) Karmali, M. A. Clin. Microbiol. Rev. 1989, 2, 15-38. 10.1021/ac0343580 CCC: $25.00
© 2003 American Chemical Society Published on Web 07/10/2003
characterized by severe cramping (abdominal pain) and diarrhea, which is initially watery but becomes grossly bloody, and occasionally vomiting or kidney failure occurs. E. coli O157:H7 can be isolated and diagnosed from foods by several microbiological methods. The U.S. Department of Agriculture (USDA) Food Safety and Inspection Service has developed a series of both presumptive and confirmatory tests for detection of E. coli O157:H7.7,8 Unlike typical E. coli, isolates of O157:H7 do not ferment sorbitol within 24 h and are negative with the MUG assay; therefore, these criteria are commonly used for selective screening and isolation. Sorbitol-MacConkey agar has been used extensively to isolate this organism from clinical specimens. Hemorrhagic colitis agar, a selective and differential medium, is used in a direct plating method to isolate O157:H7 from foods. A third procedure uses sorbitol-MacConkey medium containing potassium tellurite and cefixime. It includes an enrichment step and is a new method developed as a result of the recent foodborne outbreaks. Many other methods have been developed in an effort to replace traditional techniques that usually take two to three days for biochemical and serological characterization. There is a need for rapid assays for screening food and environmental samples for pathogens and other orgainisms. Immunomagnetic electrochemiluminescent,9 light-addressable potentiometric sensor,10,11 polymerase chain reaction (PCR),12-16 and real-time PCR17,18 have been devloped and studied for the determination of E. coli O157: H7. Immunoassay techniques, which utilize immunological reactions to measure the presence of target substance, offer sensitivity, speed, and simplicity of operation, providing potential solutions for this need. Liposomes, first discovered and reported by Bangham group in 1965,19 are simple vesicles in which an aqueous volume is entirely enclosed by either a single bilayer membrane (unilamellar) or mutiple concentric bilayers (multilamellar) of phospholipid molecules.20 The coexistence of the hydrophilic and hydrophobic compartments in liposomes makes them a versatile carrier for a wide spectrum of amphipatic, water-soluble, and lipid(7) Food Borne Pathogenic Microorganisms and Natural Toxins Handbook; USDA-CFSN: Washington, DC, 2001; Chapter 15. (8) Sharar, A. K.; Rose, B. E. Revision 4 of laboratory communication 38. Protocol for isolation and identification of Escherichia coli O157:H7., USDA-FSIS, 1996. (9) Yu, H.; Bruno, J. G. Appl. Environ. Microbiol. 1996, 62 (2), 587-592. (10) Gehring, A. C.; Patterson, D. L.; Tu, S. Anal. Biochem. 1998, 258, 293298. (11) Tu, S.; Uknails, J.; Gehring, A. J. Rapid Methods Auto. Microbiol. 1999, 7, 69-79. (12) Gooding, C. M.; Choudary, P. V. J. Dairy Res. 1997, 64, 87-93. (13) Wang, J.; Yang, R.; Guo, Z.; Qiu, M. Wei Sheng Yan Jiu 2001, 30 (5), 310312. (14) McKillip, J. L.; Jaykus, L. A.; Drake, M. J. Food Prot. 2002, 65 (11), 17751779. (15) Bopp, D. J.; Sauders, B. D.; Waring, A. L.; Ackelsberg, J.; Dumas, N.; BraunHowland, E.; Dziewulski, D.; Wallace, B. J.; Kelly, M.; Halse, T.; Musser, K. A.; Smith, P. F.; Morse, D. L.; Limberger, R. J. J. Clin. Microbiol. 2003, 41 (1), 174-180. (16) Grant, M. A. J. Food Prot. 2003, 66 (1), 18-24. (17) Heller, L. C.; Davis, C. R.; Peak, K. K.; Wingfield, D.; Cannons, A. C.; Amuso, P. T.; Cattani, J. Appl. Environ. Microbiol. 2003, 69 (3), 1844-1846. (18) Ibekwe, A. M.; Grieve, C. M. J. Appl. Microbiol. 2003, 94 (3), 421-431. (19) Bangham, A. D.; Standish, M. M.; Weissmann, G. J. Mol. Biol. 1965, 13, 253-259. (20) New, R. R. C., Ed. Liposomes: A practical approach; Oxford University Press: Oxford, U.K., 1990.
soluble molecules. Liposomes have been used in a variety of applications such as gene therapy,21 drug delivery,22,23 cosmetic skin conditioners,24 and immunodiagnostics.25-29 Unlike gold particles, latex beads, and other metal particles, for whom only the outer surface can function in the detection mechanism,28 the interiors of liposomes can be engineered to be very large to entrap a great amount of reporter molecules, such as fluorescent dyes, electroactive markers, and enzymes. The number of marker molecules that can be entrapped inside of a liposome with reasonable volume must be larger than the number possible on a solid particle’s surface28 and, therefore, must provide better signal amplification. Liposome immunoassay (LIA), using fluorescent dye-loaded liposomes whose surfaces are sensitized with the antigen of interest, has been developed for a variety of assays, particularly a flow injection automated procedure.29-34 Our previous studies have successfully demonstrated the feasibility of using antigen-tagged liposomes entrapping sulforhodamine B (SRB)dye in competitive assay format for rapid screening of aflatoxins35 and fumonisins.31,32,36 As part of this project on the development of a flow injection liposome immunoanalysis (FILIA) system for E. coli O157:H7, a multistep procedure for tagging liposomes with E. coli O157:H7 was developed. These liposomes proved useful in a preliminary development of a test-strip semiquantitative assay for E. coli O157:H7 that has potential as a rapid and inexpensive point-of-care diagnostic assay. MATERIALS AND METHODS Reagents and Materials. All inorganic chemicals and organic solvents used were reagent grade or better. Dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylglycerol (DPPG), and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE) were purchased from Avanti Polar Lipids (Alabaster, AL). N-(kMaleimidoundecanoyloxy)sulfosuccinimide ester (sulfo-KMUS), N-ethylmaleimide, hydroxylamine hydrochloride, and succinimidyl-S-acetylthioacetate (SATA) were purchased from Pierce Chemical Co. (Rockford, IL). SRB and Biotin-X-DHPE were purchased from Molecular Probes (Eugene, OR). Affinity-purified polyclonal antibody (goat anti-E. coli O157:H7), heat-killed E. coli (21) Lurquin, P. F. In Liposome Technology: Entrapment of Drugs and Other Materials; Greroriadis, G., Ed.; CRC Press: Boca Raton, FL, 1984; Vol. II, Chapter 8. (22) Brandl, M., Bachmann, D., Drechsler, M., Bauer, K. H. Drug Dev. Ind. Pharm. 1990, 16 (14), 2167-2191. (23) Vingerhoeds, M. H., Storm, G., Crommelin, D. J. A. Immunomethods 1994, 4, 259-272. (24) Rongen, H. Immunoassays: Development and application of chemiluminescent and liposome labels. Ph.D. Thesis, University of Utrecht, Utrecht, Netherlands, 1995. (25) Monroe, D. Am. Clin. Prod. Rev. 1986, 5 (12), 34-41. (26) Monroe, D. J. Liposome Res. 1990, 1 (3), 339-377. (27) Singh, A. K.; Carbonell, R. G. In Handbook of Nonmedical Applications of Liposomes; Lasic, D. D., Barenholz, Y., Eds.; CRC Press: Boca Raton, FL, 1996; pp 190-207 (28) Park, S. and Durst, R. A. Anal. Biochem. 2000, 280, 151-158. (29) Edwards, A. J.; Durst, R. A. Electroanalysis 1995, 7 (9), 838-845. (30) Rule, G. S.; Montagna, R. A.; Durst, R. A. Anal. Biochem. 1997, 244, 260269. (31) Ho, J. A.; Durst, R. A. Anal. Chim. Acta 2000, 414, 51-60. (32) Ho, J. A.; Durst, R. A. Anal. Chim. Acta 2000, 414, 61-69. (33) Lee, M. Y.; Durst, R. A. J. Agric. Food Chem. 1996, 44, 4032-4036. (34) Rule, G. S.; Palmer, D. A.; Reeves, S. G.; Durst, R. A. Anal. Proc. Inclu. Anal. Commun. 1994, 31(11), 339-340. (35) Ho, J. A.; Wauchope, R. D. Anal. Chem. 2002, 74 (7), 1493-1496. (36) Ho, J. A.; Durst, R. A. Anal. Biochem. 2003, 312, 7-13.
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O157:H7, Listeria genus specific, and Salmonella tryphimurium were purchased from Kirkegaard-Perry Laboratories, Inc. (Gaithersburg, MO). Nitrocellulose membranes were purchased from Millipore (Bedford, MA). All other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO). Methods. Preparation of Dye-Encapsulated Liposomes. Liposomes were prepared by a reversed-phase evaporation method.37-39 The lipid mixture consisted of DPPC, cholesterol, DPPG, DPPE, and Biotin-X-DHPE in a molar ratio of 5:5:0.5:0.25: 0.005. A total of 43 µ-mol of the lipid mixture was first dissolved in 4 mL of a solvent mixture consisting of 6:6:1 volume ratios of chloroform, isopropyl ether, and methanol, followed by 1-min sonication at 45 °C under nitrogen. A 0.7-mL aliquot of a warmed solution of 95 mM SRB was then added to the lipid mixture. After sonication of the solution for 3 more min with occasional swirling, the organic solvent was removed by evaporating at 45 °C, leaving a dark purple, gellike suspension of liposomes. An additional 1.2 mL of SRB was added, followed by another 3 min of sonication at 45 °C. The liposome preparation was incubated in a 45 °C water bath for 35 min before passing through the 0.4-µm polycarbonate filter to produce a homogeneous suspension of uniform size. Any unencapsulated dye or trace of organic solvent was removed from the liposome preparation by gel filtering on a 1.5 × 25-cm Sephadex G-50-150 column at room temperature, followed by dialysis (MWCO, 12-14 kDa) at 4 °C in the dark. Derivatization of Liposomes with Sulfo-KMUS. Ten milligrams of sulfo-KMUS was dissolved in DMSO solution, and the resultant mixture was added to the dialyzed liposome preparation. It was then allowed to react at room temperature for 2.5 h. The reaction was stopped by reacting with Tris-HCl (pH 7.8) for 15 min at room temperature on the shaker, followed by dialysis (MWCO, 12-14 kDa) against 0.01M HEPES buffer, pH 7.5, containing NaCl and sucrose at 4 °C in the dark. Preparation of SATA-Modified Antibodies. Two milligrams of goat anti-E. coli O157:H7 antibody (Ab) was dissolved in 0.01 M phosphate-buffered saline (PBS) at pH 7.2. A 1.0-mg sample of SATA in DMSO was added to the Ab solution. The reaction mixture was then allowed to react on a shaker for 30 min at room temperature, followed by dialysis (MWCO, 6-8 kDa) against 0.01 M PBS (pH 7.2) and EDTA (10 mM) at 4 °C for 3.5 h. Concurrently, the acetylthioacetyl-antibody was deprotected by adding hydroxylamine hydrochloride (100 µmol) to obtain free sulfhydryl groups. Subsequently the thiolated Ab was purified by gel filtration on Sephadex G-25. Fractions containing SH-derivatized protein were collected by measurement of the absorbance at 280 nm. Conjugation of Maleimide-Derivatized Liposomes with SH-Containing Antibody. The coupling of thiolated Ab to maleimide-derivatized liposomes was achieved by incubating overnight at 4°C. Unreacted sulfhydryl groups on the antibody and the unreacted sulfosuccinimidyl groups on the sulfo-KMUS were subsequently capped with N-ethylmaleimide and Tris buffer. The antibody-tagged liposomes were separated from unreacted (37) Szoka, F., Jr.; Papahadjopoulos. D. Proc. Natl. Acad. Sci. U.S.A. 1978, 75 (9), 4194-4198. (38) Szoka, F.; Olsen, F.; Heath, T.; Vail, W.; Mayhew, E.; Papahadjopoulos, D. Biochim. Biophys. Acta 1980, 601, 559-571. (39) O’Connell, J. P.; Campbell, R.-L.; Fleming, B. M.; Mercolino, T. J.; Johnson, M. D.; McLaurin, D. A. Clin. Chem. 1985, 31, 1424-1426.
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SH-derivatized antibody on a Sepharose CL-4B column equilibrated with TBS (pH 7.0) containing sucrose. The desired fraction of liposomes was collected, followed by dialysis at 4 °C in the dark for improved stability. Stability Study and Characterization of Liposomes. The intactness of lipsomes means the maintenance of their integrity, which can be determined by measuring fluorescence from sulforhodamine B before and after lysis. According to our previous studies,35 almost instantaneous lysis of liposomes was observed at room temperature when a solution of n-octyl β-D-glucopyranoside (OG) was added; total lysis of the liposomes was achieved by addition of OG to a final concentration of 50 mM. For these fluorescence tests, the SRB dye was excited at 544-nm wavelength and the fluorescent emission intensity measurements were made at 596-nm wavelength. The diameter of the liposomes was measured with a Coulter N4 particle size analyzer (Coulter Corp., Miami, FL) using the manufacturer’s method. Preparation of Test Strips and Assay Procedures. A microprocessor-controlled TLC sample applicator, Linomat IV (Camag Scientific Inc. Wrightsville Beach, NC), was used to immobilize anti-E. coli O157:H7 antibodies and anti-biotin antibodies on the plastic-backed nitrocellulose membranes. The membrane was cut into 10 × 8.8 cm sheets, which were then mounted on a mobile platform that moved at a constant rate (5 s/µL) in front of the air brush used to spray the antibody solution at concentration of 2 mg/mL, leading to a final concentrated of 4 µg of antibody/strip. The antibody-coated bands were allowed to air-dry in the hood for 5 min and further dried under vacuum (10 psi) at room temperature for 1.5 h. The coated nitrocellulose sheet was then immersed in a blocking solution consisting of 0.5% poly(vinylpyrrolidone) and 0.03% casein in Tris-buffered saline (TBS) at pH 7.0 for 60 min on a rotating shaker, followed by drying under vacuum (10 psi) at room temperature for at least 6 h. The prepared sheets were then cut into 0.5 × 8.8 cm strips using a paper cutter, producing strips with the antigen capture (AC) zone 1.5 cm and biotin capture (BC) zone 3.5 cm above the bottom of the strip. The prepared strips were packed with desiccants and stored at 4 °C until use. The format for the newly developed assay consists of immunoliposome solution, test sample, and a nitrocellulose test strip with immobilized anti-E. coli O157:H7 antibodies and anti-biotin antibodies. The assay was performed by dispensing 90 µL of the sample in PBS and 50 µL of an immunoliposome solution into a 10 × 75-mm glass test tube with gentle mixing for 30 min. A test strip was then inserted into the test tube, and the chromatographic process was begun. After the solution front reached the upper end of the test strip, it was removed and air-dried. The color intensity of each zone on the test strip was estimated visually or quantified by scanning reflectance photometer (KGW Enterprises, Inc., Elkhart, IN). Selectivity of Immunoliposomes. The evaluation of the cross-reactivity of immunoliposomes with the target E. coli O157: H7 and negative controls, such as Salmonella typhimurium and Listeria genus specific, was conducted. It was done by inserting the antibody-coated test strips into the test tubes, which contained immunoliposomes and the target antigen or negative controls. The color intensity of each zone on the test strip was estimated visually or quantified by scanning densitometry.
Table 1. Characteristics of the Liposomes mean diameter (nm) volume of liposome (µL) liposome concentration (number/mL) SRB concentration (mM) SRB molecules per liposome antibody molecules on the liposome surface stability (weeks) at 4 °C in the dark.
250 ( 29 8.18 × 10-12 1.126 × 1013 95 4.2 × 105 4800 >40
Safety Considerations. E. coli O157:H7 is a harmful bacteria that should be handled with care. Heat-killed E. coli O157:H7, other pathogenic organisms, and organic solvents for use in the modification and production of the conjugated liposomes and the performance of the assay were handled either in a laminar flow hood or chemical hood with surgical gloves. All E. coli O157:H7 or other harmful pathogen-contaminated labwares was autoclaved before being discarded. RESULTS AND DISCUSSION Preparation of Multivalent Immunoliposomes. A multistep conjugation procedure for the preparation of anti-E. coli O157:H7 antibody-tagged liposomes (immunoliposomes) was demonstrated. Sulfo-KMUS, containing an extended aliphatic spacer, is a sulfhydryl-reactive and amine-reactive heterobifunctional cross-linker, which conjugates thiolated IgG and amine group of DPPE on the liposome bilayer. The maleimide group of sulfo-KMUS is highly specific for coupling to sulfhydryl-containing molecules, thus directing the conjugation to discrete points on the second molecules. In comparison with SMCC produced immunoliposomes,28 it was concluded that sulfo-KMUS formed more stable complexes that survive for a longer period of time (>40 weeks). Characteristics of Immunoliposomes. Having a narrow size range for the tagged liposomes was extremely important in order to obtain an even capillary migration on the test strips. Extrusion of the liposome preparations through polycarbonate filters was found to reduce the size heterogeneity. Liposomes passed 20 times through a 0.4-µm polycarbonate filter had a mean diameter of 250 nm with a standard deviation of 29 nm. The 250-nm liposomes were used in all subsequent experiments. The characteristics of liposomes are listed in Table 1. With liposomes of 250-nm diameter it is possible to calculate that the average volume of a single liposome is 8.18 × 10-12 µL and the volume entrapped (assuming a bilayer thickness of 4 nm) is 7.42 × 10-12 µL. Assuming the dye concentration inside of the liposomes was equal to the original dye solution used, and comparing the fluorescence of lysed liposomes to that of standard SRB solutions, it is possible to calculate that there were ∼1.13 × 1013 liposomes/mL and that each liposome contained ∼4.20 × 105 molecules of dye. If the average surface area of the DPPC molecules is 71 Å, and that of cholesterol is 19 Å,40 it was estimated that ∼4800 molecules of anti-E. coli O157:H7 antibody were on the outer surface of each liposome given that 2.5 mol % of sulfo-KMUS-derivatized liposomal DPPE successfully reacted with SH-derivatized IgG. Although the high-load particles may cause aggregation of antigens with changes of their flow properties, it was not observed in the current study. (40) Israelchvili, J. N.; Mitchell, D. J. A. Biochim. Biophys. Acta 1975, 389, 1319.
Figure 1. Sandwich binding of E. coli O157:H7 between the immunoliposomes and anti-E. coli O157:H7 antibodies immobilized in the AC zone.
Figure 2. Scanned image of the assay performed with the different concentrations of heat-killed E. coli O157:H7. Samples A contains no E. coli O157:H7; (B) 6 × 102 cells, (C) 6 × 103 cells, (D) 6 × 104 cells, (E) 6 × 105 cells, (F) 6 × 106 cells, and (G) 6 × 107 cells.
Assay Performance. The concept of the assay (as shown in Figure 1) is to have an immobilized antibody zone (AC zone) in the membrane strip that is exposed to target antigen, E. coli O157: H7, in a sample solution. Multivalent immunoliposomes subsequently bind to the bound antigen in the AC zone, while free, unbound immunoliposomes continue to migrate to the BC zone. Thus, the color exhibited by the bound immunoliposomes on the AC zone is directly proportional to E. coli O157:H7 present in the sample, and the color exhibited on the BC zone serves as a positive control for the test trips, as shown in Figure 2. Sulforhodamine is not only a fluorophore compound but also a visible dye. Therefore, color intensity may be measured semiquantitatively by visual examination, but the use of a QuadScan reflectance photometer (KGW Enterprises, Inc.), which is equipped with a high-intensity incandescent lamp to illuminate specific zones to give a reflectance value, can provide more accurate quantitation results obtained with various concentrations of E. coli O157:H7, Analytical Chemistry, Vol. 75, No. 16, August 15, 2003
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Figure 3. Dose-response curve of heat-killed E. coli O157:H7.
as shown in Figure 3. The limit of detection estimated at 2 SD was determinated to be ∼2500 cells for heat-killed E. coli O157: H7 with a 95% confidence. To evaluate the specificity of the assay, E. coli O157:H7 and negative controls at 108-109 cells/mL were tested. As shown in Figure 4, the assays did not show any significant cross-reactivity to the nonspecific bacteria (negative controls). The detection limits of other immunobiosensing devices reported previously for E. coli O157:H7 are ∼6 × 103 cells/mL using electrochemical impedance spectroscopy,41 103 cells/mL using flow cytometry.42 However, the assay system developed here has the advantages of low cost, simplicity, and speed over other existing assay systems which can be a potential alternative method for field screening of contaminated food samples. CONCLUSIONS The newly developed assay does not need washing and incubation steps as in enzyme-linked immunosorbent assays and can be completed in 5 min. Those immunoliposomes successfully demonstrate their feasibility for use in a sandwich immunoassay that has potential as a simple, rapid, and inexpensive test for quantitative screening of food samples for E. coli O157:H7 with (41) Ruan, C.; Yang, L.; Li, Y. Anal. Chem. 2002, 74, 4814-4820. (42) Kusunoki, H.; Latiful Bari, M.; Kita, T.; SugII, S.; Uemura, T. J. Vet. Med. B 2002, 47, 551-559.
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Figure 4. Specificity of immunoliposomes to E. coli O157:H7. Scanned image of the assay performed with (A) the heat-killed of Salmonella tryphimurium (4 × 107 cells), (B) Listeria genus specific (4 × 107 cells), and (C) E. coli O157:H7 (6 × 107 cells).
densitometry. Future studies will focus on the development of the flow injection liposome immunoanalysis system and the investigation of a variety of food samples implicated in E. coli O157:H7 outbreaks. ACKNOWLEDGMENT The authors thank Dr. Richard A. Durst and Dr. Antje Ba¨eumner of Cornell University for valuable suggestions. This work was supported by National Science Council in Taiwan, ROC under Grant NSC 91-2113-M-260-011.
Received for review April 8, 2003. Accepted May 27, 2003. AC0343580
