A novel Shiga toxin 2a neutralizing antibody therapeutic with low immunogenicity and high efficacy.

Shiga toxin-producing Escherichia coli infections are difficult to treat due to the risk of antibiotic-induced stress upregulating the production of toxins, medical treatment is consequently limited to supportive care to prevent the development of hemolytic uremic syndrome (HUS). Here, we introduce a potentially therapeutic humanized mouse monoclonal antibody (Hu-mAb 2-5) targeting Stx2a, the most common Shiga toxin subtype identified from outbreaks. We demonstrate that Hu-mAb 2-5 has low immunogenicity in healthy adults ex vivo and high neutralizing efficacy in vivo, protecting mice from mortality and HUS-related tissue damage.

An in vitro combined antibiotic-antibody treatment eliminates toxicity from Shiga toxin-producing Escherichia coli.

Treating Shiga toxin-producing Escherichia coli (STEC) gastrointestinal infections is difficult. The utility of antibiotics for STEC treatment is controversial, since antibiotic resistance among STEC isolates is widespread and certain antibiotics dramatically increase the expression of Shiga toxins (Stxs), which are some of the most important virulence factors in STEC. Stxs contribute to life-threatening hemolytic uremic syndrome (HUS), which develops in considerable proportions of patients with STEC infections. Understanding the antibiotic resistance profiles of STEC isolates and the Stx induction potential of promising antibiotics is essential for evaluating any antibiotic treatment of STEC. In this study, 42 O157:H7 or non-O157 STEC isolates (including the "big six" serotypes) were evaluated for their resistance against 22 antibiotics by using an antibiotic array. Tigecycline inhibited the growth of all of the tested STEC isolates and also inhibited the production of Stxs (Stx2 in particular). In combination with neutralizing antibodies to Stx1 and Stx2, the tigecycline-antibody treatment fully protected Vero cells from Stx toxicity, even when the STEC bacteria and the Vero cells were cultured together. The combination of an antibiotic such as tigecycline with neutralizing antibodies presents a promising strategy for future STEC treatments.

Safe and effective means of detecting and quantitating Shiga-like toxins in attomole amounts.

Shiga-like toxins (verotoxins) are a class of AB5 holotoxins that are primarily responsible for the virulence associated with Shiga-like toxin producing Escherichia coli (STEC) infections. The holotoxins are composed of a pentamer of identical subunits (B subunit) responsible for delivering the catalytic subunit (A subunit) to a host cell and facilitating endocytosis of the toxin into the cell. The B subunits are not associated with toxicity. We developed a multiple reaction monitoring method based on analyzing conserved peptides, derived from the tryptic digestion of the B subunits. Stable-isotope-labeled analogues were prepared and used as internal standards to identify and quantify these characteristic peptides. We were able to detect and quantify Shiga toxins (Stx), Shiga-like toxin type 1 (Stx1) and type 2 (Stx2) subtypes, and to distinguish among most of the known subtypes. The limit of detection for digested pure standards was in the low attomole range/injection (~10 attomoles), which corresponded to a concentration of 1.7 femtomol/mL. A matrix effect was observed when dilute samples were digested in the buffer, Luria broth, or mouse plasma (LOD ~ 30 attomol/injection = 5 femtomol/mL). In addition, we determined that the procedures necessary to perform our mass spectrometry-based analysis completely inactivate the toxins present in the sample. This is a safe and effective method of detecting and quantitating Stx, Stx1, and Stx2, since it does not require the use of intact toxins.

Validation of a Cell-Based Assay for Detection of Active Shiga Toxins Produced by Escherichia coli in Water.

Shiga toxin-producing Escherichia coli (STEC) causes a wide spectrum of diseases, including hemorrhagic colitis and hemolytic uremic syndrome (HUS). Almost 5% of STEC infections result from waterborne exposures, yet there is no test listed in the EPA's current Selected Analytical Methods for the detection of active Shiga toxins (Stxs) in water. In this study, a HeLa cell-based assay is validated for the detection of metabolically active Stxs produced by STEC in water, including tap, bottled, and pond water. Active Stxs are detected even when the number of Stx-producing bacteria is less than 0.4 CFU/mL and the assay performance is not affected by background flora or chlorine in the water. This assay is not only as simple and affordable as cell-free assays but also detects active holotoxins without the use of live animals. In addition, the assay is designed for use in multi-well formats, making it ideal for high-throughput screening of water samples and therefore useful for environmental public health surveillance programs to reduce human risk of infection with STEC.

Particulate Shiga Toxin 2 in Blood is Associated to the Development of Hemolytic Uremic Syndrome in Children.

Hemolytic uremic syndrome (HUS), the leading cause of acute renal failure in children (< 3 years), is mainly related to Shiga toxins (Stx)-producing Escherichia coli (STEC) infections. STEC are confined to the gut resulting in hemorrhagic colitis, whereas Stx are delivered in blood to target kidney and brain, with unclear mechanisms, triggering HUS in 5 to 15% of infected children. Stx were found on circulating cells, free in sera (soluble Stx) or in blood cell-derived microvesicles (particulate Stx), whereby the relationship between these forms of circulating toxins is unclear. Here, we have examined 2,846 children with bloody diarrhea and found evidence of STEC infection in 5%. Twenty patients were enrolled to study the natural course of STEC infections before the onset of HUS. In patients, Stx were found to be associated to circulating cells and/or free and functionally active in sera. In most children, Stx were bound to neutrophils when high amounts of toxins were found in feces. Time-course analysis showed that Stx increased transiently in patients' sera while the decrease of toxin amount on leukocytes was observed. Notably, patients who recovered (85%) displayed different settings than those who developed HUS (15%). The distinctive feature of the latter group was the presence in blood of particulate Stx2 (Stx2 sedimented at g-forces corresponding to 1 µm microvesicles) the day before diagnosis of HUS, during the release phase of toxins from circulating cells. This observation strongly suggests the involvement of blood cell-derived particulate Stx2 in the transition from hemorrhagic colitis to HUS.

Development of novel antibodies for detection of mobile colistin-resistant bacteria contaminated in meats.

The recent discovery and rapid spread of mobile colistin-resistant gene, mcr-1, among bacteria isolated from a broad range of sources is undermining our ability to treat bacterial infections and threatening human health and safety. To prevent further transfer of colistin resistance, practical and reliable methods for mcr-1-containing bacteria are need. In this study, standards and novel polyclonal and monoclonal antibodies (mAbs) against MCR-1 were developed. Among nine mAbs, three were MCR-1 specific and six cross-reacted with both MCR-1 and MCR-2. A sandwich enzyme-linked immunosorbent assay (ELISA) was established using the polyclonal antibody as a capturer and the mAb MCR-1-7 as a detector. The assay had a limit of detection of 0.01 ng/mL for MCR-1 and 0.1 ng/mL for MCR-2 in buffer with coefficients of variation (CV) less than 15%. When applied to ground beef, chicken and pork, this ELISA identified samples inoculated with less than 0.4 cfu/g of meat, demonstrating its strong tolerance to complex food matrices. To our knowledge, this is the first immunoassay developed for MCR-1 and MCR-2. It should be useful for prompt and reliable screening of meat samples contaminated with plasmid-borne colistin-resistant bacteria, thus reducing human risk of foodborne infections with possibly no antibiotic treatment options.

An Improved Method for the Sensitive Detection of Shiga Toxin 2 in Human Serum.

Shiga toxins (Stx) released by Stx-producing E. coli (STEC) are virulence factors that are most closely 3associated with hemolytic uremic syndrome (HUS), a life-threatening complication of intestinal infections by STEC. Stx have to enter into the circulatory system before they are delivered to target organs and cause damage. The presence of Stx in sera could be a risk indicator for HUS development. However, the detection of Stx, particularly Stx2, has been difficult due to the presence of Stx2-binding components in human serum. Here, we report new ELISA-based methods for the detection of Stx1 and Stx2 in human serum and the effect of guanidinium chloride on enhancing the sensitivity for the detection of Stx2. The recovery rate for Stx2 was 62% when Stx2-spiked serum samples were treated with guanidinium chloride at a concentration of 200 mM, in contrast to 17% without guanidinium chloride treatment. The effectiveness of guanidinium chloride treatment for the detection of Stx2 in human serum was validated using sera from STEC-infected patients. Coimmunoprecipitation results indicated a specific physical interaction between Stx2 and the human serum amyloid P component (HuSAP) in human serum samples. Our in vitro study demonstrated that the inhibition from HuSAP alone for the detection of Stx2 was only 20%, much less than 69.6% from human serum at Stx2 level 10 ng/mL, suggesting that there may be other factors that bind Stx2 in human serum. This study indicates that treatment of serum samples with guanidinium chloride may be useful for the early and sensitive detection of Stx2 in sera of STEC-infected patients, so preventive measures can be adopted in a timely manner.

Novel monoclonal antibodies against Stx1d and 1e and their use for improving immunoassays.

Shiga toxins (Stxs) are major causative agents for bloody diarrhea and hemolytic uremic syndrome, a life-threatening disease in humans. No effective treatment is available. Early detection of Stxs in clinical samples is critical for disease management. As bacteria evolve, new Stxs are produced; therefore, methods used to identify them need to be improved as well. In this study, new monoclonal antibodies (mAbs) against Stx1d and 1e were developed and used to improve a commercial Stx1 kit. Incorporation of the new mAbs into the Abraxis Stx1 kit not only increased the assay sensitivity to Stx1d, but the assay was conferred the ability to detect Stx1e, a newly identified subtype of Stx1 produced by an atypical Stx-producing bacterial strain, Enterobacter cloacae M12X01451, isolated from a clinical specimen. This toxin was not detectable using existing commercial kits. The signal to noise ratio (s/n) of the new assay was increased 3-fold for Stx1d, and 44-fold for Stx1e at toxin concentration of 10ng/mL. The limit of detection (LOD) was 10pg/mL for Stx1a, and 100pg/mL for Stx1c, 1d and 1e. When used for bacterial strains, the sensitivity of the new assay was improved 2.5- to 60-fold depending on subtypes produced. In summary, high affinity mAbs against Stx1d and 1e were developed and incorporation of these mAbs into the Stx1 kit significantly enhanced the assay sensitivity and broadened the subtype-specificity. This improvement should be useful for reducing product recalls and disease mistreatment due to failures of detecting less common but clinically important subtypes of Stxs.

Detection of Abrin Holotoxin Using Novel Monoclonal Antibodies.

Abrin, a member of the ribosome-inactivating protein family, is produced by the Abrus precatorius plant. Having the potential to pose a severe threat to both human and animal health, abrin is classified as a Select Agent by the U.S. Department of Health and Human Services. However, an immunoassay that is specific for intact abrin holotoxin has not yet been reported. In this study, seven new monoclonal antibodies (mAbs), designated as Abrin-1 through Abrin-7 have been developed. Isotyping analyses indicate these mAbs have IgG1, IgG2a, or IgG2b heavy-chains and kappa light-chains. Western blot analyses identified two abrin A-chain specific mAbs, Abrin-1 and Abrin-2, and four B-chain specific mAbs (Abrin-3, -5, -6, and -7). A sandwich enzyme-linked immunosorbent assay (ELISA), capable of detecting a mixture of abrin isoforms and agglutinins was developed using B-chain specific Abrin-3 for capture and A-chain specific Abrin-2 as detector. The ELISA is highly sensitive and detects 1 ng/mL of the abrin holotoxin in phosphate-buffered saline, nonfat milk, and whole milk, significantly below concentrations that would pose a health concern for consumers. This ELISA also detects native abrin in plant extracts with a very low background signal. The new abrin mAbs and ELISA should be useful for detecting this potent toxin in the milk supply chain and other complex matrices.

New Monoclonal Antibodies against a Novel Subtype of Shiga Toxin 1 Produced by Enterobacter cloacae and Their Use in Analysis of Human Serum.

Shiga toxin (Stx) is a major virulence factor of several bacterial pathogens that cause potentially fatal illness, including Escherichia coli and Shigella spp. The continual emergence of new subtypes of Stxs presents challenges for the clinical diagnosis of infections caused by Stx-producing organisms. Here, we report the development of four new monoclonal antibodies (MAbs) against Stx1e, a novel subtype of Stx1 that was produced by an Enterobacter cloacae strain and had limited reactivity with existing anti-Stx1 antibodies. Western blot analysis indicates that these MAbs were Stx1 specific, bound to the A subunit, and had distinct preferences for subtypes of Stx1. Of the four MAbs, Stx1e-2 was capable of partially neutralizing cytotoxicities derived from Stx1e in Vero cells. Enzyme-linked immunosorbent assays assembled with these high-affinity MAbs detected Stx1e at concentrations as low as 4.8 pg/ml in phosphate-buffered saline and 53.6 pg/ml in spiked human serum samples and were also capable of distinguishing Stx1e-producing strains in enriched cultures. These assays may therefore have clinical value in diagnosing Stx1e-producing bacterial infection. Additionally, characteristics of Stx1e, such as the origin of stx1e genes, conditions for toxin expression, receptor binding, and cytotoxicity, were investigated with the new antibodies developed in this study. This information should be useful for further understanding the clinical significance and prevalence of Stx1e-harboring E. cloacae and other organisms. IMPORTANCE Stxs are among the most clinically important virulence factors of Shigella and enterohemorrhagic Escherichia coli. There are many varieties of Stx, and although Stx1a and Stx2a are the most common and widely distributed types of Stx, new variants of Stx are continually emerging. These new variants of Stx can be challenging to detect, since most Stx detection kits are optimized for the detection of Stx1a and Stx2a. Stx1e, recently discovered in an atypical host (Enterobacter cloacae), is undetectable by many Stx assays. To formulate new assays for the detection of Stx1e, we generated four new MAbs that recognize this Stx subtype. Using these antibodies, we generated an assay capable of detecting Stx1e at low picogram-per-milliliter concentrations. This assay is also compatible with a human serum matrix, suggesting that it may have utility for the clinical detection and diagnosis of Stx1e-associated infections.

A New Immunoassay for Detecting All Subtypes of Shiga Toxins Produced by Shiga Toxin-Producing E. coli in Ground Beef.

BACKGROUND: Shiga toxin (Stx) is a common virulence factor of all Shiga toxin producing E. coli (STEC) that cause a wide spectrum of disease, including hemorrhagic colitis and hemolytic uremic syndrome (HUS). Although several commercial kits are available for detection of Stx produced by STEC, none of them are capable of recognizing all subtypes of Stxs, which include three subtypes of Stx1 and seven subtypes of Stx2. METHODS AND FINDINGS: New monoclonal and polyclonal antibodies against Stx1 and Stx2 were developed. A universal sandwich ELISA capable of detecting all known subtypes of Stx1 and Stx2 was established using a pool of newly developed antibodies. To precisely monitor the sensitivity of the assay for each subtype of Stxs, recombinant toxoids were created and used as standards in ELISAs. Because of the high affinity of the antibodies incorporated, the ELISA assay is highly sensitive with a limit of detection for the different subtypes of Stx1a and Stx2a between 10 and 50 pg/mL in phosphate buffered saline (PBS). The assay was also able to identify STEC based on the production of Stxs using the supernatants of culture fluids or even single colonies on agar plates without lengthy enrichment in liquid medium. When applied to ground beef samples, this newly developed ELISA was capable of distinguishing beef samples spiked with a single bacterial cell. CONCLUSIONS: A highly sensitive and universal assay for all subtypes of Stx1 and Stx2 was developed. It has significantly improved upon the current technologies by avoiding false negative results due to the narrow detection range of the assay. The assay developed in this study can be useful for prompt detection of new and emerging serotypes and screening ground beef samples for contamination of STEC at an early stage in the food supply chain, thus avoiding the need for possible recall.

Phage-mediated Shiga toxin (Stx) horizontal gene transfer and expression in non-Shiga toxigenic Enterobacter and Escherichia coli strains.

Enterobacter cloacae M12X01451 strain recently identified from a clinical specimen produces a new Stx1 subtype (Stx1e) that was not neutralized by existing anti-Stx1 monoclonal antibodies. Acquisition of stx by Ent. cloacae is rare and origin/stability of stx1e in M12X01451 is not known. In this study, we confirmed the ability of Stx1a- and Stx1e-converting phages from an Escherichia coli O157:H7 strain RM8530 and M12X01451 respectively to infect several E. coli and Ent. cloacae strains. stx1e was detected in 97.5% and 72.5% of progenies of strains lysogenized by stx1e phage after 10 (T10) and 20 (T20) subcultures, versus 65% and 17.5% for stx1a gene. Infection of M12X01451 and RM8530 with each other's phages generated double lysogens containing both phages. stx1a was lost after T10, whereas the stx1e was maintained even after T20 in M12X01451 lysogens. In RM8530 lysogens, the acquired stx1e was retained with no mutations, but 20% of stx1a was lost after T20 ELISA and western blot analyses demonstrated that Stx1e was produced in all strains lysogenized by stx1e phage; however, Stx1a was not detected in any lysogenized strain. The study results highlight the potential risks of emerging Stx-producing strains via bacteriophages either in the human gastrointestinal tract or in food production environments, which are matters of great concern and may have serious impacts on human health.

Immuno-PCR Assay for Sensitive Detection of Proteins in Real Time.

The immuno-PCR (IPCR) assay combines the versatility and robustness of immunoassays with the exponential signal amplification power of the polymerase chain reaction (PCR). Typically, IPCR allows a 10-1,000-fold increase in sensitivity over the analogous enzyme-linked immunosorbent assay (ELISA). This is achieved by replacing the signal-producing antibody-enzyme conjugate of an ELISA with an antibody-DNA conjugate that serves as a marker for PCR amplification. The amplification power of the PCR allows for the detection of even single molecules of nucleic acid templates, making it well suited for a broad range of applications. Here, we describe the application of an IPCR assay for detection of trace amount of antigens using ricin as an example.

New Stx2e Monoclonal Antibodies for Immunological Detection and Distinction of Stx2 Subtypes.

BACKGROUND: Stx2e is a primary virulence factor in STEC strains that cause edema disease in neonatal piglets. Though Stx2a and Stx2e are similar, many antibody-based Stx detection kits are designed to detect Stx2a and do not recognize the Stx2e subtype. METHODS AND FINDINGS: Four monoclonal antibodies against Stx2e were developed and characterized. Two of these mAbs recognize the B subunit of Stx2e, Stx2f, and to a lesser extent, Stx2b, Stx2c, and Stx2d. The other two mAbs recognize the A subunit of Stx2e, and cross-react with all Stx2 subtypes except Stx2f. The most sensitive sandwich ELISA using these mAbs has a limit of detection for Stx2e of 11.8 pg/mL. The ability of the neutralizing antibody Stx2e-2 to block Stx2e-receptor binding in Vero cells was visualized using immunofluorescence. Combinations of these and previously developed mAbs permit ELISA-based differentiation between closely related Stx2a, Stx2c, and Stx2d (using mAbs Stx2-5/2-1, Stx2-5/2e-2, and Stx2e-3/2e-2, respectively). CONCLUSIONS: The sensitive immunoassays developed in this study should augment our capacity to detect Stx2e in porcine environments and biological samples. Moreover, immunoassays that can distinguish between the closely related Stx2a, Stx2c, and Stx2d subtypes can be useful in quickly analyzing Stx subtypes in samples containing more than one strain of STEC.

Mass Spectrometry-Based Method of Detecting and Distinguishing Type 1 and Type 2 Shiga-Like Toxins in Human Serum.

Shiga-like toxins (verotoxins) are responsible for the virulence associated with a variety of foodborne bacterial pathogens. Direct detection of toxins requires a specific and sensitive technique. In this study, we describe a mass spectrometry-based method of analyzing the tryptic decapeptides derived from the non-toxic B subunits. A gene encoding a single protein that yields a set of relevant peptides upon digestion with trypsin was designed. The (15)N-labeled protein was prepared by growing the expressing bacteria in minimal medium supplemented with (15)NH4Cl. Trypsin digestion of the (15)N-labeled protein yields a set of (15)N-labeled peptides for use as internal standards to identify and quantify Shiga or Shiga-like toxins. We determined that this approach can be used to detect, quantify and distinguish among the known Shiga toxins (Stx) and Shiga-like toxins (Stx1 and Stx2) in the low attomole range (per injection) in complex media, including human serum. Furthermore, Stx1a could be detected and distinguished from the newly identified Stx1e in complex media. As new Shiga-like toxins are identified, this approach can be readily modified to detect them. Since intact toxins are digested with trypsin prior to analysis, the handling of intact Shiga toxins is minimized. The analysis can be accomplished within 5 h.

New high-affinity monoclonal antibodies against Shiga toxin 1 facilitate the detection of hybrid Stx1/Stx2 in vivo.

BACKGROUND: Shiga toxin-producing E. coli (STEC) are a group of common and potentially deadly intestinal pathogens expressing Shiga toxin (Stx) as a primary virulence factor. Of the two types of Stx, Stx2 is responsible for more severe symptoms during infection, while Stx1 is almost identical to the Shiga toxin from Shigella dysenteriae, a ubiquitous pathogen in developing countries. Although antibodies against Stx1 have been reported, few have reached the affinity needed for assembling highly sensitive immunoassays. Sensitive and affordable immunoassays for Stx1 and Stx2 could help improve detection of STEC in livestock, food, the environment, and in clinical samples resulting in improved food safety and human health. METHOD AND FINDINGS: Three new monoclonal antibodies (mAbs) against the B subunit of Stx1 were generated using recombinant toxoid Stx1E167Q and hybridoma technology. These new mAbs recognize all subtypes of Stx1, but do not cross-react with any subtype of Stx2. In addition, they exhibited the ability to neutralize Stx1 toxicity in Vero cell assays. An optimized sandwich ELISA using of a pair of these mAbs had a limit of detection of 8.7 pg/mL, which is superior to any existing assay of this kind. Using one of these Stx1 mAbs in concert with Stx2 mAbs, the presence of hybrid Stx1/Stx2 toxin in the culture media of STEC strains that express both Stx1 and Stx2 was demonstrated. CONCLUSIONS: These new mAbs provide a mix of availability, utility, versatility, and most importantly, increased sensitivity for detection of Stx1. There are numerous potential applications for these mAbs, including low-cost detection assays and therapeutic use. Analysis of hybrid Stx1/2 could provide new insights on the structure, activity, and cellular targets of Shiga toxins.

Development of monoclonal antibodies and immunoassays for sensitive and specific detection of Shiga toxin Stx2f.

BACKGROUND: Shiga toxin 2 (Stx2) is a major virulence factor in gastrointestinal diseases caused by Escherichia coli. Although Stx2a (prototypical Stx2) is well-studied, all seven subtypes of Stx2 have been associated with disease in mammals. Several subtypes of Stx2, including Stx2f, are difficult to detect immunologically. METHODS AND FINDINGS: Four novel monoclonal antibodies (mAbs) against the Stx2f subtype were produced and characterized. These mAbs react exclusively to the Stx2f A subunit, and do not cross-react with other subtypes of Stx2. A Stx2f-specific sandwich ELISA was established and a limit of detection of 0.123 ng/mL was obtained using one pair of the mAbs. The receptor preference of Stx2f was confirmed using this sandwich ELISA. Three out of four mAbs can partially neutralize the toxicity of Stx2f in a cell-based assay. These mAbs were also demonstrated to be highly specific and reactive when applied to colony immunoblot assays. CONCLUSIONS: Novel mAbs specific to Stx2f were developed for the first time, providing new assets for the STEC community. Immunoassays with improved sensitivity and specificity will be useful for the detection of Stx2f present in food, environmental, and clinical samples.

Mouse in vivo neutralization of Escherichia coli Shiga toxin 2 with monoclonal antibodies.

Shiga toxin-producing Escherichia coli (STEC) food contaminations pose serious health concerns, and have been the subject of massive food recalls. STEC has been identified as the major cause of the life-threatening complication of hemolytic uremic syndrome (HUS). Besides supportive care, there currently are no therapeutics available. The use of antibiotics for combating pathogenic E. coli is not recommended because they have been shown to stimulate toxin production. Clearing Stx2 from the circulation could potentially lessen disease severity. In this study, we tested the in vivo neutralization of Stx2 in mice using monoclonal antibodies (mAbs). We measured the biologic half-life of Stx2 in mice and determined the distribution phase or t(1/2) α to be 3 min and the clearance phase or t(1/2) ß to be 40 min. Neutralizing mAbs were capable of clearing Stx2 completely from intoxicated mouse blood within minutes. We also examined the persistence of these mAbs over time and showed that complete protection could be passively conferred to mice 4 weeks before exposure to Stx2. The advent of better diagnositic methods and the availability of a greater arsenal of therapeutic mAbs against Stx2 would greatly enhance treatment outcomes of life threatening E. coli infections.

A polyclonal antibody based immunoassay detects seven subtypes of Shiga toxin 2 produced by Escherichia coli in human and environmental samples.

BACKGROUND: Shiga toxin-producing Escherichia coli (STEC) are frequent causes of severe human diseases ranging from diarrhea to hemolytic uremic syndrome. The existing strategy for detection of STEC relies on the unique sorbitol-negative fermentation property of the O157 strains, the most commonly identified serotype has been E. coli O157. It is becoming increasingly evident, however, that numerous non-O157 STEC serotypes also cause outbreaks and severe illnesses. It is necessary to have new methods that are capable of detecting all STEC strains. METHODS AND FINDINGS: Here we describe the development of a sandwich ELISA assay for detecting both O157 and non-O157 STECs by incorporating a novel polyclonal antibody (pAb) against Stx2. The newly established immunoassay was capable of detecting Stx2a spiked in environmental samples with a limit of detection between 10 and 100 pg/mL in soil and between 100 and 500 pg/mL in feces. When applied to 36 bacterial strains isolated from human and environmental samples, this assay detected Stx2 in all strains that were confirmed to be stx2-positive by real-time PCR, demonstrating a 100% sensitivity and specificity. CONCLUSIONS: The sandwich ELISA developed in this study will enable any competent laboratory to identify and characterize Stx2-producing O157 and non-O157 strains in human and environmental samples, resulting in rapid diagnosis and patient care. The results of epitope mapping from this study will be useful for further development of a peptide-based antibody and vaccine.