antibody against tcdb gtd  (R&D Systems)

Journal: Infection and Immunity

Article Title: Mouse monoclonal antibodies against Clostridioides difficile toxins TcdA and TcdB target diverse epitopes for neutralization

doi: 10.1128/iai.00139-25

Figure Lengend Snippet: Neutralization of TcdA or TcdB by antibodies on Vero cells. ( A ) Neutralization results for TcdA with mAbs. ( B ) EC50 results from analysis of neutralization data. ( C ) Neutralization results for TcdB with mAbs. Technical replicates were averaged for analysis in GraphPad Prism with least squares fit of the model.

Article Snippet: Primary antibodies against TcdA (1:1,000, NB600-1066, Novus Biologicals), TcdB (20B3; 43), and GAPDH (1:5,000, 14C10, Cell Signaling) were diluted in 5% milk-TBS-T and incubated with the membranes overnight at 4°C.

Techniques: Neutralization

Journal: Infection and Immunity

Article Title: Mouse monoclonal antibodies against Clostridioides difficile toxins TcdA and TcdB target diverse epitopes for neutralization

doi: 10.1128/iai.00139-25

Figure Lengend Snippet: Effect of mouse neutralizing antibodies on binding of TcdA and TcdB. Panels show binding of TcdA to Caco-2 and Vero cells (A), or TcdB to Caco-2, Vero, and A549 cells (B) after treatment with individual TcdA- and TcdB-specific mAbs and Fabs. Toxin-only controls are shown in red, and all antibody-containing reactions are normalized to their corresponding controls. Assays were performed in triplicate and analyzed in GraphPad Prism by one-way ANOVA using Dunnett’s multiple comparisons test. ( P * <0.05, ** <0.01, *** 0.0001)

Article Snippet: Primary antibodies against TcdA (1:1,000, NB600-1066, Novus Biologicals), TcdB (20B3; 43), and GAPDH (1:5,000, 14C10, Cell Signaling) were diluted in 5% milk-TBS-T and incubated with the membranes overnight at 4°C.

Techniques: Binding Assay

Journal: Infection and Immunity

Article Title: Mouse monoclonal antibodies against Clostridioides difficile toxins TcdA and TcdB target diverse epitopes for neutralization

doi: 10.1128/iai.00139-25

Figure Lengend Snippet: Antibodies protect human epithelial cell lines from depolarization by TcdA (HT29 cells, pH 5.0) or TcdB (A549 cells, pH 7.2), measured by changes in DiBAC4(3) fluorescence. ( A ) TcdA (10 nM, black) with 10 nM of mCDIFA-184-9 (blue), or mCDIFA-205-7 (purple), mCDIFA-230-2 (orange), or mCDIFA-248-25 (green). TcdA-L1108K (pore-formation mutant) is shown in red. ( B ) TcdB (1 nM, black) with 10 nM of mCDIFB-6-30 (green) or mCDIFB-8-26 (purple). TcdB-L1106K is in red. mCDIFA-60-22 and mCDIFB-56-15 were not included, since they block binding of the cell surface. Unrelated antibodies do not protect from depolarization, as shown in controls comparing ( C ) TcdA (10 nM, black) with 10 nM of mCDIFB-8-26 (blue) or mCDIFA-230-2 (orange), and ( D ) TcdB (1 nM, black) with 10 nM of mCDIFA-248-25 (green) or mCDIFB-8-26 (purple). Fluorescence is normalized to a no-toxin control, and all traces represent the average of three independent replicates.

Article Snippet: Primary antibodies against TcdA (1:1,000, NB600-1066, Novus Biologicals), TcdB (20B3; 43), and GAPDH (1:5,000, 14C10, Cell Signaling) were diluted in 5% milk-TBS-T and incubated with the membranes overnight at 4°C.

Techniques: Fluorescence, Mutagenesis, Blocking Assay, Binding Assay, Control

Journal: Infection and Immunity

Article Title: Mouse monoclonal antibodies against Clostridioides difficile toxins TcdA and TcdB target diverse epitopes for neutralization

doi: 10.1128/iai.00139-25

Figure Lengend Snippet: Protection of Rac1 from toxin-mediated glucosylation in Caco-2 cells. TcdA (10 nM) or TcdB (10 nM) were pre-incubated with 10-fold molar excess of mAb before cell intoxication. Results are normalized to total Rac1 and represent three independent replicates ( P **** <0.0001).

Article Snippet: Primary antibodies against TcdA (1:1,000, NB600-1066, Novus Biologicals), TcdB (20B3; 43), and GAPDH (1:5,000, 14C10, Cell Signaling) were diluted in 5% milk-TBS-T and incubated with the membranes overnight at 4°C.

Techniques: Incubation

Journal: Infection and Immunity

Article Title: Mouse monoclonal antibodies against Clostridioides difficile toxins TcdA and TcdB target diverse epitopes for neutralization

doi: 10.1128/iai.00139-25

Figure Lengend Snippet: Binding epitopes of TxdA- and TcdB-specific monoclonal antibodies mapped onto the three-dimensional structures of the toxins. ( A ) Three-dimensional structure of TcdA (4R04.pdb ) with identified monoclonal antibody bound epitopes illustrated. Red—N-terminal (glucosyltransferase) domain, orange—proteolytic domain, green—delivery domain. ( B ) Three-dimensional structure of TcdB (6OQ5.pdb ) with identified monoclonal antibody bound epitopes illustrated. Red—N-terminal (glucosyltransferase) domain, orange—proteolytic domain, green—delivery domain. Sequence segments showing decreased deuterium uptake in the presence of the antibodies are shown in yellow. Starting and ending residues of each segment are indicated in the expanded views. Structural illustrations were rendered using Discovery Studio Visualizer 4.5.

Article Snippet: Primary antibodies against TcdA (1:1,000, NB600-1066, Novus Biologicals), TcdB (20B3; 43), and GAPDH (1:5,000, 14C10, Cell Signaling) were diluted in 5% milk-TBS-T and incubated with the membranes overnight at 4°C.

Techniques: Binding Assay, Bioprocessing, Sequencing

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Genomic and phenotypic characterization of a Clostridioides difficile strain of the epidemic ST37 type from China

doi: 10.3389/fcimb.2024.1412408

Figure Lengend Snippet: Size of PaLoc genes in reference strain and A-B+ C. difficile strains.

Article Snippet: After being washed with PBST, the plates were further incubated with 50 μl of HRP-Chicken anti-Tcd A antibody (1: 5000 dilution, Gallus Immunotech, USA) or 50 μl HRP-Chicken anti-TcdB antibody (1: 2500 dilution, Gallus Immunotech, USA) per well at 37°C for 1 hour.

Techniques:

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Genomic and phenotypic characterization of a Clostridioides difficile strain of the epidemic ST37 type from China

doi: 10.3389/fcimb.2024.1412408

Figure Lengend Snippet: Phylogenetic tree of A-B+ C. difficile T cdB subtypes. A neighbor-joining phylogenetic tree of TcdB amino acid sequences from the A-B+ strains examined in this study and other strains representing the various subtypes of TcdB. Xy06 TcdB is clustered with several A-B+ strains that encode TcdB3 (light blue). The scale bar indicates 0.020 amino acid substitutions per site. Bootstrap values of 50 or greater are displayed.

Article Snippet: After being washed with PBST, the plates were further incubated with 50 μl of HRP-Chicken anti-Tcd A antibody (1: 5000 dilution, Gallus Immunotech, USA) or 50 μl HRP-Chicken anti-TcdB antibody (1: 2500 dilution, Gallus Immunotech, USA) per well at 37°C for 1 hour.

Techniques:

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Genomic and phenotypic characterization of a Clostridioides difficile strain of the epidemic ST37 type from China

doi: 10.3389/fcimb.2024.1412408

Figure Lengend Snippet: Toxin production in strains Xy06 and Xy07. TcdA (A) and TcdB (B) concentrations of C. difficile culture supernatants at different time points. (C) TcdB concentrations of C. difficile culture supernatants were compared at the same time point. *P < 0.05 (t-test). (D) Cytotoxicity of C. difficile culture supernatants. C. difficile culture supernatants (16 μl) collected at 72 hours post-inoculation in TY medium were added to CT26 cells seeded in 96-well plates. CT26 cell rounding was visualized by phase-contrast microscopy (40X) at 20 hours post-treatment. (E) Percentage of round cells at different time points (****P<0.0001, one-way ANOVA). Data are presented as mean ± SEM (n = triplicate). ns- not significant.

Article Snippet: After being washed with PBST, the plates were further incubated with 50 μl of HRP-Chicken anti-Tcd A antibody (1: 5000 dilution, Gallus Immunotech, USA) or 50 μl HRP-Chicken anti-TcdB antibody (1: 2500 dilution, Gallus Immunotech, USA) per well at 37°C for 1 hour.

Techniques: Microscopy