A High-Throughput, Precipitating Colorimetric Sandwich ELISA Microarray for Shiga Toxins

Gehring, Andrew G.; He, Xiaohua; Fratamico, Pina M.; Lee, Joseph; Bagi, Lori K.; Brewster, Jeffrey D.; Paoli, George C.; He, Yiping; Xie, Yanping; Skinner, Craig; Barnett, Charlie; Harris, Douglas W.

doi:10.3390/toxins6061855

Cited by 12 publications

(3 citation statements)

References 26 publications

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“…Furthermore, the LOD lies in the range of ~10 to ~30 ng/mL (Fig. 3A and B) and is therefore comparable to established methods, such as ELISA LOD of ~30 ng/mL 33 or Vero cell cytotoxicity assay LOD of ~10-32 ng/mL 34 . Therefore, the combination of simplicity, costeffectiveness, and sensitivity are clear advantages of the novel FRET-based assay.…”

Section: Discussionsupporting

confidence: 70%

Rapid enzymatic detection of Shigatoxin-producingE. coliusing fluorescence-labeled oligonucleotide substrates

Ramming,

Lang,

Hauf

et al. 2023

Preprint

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Shigatoxin-producingE. coli(STEC) are important human pathogens causing disease ranging from diarrhea to severe hemolytic uremic syndrome. As STEC are transmitted via animals, food, and water, and may produce large outbreaks, their timely and qualified detection including isolate recovery is of high importance, but challenging and labor-intense. Thus, the availability of an easy-to-perform rapid test would be a tremendous advance. Since the common feature and major virulence factor of otherwise multifaceted STEC is the Shiga toxin (Stx), we developed a detection method for Stx, specifically for its catalytic RNA-N-glycosidase activity targeting the Sarcin Ricin Loop (SRL) of 28S ribosomal RNA. To this end, synthetic ssDNA substrates mimicking the SRL were designed and linked to a fluorophore and quencher pair, which conferred a fluorescence signal after cleavage by Stx. Optimal results using bacterial culture supernatants or single colonies were achieved for substrateStxSense 4following 30 to 60 minutes incubation. Importantly, different Stx1 and Stx2 subtypes, diverse STEC serotypes, andShigellawere detected. In conclusion, the assay offers rapid and facile detection of STEC based on a real-time readout for Stx activity. Therefore, it may improve STEC risk evaluation, therapy decisions, outbreak and source detection, and simplify research for antimicrobials.

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Section: Discussionsupporting

confidence: 70%

Rapid enzymatic detection of Shigatoxin-producingE. coliusing fluorescence-labeled oligonucleotide substrates

Ramming,

Lang,

Hauf

et al. 2023

Preprint

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“…2 c upper panel), consistent with other reports using homogeneous proximity ligation assays [ 12 , 19 , 22 ]. This is often referred to as the “high-dose hook effect” and has been observed in other antibody based assays [ 33 – 35 ]. As a result, the assay had a linear signal response range of a 1–2 order of magnitude (Fig.…”

Section: Resultsmentioning

confidence: 93%

SH2-PLA: a sensitive in-solution approach for quantification of modular domain binding by proximity ligation and real-time PCR

et al. 2015

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BackgroundThere is a great interest in studying phosphotyrosine dependent protein-protein interactions in tyrosine kinase pathways that play a critical role in many aspects of cellular function. We previously established SH2 profiling, a phosphoproteomic approach based on membrane binding assays that utilizes purified Src Homology 2 (SH2) domains as a molecular tool to profile the global tyrosine phosphorylation state of cells. However, in order to use this method to investigate SH2 binding sites on a specific target in cell lysate, additional procedures such as pull-down or immunoprecipitation which consume large amounts of sample are required.ResultsWe have developed PLA-SH2, an alternative in-solution modular domain binding assay that takes advantage of Proximity Ligation Assay and real-time PCR. The SH2-PLA assay utilizes oligonucleotide-conjugated anti-GST and anti-EGFR antibodies recognizing a GST-SH2 probe and cellular EGFR, respectively. If the GST-SH2 and EGFR are in close proximity as a result of SH2-phosphotyrosine interactions, the two oligonucleotides are brought within a suitable distance for ligation to occur, allowing for efficient complex amplification via real-time PCR. The assay detected signal across at least 3 orders of magnitude of lysate input with a linear range spanning 1–2 orders and a low femtomole limit of detection for EGFR phosphotyrosine. SH2 binding kinetics determined by PLA-SH2 showed good agreement with established far-Western analyses for A431 and Cos1 cells stimulated with EGF at various times and doses. Further, we showed that PLA-SH2 can survey lung cancer tissues using 1 μl lysate without requiring phospho-enrichment.ConclusionsWe showed for the first time that interactions between SH2 domain probes and EGFR in cell lysate can be determined in a microliter-scale assay using SH2-PLA. The obvious benefit of this method is that the low sample requirement allows detection of SH2 binding in samples which are difficult to analyze using traditional protein interaction assays. This feature along with short assay runtime makes this method a useful platform for the development of high throughput assays to determine modular domain–ligand interactions which could have wide-ranging applications in both basic and translational cancer research.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-015-0169-1) contains supplementary material, which is available to authorized users.

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“…OD 600 measurements of both strains were internally collected every 2 h for 48 h. The supernatants of E. coli E4 were collected every 6 h from 30 to 48 h. The shiga toxins were analyzed via colorimetric enzyme-linked immunosorbent assay (ELISA) immunoassay previously described by Gehring et al, with a tiny modification. 22 All wells were filled with 200 μL of phosphatebuffered saline containing 0.05% Tween 20 (PBST) and immediately emptied by rapidly inverting the plate. Supernatants (100 μL) of E. coli E4 were added to wells for 30 min.…”

Section: ■ Materials and Methodsmentioning

confidence: 99%

Impact of Cyanocobalamin and Methylcobalamin on Inflammatory Bowel Disease and the Intestinal Microbiota Composition

Zhu

Xiang

Feng

et al. 2018

J. Agric. Food Chem.

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Patients with inflammatory bowel disease (IBD) are usually advised to supplement various types of vitamin B12, because vitamin B12 is generally absorbed in the colon. Thus, in the current study, the influence of cyanocobalamin (CNCBL) or methylcobalamin (MECBL) ingestion on IBD symptoms will be investigated. Then, whether and how the application of various cobalamins would modify the taxonomic and functional composition of the gut microbiome in mice will be examined carefully. Dextran-sulfate-sodium-induced IBD mice were treated with MECBL or CNCBL; disease activity index (DAI) scores and intestinal inflammatory conditions of mice were evaluated. Fecal samples were collected; microbiota composition was determined with a 16s rRNA analysis; functional profiles were predicted by phylogenetic investigation of communities by reconstruction of unobserved states (PICRUSt); and short-chain fatty acids were measured. The consequence of higher relative abundances of Enterobacteriaceae and isomeric short-chain fatty acids by cobalamin treatment revealed that a high concentration of CNCBL but not MECBL supplementation obviously aggravated IBD. A microbial ecosystem rich in Escherichia/Shigella and low in Lactobacillus, Blautia, and Clostridium XVIII was observed in IBD mice after a high concentration of CNCBL supplementation. In cobalamin-dependent enzymes, CNCBL was more efficient in the adenosylcobalamin system than MECBL and vice versa in the MECBL system. The distinct effects of various cobalamins were associated with the distribution and efficiency of vitamin-B12-dependent riboswitches. CNCBL had a strong inhibitory effect on all riboswitches, especially on btuB and pocR riboswitches from Enterobacteriaceae. CNCBL aggravated IBD via enhancing the proportion of Enterobacteriaceae organisms through riboswitch and enzyme systems. The present study provides a critical reference for offering a suitable amount and type of cobalamin during a symbiotic condition.

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A High-Throughput, Precipitating Colorimetric Sandwich ELISA Microarray for Shiga Toxins

Cited by 12 publications

References 26 publications

Rapid enzymatic detection of Shigatoxin-producingE. coliusing fluorescence-labeled oligonucleotide substrates

Rapid enzymatic detection of Shigatoxin-producingE. coliusing fluorescence-labeled oligonucleotide substrates

SH2-PLA: a sensitive in-solution approach for quantification of modular domain binding by proximity ligation and real-time PCR

Impact of Cyanocobalamin and Methylcobalamin on Inflammatory Bowel Disease and the Intestinal Microbiota Composition

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