Clostridium Difficile Toxin A Antibody (PCG4.1) - BSA Free

Immunocytochemistry/Immunofluorescence: Clostridium Difficile Toxin A Antibody (PCG4.1) [NB600-1066] - TcdA colocalizes with PACSIN2 during entry in Caco-2 cells. Immunofluorescence assays were performed as described in Fig 1B. At indicated time points, cells were fixed, stained for PACSIN2 and analyzed by confocal microscopy. Merged images show PACSIN2 in red, toxin in green and colocalization in yellow. The images shown are representative of multiple fields imaged from three independent experiments. Image collected and cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.ppat.1006070), licensed under a CC-BY license.

Immunocytochemistry/Immunofluorescence: Clostridium Difficile Toxin A Antibody (PCG4.1) [NB600-1066] - TcdA and TcdB utilize distinct endocytic mechanisms to intoxicate colonic epithelial cells. TcdA does not colocalize with clathrin heavy chain during cell entry. Caco-2 cells on glass coverslips were chilled at 10C for 45 min and then exposed to media containing 50 nM TcdA-546 or buffer (no toxin control). The toxin was allowed to bind to cells for 45 min at 10C. Unbound toxin was removed, and cells were shifted to 37C to allow internalization of toxin for the times shown. At each time point, cells were washed once with pre-warmed PBS, fixed and stained for CHC, and imaged by confocal microscopy. Merged images show clathrin in red and toxin in green. Image collected and cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.ppat.1006070), licensed under a CC-BY license.

Immunocytochemistry/Immunofluorescence: Clostridium Difficile Toxin A Antibody (PCG4.1) [NB600-1066] - Depletion of PACSIN2 inhibits TcdA entry in Caco-2 cells. Caco-2 cells expressing non-targeting shRNA (Ctrl shRNA) or shRNA 982 targeting PACSIN2 were incubated with 50 nM TcdA-546 at 10C for 45 min. Unbound toxins were removed and cells were shifted to 37C to allow internalization. After 20 min, cells were washed, fixed, stained for PACSIN2 and imaged by confocal microscopy. PACSIN2 and TcdA staining from ctrl shRNA and sh982 expressing cells are shown. The images shown are representative of multiple fields imaged from three independent experiments. Image collected and cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.ppat.1006070), licensed under a CC-BY license.

Immunocytochemistry/ Immunofluorescence: Clostridium Difficile Toxin A Antibody (PCG4.1) - BSA Free [NB600-1066] - PACSIN2 is required for TcdA uptake & toxin-induced rounding in wildtype MEF cells. (C), (D) & (E). Depletion of PACSIN2 reduces TcdA uptake in MEF cells. Wildtype MEF cells expressing luciferase (luc) or PACSIN2 siRNA incubated with 50 nM TcdA-546 at 10°C for 45 min. Cells allowed to warm up to 37°C for 2 min & then washed to remove unbound toxins & incubated with fresh media prewarmed to 37°C. Bound toxins allowed to internalize for 9 min at 37°C. Cells then fixed, stained for PACSIN2 & imaged by confocal microscopy. PACSIN2 & TcdA-546 staining from each condition shown in (C). Scale bars, 10 μm. The images shown representative of multiple fields imaged from two independent experiments. (D) Comparison of mean fluorescence intensities of PACSIN2 between luc & PACSIN2 siRNA transfected cells. Data represent mean & SD of 101 individual cells. Student’s t test ***p<0.0001. (E) Comparison of mean fluorescence intensities of TcdA-546 between luc & PACSIN2 siRNA transfected cells. Data represent mean & SD of 101 individual cells. Student’s t test ***p<0.0001. Cells chosen at random for intensity analyses. Image collected & cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.ppat.1006070), licensed under a CC0-1.0 license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: Clostridium Difficile Toxin A Antibody (PCG4.1) - BSA Free [NB600-1066] - TcdA colocalizes with PACSIN2 in wildtype MEF cells.(A) Wildtype MEFs on glass coverslips were allowed to bind 50 nM TcdA-546 for 45 min at 10°C, & cells were shifted to 37°C to allow internalization of toxin for 3 min. Cells were fixed, stained for PACSIN2, & analyzed by confocal microscopy. Merged images show PACSIN2 in red, toxin in green, & colocalization in yellow. Scale bars, 10 μm. The images shown are representative of multiple fields imaged from two independent experiments. (B) Immunofluorescence assays were performed as described in (A), but cells were stained for cav1 in addition to PACSIN2. Merged images show PACSIN2 in red, toxin in green, & cav1 in blue. Yellow puncta in merged images denote TcdA- & PACSIN2-positive structures. Pink punta denote caveolae-associated PACSIN2. Scale bars, 10 μm. (C) Pearson’s correlation coefficient to assess the extent of colocalization between PACSIN2, cav1 & TcdA-546 after 3 min toxin uptake. Data represent mean & SD of 31 individual cells chosen at random. Image collected & cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.ppat.1006070), licensed under a CC0-1.0 license. Not internally tested by Novus Biologicals.

Immunocytochemistry/ Immunofluorescence: Clostridium Difficile Toxin A Antibody (PCG4.1) - BSA Free [NB600-1066] - TcdA colocalizes with PACSIN2 in wildtype MEF cells.(A) Wildtype MEFs on glass coverslips were allowed to bind 50 nM TcdA-546 for 45 min at 10°C, & cells were shifted to 37°C to allow internalization of toxin for 3 min. Cells were fixed, stained for PACSIN2, & analyzed by confocal microscopy. Merged images show PACSIN2 in red, toxin in green, & colocalization in yellow. Scale bars, 10 μm. The images shown are representative of multiple fields imaged from two independent experiments. (B) Immunofluorescence assays were performed as described in (A), but cells were stained for cav1 in addition to PACSIN2. Merged images show PACSIN2 in red, toxin in green, & cav1 in blue. Yellow puncta in merged images denote TcdA- & PACSIN2-positive structures. Pink punta denote caveolae-associated PACSIN2. Scale bars, 10 μm. (C) Pearson’s correlation coefficient to assess the extent of colocalization between PACSIN2, cav1 & TcdA-546 after 3 min toxin uptake. Data represent mean & SD of 31 individual cells chosen at random. Image collected & cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.ppat.1006070), licensed under a CC0-1.0 license. Not internally tested by Novus Biologicals.

• Reactivity: Ba
• Applications: WB, ELISA, ICC/IF
• Clone: PCG4.1
• Clonality: Monoclonal
• Host: Mouse
• Conjugate: Unconjugated
• Format: BSA Free
• Concentration: 1.16 mg/ml

Clostridium Difficile Toxin A Antibody (PCG4.1) - BSA Free Summary

• Immunogen: Full length protein A (C. difficile).
• Localization: Secreted
• Specificity: C. difficile Toxin A. Does not cross react with C. difficile Toxin B.
• Isotype: IgG2a Kappa
• Clonality: Monoclonal
• Host: Mouse
• Purity: Protein A purified

Applications/Dilutions

• Dilutions:
  • ELISA 1:100-1:2000
  • Immunocytochemistry/ Immunofluorescence
  • Western Blot 1:100-1:2000
• Application Notes: WB reactivity reported in (PMID: 28346491). Works with stool samples.

Packaging, Storage & Formulations

• Storage: Store at 4C short term. Aliquot and store at -80C long term. Avoid freeze-thaw cycles.
• Buffer: 10mM Sodium Phosphate (pH 7.4) and 0.15M NaCl
• Preservative: 0.05% Sodium Azide
• Concentration: 1.16 mg/ml
• Purity: Protein A purified

Alternate Names for Clostridium Difficile Toxin A Antibody (PCG4.1) - BSA Free

• TcdA
• ToxA
• Toxin A

Background

Clostridium difficile, or C. difficile, a gram positive anaerobic bacterium, is the major causative agent of colitis and diarrhoea that can occur following antibiotic intake. C. difficile infection is one of the most common hospital acquired infections. When C. difficile colonize the gut, they release two potent toxins, toxin A and toxin B, which bind to certain receptors in the lining of the colon and ultimately cause diarrhoea and inflammation of the large intestine.

Limitations

This product is for research use only and is not approved for use in humans or in clinical diagnosis. Primary Antibodies are guaranteed for 1 year from date of receipt.

Publications for Clostridium Difficile Toxin A Antibody (NB600-1066)(24)

Publications using NB600-1066

• Nabukhotna, K;Kordus, SL;Shupe, JA;Cano Rodríguez, R;Smith, A;Bohannon, JK;Washington, MK;Lacy, DB; Purified CDT toxins and a clean deletion within the CDT locus provide novel insights into the contribution of binary toxin in cellular inflammation and Clostridioides difficile infection PLoS pathogens 2024-09-19 [PMID: 39298531]
• Wang S, Heuler J, Wickramage I, Sun X Genomic and Phenotypic Characterization of the Nontoxigenic Clostridioides difficile Strain CCUG37785 and Demonstration of Its Therapeutic Potential for the Prevention of C. difficile Infection Microbiology spectrum 2022-03-22 [PMID: 35315695] (ELISA, Bacteria)
  
  Details:
  Clostridioides difficile
• Zhu D, Wang S, Sun X. FliW and CsrA Govern Flagellin (FliC) Synthesis and Play Pleiotropic Roles in Virulence and Physiology of Clostridioides difficile R20291 Frontiers in microbiology 2021-10-05 [PMID: 34675903]
• Zhu D, Patabendige HMLW, Tomlinson BR et al. Cwl0971, a novel peptidoglycan hydrolase, plays pleiotropic roles in Clostridioides difficile R20291 Environmental microbiology 2021-04-24 [PMID: 33893759]
• Ahmed UKB, Shadid TM, Larabee JL, Ballard JD. Combined and Distinct Roles of Agr Proteins in Clostridioides difficile 630 Sporulation, Motility, and Toxin Production mBio 2021-01-14 [PMID: 33443122]
• Oliveira PH, Ribis JW, Garrett EM et al. Epigenomic characterization of Clostridioides difficile finds a conserved DNA methyltransferase that mediates sporulation and pathogenesis Nat Microbiol 2019-11-25 [PMID: 31768029] (WB, Bacteria)
• Edwards AN, Krall EG, McBride SM RstA Regulation of Clostridioides difficile Toxin Production and Sporulation in Phenotypically Diverse Strains bioRxiv 2019-09-18 [PMID: 31659010] (WB, C. difficile)
• Edwards A, Anjuwon-Foster B, McBride S. RstA is a Major Regulator of Clostridioides difficile Toxin Production and Motility. bioRxiv 2018-09-13 [PMID: 30862746] (WB, Bacteria)
• Daou N, Wang Y, Levdikov VM et al. Impact of CodY protein on metabolism, sporulation and virulence in Clostridioides difficile ribotype 027 PLoS ONE 2019-01-30 [PMID: 30699117] (ELISA, Bacteria)
• Woods E, Edwards A, McBride S. The C. difficile clnRAB operon initiates adaptations to the host environment in response to LL-37 PLoS Pathog. [PMID: 30125334] (WB, Bacteria)
• Anjuwon-Foster BR, Maldonado-Vazquez N, Tamayo R. Et al. Characterization of Flagellum and Toxin Phase Variation in Clostridioides difficile Ribotype 012 Isolates J Bacteriol 2018-05-09 [PMID: 29735765] (WB, Bacteria)
  
  Details:
  Citation using the DyLight 800 version of this antibody.
• Kroh HK, Chandrasekaran R, Rosenthal K et al. Use of a neutralizing antibody helps identify structural features critical for binding of Clostridium difficile toxin TcdA to the host cell surface J. Biol. Chem. 2017-07-13 [PMID: 28705932] (Bacteria)
• Anjuwon-Foster BR, Tamayo R. A genetic switch controls the production of flagella and toxins in Clostridium difficile. PLoS Genet. 2017-03-27 [PMID: 28346491] (WB, Bacteria)
• Chandrasekaran R, Kenworthy AK, Lacy DB et al. Clostridium difficile Toxin A Undergoes Clathrin-Independent, PACSIN2-Dependent Endocytosis PLoS Pathog. 2016-12-11 [PMID: 27942025] (WB, Human)
• Childress KO, Edwards AN, Nawrocki KL et al. The Phosphotransfer Protein CD1492 Represses Sporulation Initiation in Clostridium difficile. Infect. Immun. 2016-12-01 [PMID: 27647869] (WB, Hamster)
• Edwards AN, Tamayo R, McBride SM. A Novel Regulator Controls Clostridium difficile Sporulation, Motility and Toxin Production. Mol. Microbiol. 2016-02-24 [PMID: 26915493] (WB, Bacteria)
• Willing SE, Richards EJ, Sempere L et al. Increased toxin expression in a Clostridium difficile mfd mutant. BMC Microbiol. 2015-12-18 [PMID: 26679502] (WB, Bacteria)
  
  Details:
  Clostridium Difficile Toxin A antibody (clone PCG4.1) was used for WB assays in experiments involving Clostridium difficile mfd mutant. The antibody was used at 1:1000 dilution in 3 % milk powder and detected using HRP-conjugated goat anti-mouse secondary antibody .
• McKee RW, Mangalea MR, Purcell EB et al. The Second Messenger Cyclic Di-GMP Regulates Clostridium difficile Toxin Production by Controlling Expression of sigD. J Bacteriol. 2013-11-01 [PMID: 24039264] (WB, Bacteria)
• Ahmed UKB The Accessory Gene Regulator (Agr) System and Regulatory Network in Clostridioides Difficile Thesis 1905-07-13
• Kolling GL, Wu M, Alcantara-Warren C et al. Lactic acid production by Streptococcus thermophilus alters Clostridium difficile infection and in vitro Toxin A production Gut Microbes 2012-11-01 [PMID: 22895082] (WB, Bacteria)
• Aubry A, Hussack G, Chen W et al. Modulation of Toxin Production by the Flagellar Regulon in Clostridium difficile Infect Immun 2012-10-01 [PMID: 22851750] (ELISA, WB, Bacteria)
• Vohra P, Poxton IR. Comparison of toxin and spore production in clinically relevant strains of Clostridium difficile. Microbiology;157(Pt 5):1343-53. 2011-05-01 [PMID: 21330434] (ELISA)
• Goulding D, Thompson H, Emerson J et al. Distinctive profiles of infection and pathology in hamsters infected with Clostridium difficile strains 630 and B1. Infect Immun;77(12):5478-85. 2009-12-01 [PMID: 19752031]
• Dissanayake SK, Olkhanud PB, O'Connell MP et al. Wnt5A Regulates Expression of Tumor-Associated Antigens in Melanoma via Changes in Signal Transducers Activators of Transcription 3 Phosphorylation. Cancer Res;68(24):10205-10214. 2008-01-01 [PMID: 19074888]

FAQs for Clostridium Difficile Toxin A Antibody (NB600-1066). (Showing 1 - 3 of 3 FAQs).

1. I am seeking monoclonal antibodies specific for the C Terminal binding regions of clostridum difficile toxins A and B (aa1852-2366)?
   • I would recommend catalog # NB100-62473. Although it is a polyclonal, it is the only one that we know binds to the C-Terminal portion of the protein. The other antibodies we have against both targets are synthesized from the full length sequence. We do not routinely epitope map our antibodies so we cannot be certain where they are binding. This polyclonal is synthesized against a small 10 amino acid region of the protein so it should be highly specific to that location.
2. Was NB-600-1066 Clostridium Difficile Toxin A antibody PCG4.1 purified from ascites or tissue culture supernatant? We are in search of an ELISA grade anti- Tcd A antibody that does not contain other antibodies.
   • Our Clostridium Difficile Toxin A Antibody catalog number NB600-1066 was protein A purified from mouse ascites.
3. Two questions: 1. Do you have any available data regarding the titer of NB600-1066, Anti C. difficile toxin A mAB PGC4 in ELISA? 2. I see that you sell other C. difficile toxin A and B antibodies. Is there any available ELISA data on those antibodies? And are they free from other nonspecific antibodies? Or are all of your mABs purified from ascites?
   • 1. We do not have an in-house ELISA titer, but I have two publications that have used NB600-1066 in ELISA applications: PMID 21330434 and PMID 22851750. 2. For our other Clostridium Difficile products, the ELISA testing data came from either publications or from the clone inventors. We unfortunately do not have any in-house ELISA data for these products. Our mABs can be purified from either tissue culture supernatant or ascites. We try to include the source on our datasheet, but some of them are missing this information. If you have a specific product, we can always look this information up for you. Since our mABs are either sold unpurified or protein A/G purified, there is a possibility for some non-specific antibodies to be present. We unfortunately do not test our products for the presence of other antibodies.