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: 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: Toxin-antibody Fab complexes visualized by negative-stain electron microscopy. TcdA1-1832 with ( A ) mCDIFA-248-25, ( B ) mCDIFA-230-2, ( C ) mCDIFA-184-9, or ( D ) mCDIFA-205-7. Full length TcdA with ( E ) mCDIFA-60-22. TcdB1-1810 with ( F ) mCDIFB-8-26, ( G ) mCDIFB-6-30, or ( H ) mCDIFB-56-15. Each image is a single class average with a representative view of the complex (scale bars 100 Å). (Right) Crystal structure of TcdA1-1832 (4R04.pdb) with the GTD in red, the auto-processing domain in blue, and the delivery domain in green.

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: Staining, Electron Microscopy

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: Cryo-electron microscopy of TcdA in complex with mCDIF248-25 Fab. ( A ) Model of complex (3.23 Å resolution), rotated 90 degrees to visualize Fab orientation at the end of the TcdA delivery domain (TcdA blue, mCDIF248-25 Fab green). ( B ) Residues involved in hydrogen bonding in the toxin-antibody epitope are highlighted in yellow (TcdA), red (Fab heavy chain), and orange (Fab light chain).

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: Cryo-Electron Microscopy

Journal: Antibiotics

Article Title: High Serum Levels of Toxin A Correlate with Disease Severity in Patients with Clostridioides difficile Infection

doi: 10.3390/antibiotics10091093

Figure Lengend Snippet: Representative images and the standard curve of ( A ) TcdA and ( B ) TcdB dot plots necessary for determining the concentration of the toxins in the blood of CDI patients. Toxin concentrations are reported as the mean value of triplicate dots ± standard deviation (SD) normalized to control (i.e., 0 pg/mL); a.d.u., arbitrary densitometric unit.

Article Snippet: The membrane was hybridized overnight at 4 °C with the anti- Clostridioides difficile TcdA (MCA2597; Bio-Rad, Hercules, CA, USA) or the anti- Clostridioides difficile TcdB (MCA4737; Bio-Rad, Hercules, CA, USA) primary antibodies diluted 1:20 in 5% non-fat dry milk dissolved in TTBS ( w / v ).

Techniques: Concentration Assay, Standard Deviation, Control

Journal: Antibiotics

Article Title: High Serum Levels of Toxin A Correlate with Disease Severity in Patients with Clostridioides difficile Infection

doi: 10.3390/antibiotics10091093

Figure Lengend Snippet: Mean laboratory findings and mean toxemia at CDI diagnosis (T0), and at 4 (T4) and 10 days (T10) after CDI diagnosis of the 35 CDI patients included in the study. SD: standard deviation.

Article Snippet: The membrane was hybridized overnight at 4 °C with the anti- Clostridioides difficile TcdA (MCA2597; Bio-Rad, Hercules, CA, USA) or the anti- Clostridioides difficile TcdB (MCA4737; Bio-Rad, Hercules, CA, USA) primary antibodies diluted 1:20 in 5% non-fat dry milk dissolved in TTBS ( w / v ).

Techniques: Biomarker Discovery

Journal: Antibiotics

Article Title: High Serum Levels of Toxin A Correlate with Disease Severity in Patients with Clostridioides difficile Infection

doi: 10.3390/antibiotics10091093

Figure Lengend Snippet: Clinical features, comorbidities, laboratory findings, and outcome of the 18 cases with mild CDI and the 17 cases with severe CDI before diagnosis, at T0, T4, and T10. RR: Risk ratio. CI: Confidence interval. SD: Standard deviation. CCI: Charlson Co-morbidity Index.

Article Snippet: The membrane was hybridized overnight at 4 °C with the anti- Clostridioides difficile TcdA (MCA2597; Bio-Rad, Hercules, CA, USA) or the anti- Clostridioides difficile TcdB (MCA4737; Bio-Rad, Hercules, CA, USA) primary antibodies diluted 1:20 in 5% non-fat dry milk dissolved in TTBS ( w / v ).

Techniques: Biomarker Discovery, Standard Deviation, Bacteria

Journal: Antibiotics

Article Title: High Serum Levels of Toxin A Correlate with Disease Severity in Patients with Clostridioides difficile Infection

doi: 10.3390/antibiotics10091093

Figure Lengend Snippet: Changes of TcdA plasma levels in mild and severe CDI cases. TcdA levels significantly decreased at 10 days (T10) after CDI diagnosis in mild cases compared to levels measured at T0 and T4. Patients whose TcdA levels at T0 were 0 pg/μL were not included in these graphs. One way-ANOVA test, * p < 0.05.

Article Snippet: The membrane was hybridized overnight at 4 °C with the anti- Clostridioides difficile TcdA (MCA2597; Bio-Rad, Hercules, CA, USA) or the anti- Clostridioides difficile TcdB (MCA4737; Bio-Rad, Hercules, CA, USA) primary antibodies diluted 1:20 in 5% non-fat dry milk dissolved in TTBS ( w / v ).

Techniques: Clinical Proteomics, Biomarker Discovery