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Microbead-Based Immunoassay for Simultaneous Detection of Shiga Toxins and Isolation of Escherichia Coli O157 in Foods

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Microbead-Based Immunoassay for Simultaneous Detection of Shiga Toxins and Isolation of Escherichia Coli O157 in Foods 373 Journal of Food Protection, Vol. 74, No. 3, 2011, Pages 373–379 doi:10.4315/0362-028X.JFP-10-344 Microbead-Based Immunoassay for Simultaneous Detection of Shiga Toxins and Isolation of Escherichia coli O157 in Foods LAURIE M. CLOTILDE,1 CLAY BERNARD IV,1 GARY L. HARTMAN,2 DAVID K. LAU,2 AND J. MARK CARTER1* 1Western Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, Albany, California 94710; and 2 U.S. Food and Drug Administration, Alameda, California 94502, USA Downloaded from http://meridian.allenpress.com/jfp/article-pdf/74/3/373/1685091/0362-028x_jfp-10-344.pdf by guest on 30 September 2021 MS 10-344: Received 9 August 2010/Accepted 12 November 2010 ABSTRACT Shiga toxin–producing Escherichia coli (STEC) is a significant foodborne pathogen with great economic consequences. There has been an increased food safety concern with this organism since outbreaks of human illnesses caused by this pathogen were first reported in 1982. Therefore, developing a reliable, sensitive, and rapid assay capable of detecting E. coli O157 and the main toxins produced by STEC (i.e., Shiga toxins 1 [Stx1] and 2 [Stx2]) will directly benefit regulatory agencies by minimizing analysis time. Here, we use Luminex technology to detect multiple analytes in a single 50-ml sample. Using commercially available monoclonal antibodies coupled to carboxylated magnetic microbeads, we developed an immunoassay capable of simultaneously serotyping E. coli O157 and detecting Stx1 and/or Stx2. The specificity and sensitivity of this immunoassay was tested against a collection of 34 E. coli isolates belonging to various O serogroups phenotypically different for Stx. The results were compared with microplate sandwich enzyme-linked immunosorbent assay (ELISA), and no cross-reactivity was observed for any of the monoclonal antibodies used. An increased sensitivity up to 1,000 times was observed in the microbead-based immunoassay when compared with the microplate sandwich ELISA. The results indicate that Luminex technology has the potential to simultaneously detect multiple targets without loss of specificity and/or sensitivity. A blind experiment was conducted with 48 samples of ground beef, lettuce, and milk spiked with #2 CFU/g E. coli. All the samples were correctly identified, with no false positives or false negatives. This microbead-based immunoassay could be extended to simultaneously detect additional foodborne pathogens and their toxic markers. Shiga toxin–producing Escherichia coli (STEC) com- In recent years, research has turned toward molecular prises significant foodborne pathogens with high economic technologies to meet the need for rapid and multiplexed impact. In the United States, recent annual cost estimates for detection. For example, two-dimensional microarrays have acute care ranged from $1 to $2 billion, based on the the ability to simultaneously detect hundreds of specific assumption that E. coli O157 caused 73,000 illnesses and 61 probes, but suspension microbead arrays such as Luminex deaths, and non-O157 STEC caused more than 36,000 can offer more flexibility and cost-effectiveness (16). illnesses (34, 41). Although beef products, especially in the Briefly, Luminex technology is a multiplexed microbead- ground form, remain the main source of infection (47), other based sensing system capable of analyzing (theoretically) up documented sources include green leafy vegetables (8, 9), to 100 different binding reactions per sample (14, 15). This and dairy products (10, 11) contaminated with STEC- technology utilizes microbeads internally stained with infected cattle feces (48). The pathogenesis of human different ratios of red and infrared fluorophores to create infections with STEC depends on the production of AB5 100 unique spectral addresses (27). Assays using this toxins called Shiga toxins (Stx1 and Stx2) (36, 39). STEC (5, technology can be designed in a sandwich format, wherein 32, 52) have often been linked to outbreaks of human antigens are captured by monoclonal antibodies (mAb) that illnesses, and most of these are traced to consumption of E. are covalently immobilized on the surface of the micro- coli O157–contaminated foods (7, 11, 51). Currently, there beads. The bound analytes are detected with secondary mAb are no commercially available kits capable of detecting E. labeled with biotin and a phycoerythrin reporter. The coli O157 and its two main virulence factors (i.e., Stx1 and Luminex analyzer instrument is then used to quantify the Stx2) simultaneously in foods. Thus, this study focused on amount of bound analyte and to qualify the mixture of this specific STEC serotype as well as the main virulence unique microbeads, allowing multiplexed interrogation. The factors. specificity of the mAb used allows differentiation of closely related analytes, as evidenced earlier by the serotyping of bacteria (2) and viruses (24). A microbead-based immuno- * Author for correspondence. Tel: 510-559-6053; Fax: 510-559-6429; assay presents several advantages (26, 37, 38), which E-mail: [email protected]. include (i) multiplex format, (ii) reduced cost of analysis as 374 CLOTILDE ET AL. J. Food Prot., Vol. 74, No. 3 the degree of multiplexing increases, (iii) rapid data TABLE 1. Shiga toxin–producing Escherichia colia acquisition, and (iv) excellent sensitivity and specificity. b b Strain no. Serotype Stx1 Stx2 In addition, the large surface area of the microbeads allows coupling of up to 100,000 capture mAb per microbead, thus 1 O26:H11 22 allowing high density of capture mAb, with increased 2 O26 22 antigen binding and enhanced sensitivity of the immuno- 3 O26 z 2 z assay (33). 4 O45:H2 2 5 O45:H61 22 In this article, we describe a microbead-based immu- 6 O45 z 2 noassay for the simultaneous detection of E. coli O157, 7 O45 z 2 Stx1, and/or Stx2. We first compared the specificity and 8 O45 2 z sensitivity of Luminex technology with that of the standard 9 O91 z 2 sandwich enzyme-linked immunosorbent assay (ELISA). 10 O91 2 z Then, the test procedure was evaluated with ground beef, 11 O91 22 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/74/3/373/1685091/0362-028x_jfp-10-344.pdf by guest on 30 September 2021 lettuce, and milk samples spiked with individual serogroups 12 O103:H8 22 13 O103:H38 22 of E. coli expressing various combinations of Stx1 and Stx2. 14 O103 z 2 MATERIALS AND METHODS 15 O111:H8 z 2 16 O111:H21 22 mAb and analytes. The mAb used were anti-Stx1 (Sifin 17 O111:H21 22 GmbH, Berlin, Germany), anti-Stx2 (Sifin GmbH), and anti–E. coli 18 O121:H19 2 z O157 lipopolysaccharide (LPS; OEM Concepts, Saco, ME). When 19 O121:NMc 2 z we prescreened via capture ELISA, we identified only one good- 20 O121:NM 2 z quality mAb against Stx1, which happened to be specific for the B 21 O121 2 z subunit (data not shown). As the tertiary structure of Stx protein 22 O121 22 includes five copies of the B subunit, the anti-Stx1B mAb works for 23 O128:H2 22 both capture and detection. This same strategy applies to the anti– 24 O128:H7 22 E. coli O157 LPS mAb. Even after partial lysis by polymyxin, 25 O128:H21 22 bacteria exhibit many copies of surface-accessible LPS, so the 26 O145:H28 22 same mAb can be used for both capture and detection. For Stx2, 27 O145:NM 2 z two different mAb were used, one specific to the A subunit for 28 O145 2 z detection and one specific to the B subunit for capture. The anti- 29 O157:H7 22 Stx2 mAb used had been previously tested against Stx2 and 30 O157:H7 2 z its variants (55). The results showed it recognized Stx2c, Stx2d, 31 O157:H7 zz Stx2d-activatable, and Stx2e, but not Stx2f, which is only rarely 32 O157:H7 zz associated with human disease (18, 45). Detector mAb were 33 O157:H7 zz biotinylated with the EZ-Link Sulfo-NHS-Long Chain-Biotin kit 34 O157:H7 zz (Pierce, Rockford, IL), according to the manufacturer’s instruc- a tions. With regard to analytes, #2 CFU/ml or g of each E. coli Drs. Robert Mandrell and Beatriz Quin˜ones (USDA-ARS, strain tested were grown in 50 ml of brain heart infusion broth Produce Safety Microbiology Research Unit, Albany, CA) (BHIB; Oxoid, Ltd., Basingstoke, UK) or spiked into 50 g of food. provided these E. coli strains. Unless otherwise specified, only Samples were then incubated overnight (15 h) in a shaking the serogroup of those strains were identified by serological identification. incubator (100 rpm) at 37uC. After enrichment, the samples were b serially diluted to determine the sensitivity of the immunoassay. The Stx genotypes were previously determined by PCR (data not Bacterial counts for each sample were established by plate count shown) by Drs. Robert Mandrell and Beatriz Quin˜ones, and the on tryptic soy agar (Oxoid, Ltd.). The 50-ml samples were then phenotypes were assessed by a verocytotoxicity assay (data treated with 2.5 ml of 50,000 U/ml polymyxin B?sulfate (Axxora, shown in Table 1). c The strain was not motile for the H antigen, i.e. ‘‘NM.’’ L.L.C., San Diego, CA) for 30 min in a shaking incubator (100 rpm) at 37uC. Highly concentrated (i.e., undiluted) samples were used to establish the point at which binding sites on the 0.1% Tween 20 (PBS-T) was used for washes. Reagent-grade microbeads were fully saturated and to explore immunoassay chemicals (i.e., 2,29-azino-bis(3-ethylbenzthiazoline-6-sulfonic ac- cross-reactivity and nonspecific binding. Samples were tested by id) [ABTS], H2O2, KCl, Na3 citrate, NaCl, and Tween 20) were both conventional microplate sandwich ELISA and by microbead- purchased from Sigma Chemical Co.
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