Development of Monoclonal Antibodies and Immunoassays for Sensitive and Specific Detection of Shiga Toxin Stx2f

Skinner, Craig; Patfield, Stephanie; Stanker, Larry H.; He, Xiaohua

doi:10.1371/journal.pone.0076563

Cited by 22 publications

(49 citation statements)

References 42 publications

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“…Strains producing subtype Stx2a, Stx2c, or Stx2d are often associated with development of hemorrhagic colitis and HUS (2). In contrast, Stx2b (3), Stx2e (4), Stx2f (5), and Stx2g (6) are scarcely associated with serious human disease. STEC strains isolated from red deer, sheep, and goat products were reported to carry stx2b (7), and those from bovine products carried stx2g (8).…”

Section: Introductionmentioning

confidence: 99%

Shiga Toxin Subtypes and Virulence Genes in <i>Escherichia coli</i> Isolated from Cattle

et al. 2017

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SUMMARY: Subtypes of stx1 and stx2 in 45 Shiga toxin-producing Escherichia coli (STEC) strains isolated from cattle were investigated by PCR. Only subtype stx1a was detected among all the stx1-positive strains. The major stx2 subtype was stx2a followed by stx2d, stx2c, stx2b, and stx2g in decreasing order of frequency. stx2c was found in strains of serotypes O157 and O174. stx2d was found in 11 strains. These strains were confirmed by DNA sequencing to carry both the activatable tail and the END motif; all were eae-negative, and 3 contained stx2d as the only stx. stx2g was found in 2 strains in association with stx2a, estA1, and astA. In addition, 7 hybrid strains of shigatoxigenic and enterotoxigenic E. coli (STEC/ETEC) were found to harbor one or both of stx1a and stx2a (stx1a/stx2a ) and estA1. Among 27 serotypes of STEC strains isolated from cattle, O157:H7 and O109:H-strains were eae-positive. Other putative adhesin genes, such as saa, iha, espP, and lpfA O113 were detected in more than 12 serotypes.

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Section: Introductionmentioning

confidence: 99%

Shiga Toxin Subtypes and Virulence Genes in <i>Escherichia coli</i> Isolated from Cattle

et al. 2017

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“…None of the Stx2 subtypes were recognized in these tests, reaffirming the assay's specificity to Stx1 (Figure 2C). The presence of Stx2 in these media was confirmed by Vero cell assays [28]. To determine whether the three mAbs can recognize all subtypes of Stx1, we used culture supernatants from STEC strains containing Stx1a (RM13506, RM11768), Stx1c/Stx2b (AA1, FF6), and Stx1d (RM13149, II9) as toxin sources and performed sandwich ELISA using a single mAb for both capture and detection, with the detection antibody being biotinylated.…”

Section: Resultsmentioning

confidence: 99%

“…The purification of monoclonal antibody was conducted as described [28]. Briefly, 400 mL of antibody-containing media (hybridoma cells grown for 2–3 days) was passed through a Protein G column (GE Healthcare).…”

Section: Methodsmentioning

confidence: 99%

New High-Affinity Monoclonal Antibodies against Shiga Toxin 1 Facilitate the Detection of Hybrid Stx1/Stx2 In Vivo

et al. 2014

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BackgroundShiga 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 FindingsThree 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.ConclusionsThese 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.

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“…Stx diagnostic PCRs were performed as described previously (29). Previously described primers and reaction conditions were used (8).…”

Section: Methodsmentioning

confidence: 99%

“…Vero (African green monkey adult kidney) cells (ATCC CCL-81) were obtained from the American Type Culture Collection (Manassas, VA) (30). Vero cells were prepared and grown as described previously (29). Briefly, the medium used for Vero cell propagation and growth (in a humidified cell culture incubator at 37°C with 5% CO 2 ), was Dulbecco's modified Eagle's medium (DMEM) (Invitrogen) with 10% fetal bovine serum (FBS) (Invitrogen).…”

Section: Methodsmentioning

confidence: 99%

An In Vitro Combined Antibiotic-Antibody Treatment Eliminates Toxicity from Shiga Toxin-Producing Escherichia coli

Skinner

Zhang

Patfield

et al. 2015

Antimicrob Agents Chemother

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bTreating 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.

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Development of Monoclonal Antibodies and Immunoassays for Sensitive and Specific Detection of Shiga Toxin Stx2f

Cited by 22 publications

References 42 publications

Shiga Toxin Subtypes and Virulence Genes in <i>Escherichia coli</i> Isolated from Cattle

Shiga Toxin Subtypes and Virulence Genes in <i>Escherichia coli</i> Isolated from Cattle

New High-Affinity Monoclonal Antibodies against Shiga Toxin 1 Facilitate the Detection of Hybrid Stx1/Stx2 In Vivo

An In Vitro Combined Antibiotic-Antibody Treatment Eliminates Toxicity from Shiga Toxin-Producing Escherichia coli

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