Anti-C. difficile TcdA-A2 VHH Single Domain Antibody is a recombinant protein produced in E. coli.
Figure 1 Antitoxin VHHs recognize conformational (A4.2, A5.1, A20.1, and A26.8) and linear (A19.2) epitopes.
ELISA demonstrates that purified anti-TcdA VHHs recognize both native toxins and recombinant RBD fragments. Wells were coated with equimolar concentrations of the proteins shown on the x axis. VHHs were added at 2 g/ml. The B5.2 VHH bound TcdA.
Hussack, G., Arbabi-Ghahroudi, M., van Faassen, H., Songer, J. G., Ng, K. K. S., MacKenzie, R., & Tanha, J. (2011). Neutralization of Clostridium difficile toxin A with single-domain antibodies targeting the cell receptor binding domain. Journal of Biological Chemistry, 286(11), 8961-8976.
Figure 2 Antitoxin VHHs recognize conformational (A4.2, A5.1, A20.1, and A26.8) and linear (A19.2) epitopes.
ELISA illustrating the binding of various concentrations of purified VHHs to immobilized TcdA, respectively.
Hussack, G., Arbabi-Ghahroudi, M., van Faassen, H., Songer, J. G., Ng, K. K. S., MacKenzie, R., & Tanha, J. (2011). Neutralization of Clostridium difficile toxin A with single-domain antibodies targeting the cell receptor binding domain. Journal of Biological Chemistry, 286(11), 8961-8976.
Figure 3 Antitoxin VHHs recognize conformational (A4.2, A5.1, A20.1, and A26.8) and linear (A19.2) epitopes.
ELISA on TcdA treated with various temperatures for 30 min before probing with VHHs. The dotted line represents the TcdA midpoint thermal unfolding temperature (Tm) of 55 °C. At treatment temperatures above the TcdA Tm, binding of four out of five TcdA-specific VHHs was abolished. A19.2 bound TcdA exposed to temperatures above its Tm, confirming that denatured TcdA was coated in the wells and that A19.2 recognized a linear epitope.
Hussack, G., Arbabi-Ghahroudi, M., van Faassen, H., Songer, J. G., Ng, K. K. S., MacKenzie, R., & Tanha, J. (2011). Neutralization of Clostridium difficile toxin A with single-domain antibodies targeting the cell receptor binding domain. Journal of Biological Chemistry, 286(11), 8961-8976.
Figure 4 VHHs bind TcdA with high affinity.
SPR sensorgrams of TcdA-specific VHHs A20.1 binding to immobilized TcdA
Hussack, G., Arbabi-Ghahroudi, M., van Faassen, H., Songer, J. G., Ng, K. K. S., MacKenzie, R., & Tanha, J. (2011). Neutralization of Clostridium difficile toxin A with single-domain antibodies targeting the cell receptor binding domain. Journal of Biological Chemistry, 286(11), 8961-8976.
Figure 5 Potent neutralization of TcdA-induced cell rounding in vitro.
Summary of neutralization assays. The final concentration of VHHs in each assay well was 1000 nM, and VHHs were added as singles, pairs, or triplet combinations. White barsrepresent single VHHs or PBS control; gray barsrepresent paired combinations, and black barsrepresent triplet combinations. Percent inhibition of cell rounding is relative to cells receiving TcdA only. Combinations of VHHs (i.e. pairs and triples) increased toxin neutralizing efficacy.
Hussack, G., Arbabi-Ghahroudi, M., van Faassen, H., Songer, J. G., Ng, K. K. S., MacKenzie, R., & Tanha, J. (2011). Neutralization of Clostridium difficile toxin A with single-domain antibodies targeting the cell receptor binding domain. Journal of Biological Chemistry, 286(11), 8961-8976.
Figure 6 Potent neutralization of TcdA-induced cell rounding in vitro.
Summary of neutralization assays. The final concentration of VHHs in each assay well was 10 nM, and VHHs were added as singles, pairs, or triplet combinations. White barsrepresent single VHHs or PBS control; gray barsrepresent paired combinations, and black barsrepresent triplet combinations. Percent inhibition of cell rounding is relative to cells receiving TcdA only. Combinations of VHHs (i.e. pairs and triples) increased toxin neutralizing efficacy.
Hussack, G., Arbabi-Ghahroudi, M., van Faassen, H., Songer, J. G., Ng, K. K. S., MacKenzie, R., & Tanha, J. (2011). Neutralization of Clostridium difficile toxin A with single-domain antibodies targeting the cell receptor binding domain. Journal of Biological Chemistry, 286(11), 8961-8976.
Figure 7 Potent neutralization of TcdA-induced cell rounding in vitro.
Summary of neutralization assays. The final concentration of VHHs in each assay well was 0.1 nM, and VHHs were added as singles, pairs, or triplet combinations. White barsrepresent single VHHs or PBS control; gray barsrepresent paired combinations, and black barsrepresent triplet combinations. Percent inhibition of cell rounding is relative to cells receiving TcdA only. Combinations of VHHs (i.e. pairs and triples) increased toxin neutralizing efficacy.
Hussack, G., Arbabi-Ghahroudi, M., van Faassen, H., Songer, J. G., Ng, K. K. S., MacKenzie, R., & Tanha, J. (2011). Neutralization of Clostridium difficile toxin A with single-domain antibodies targeting the cell receptor binding domain. Journal of Biological Chemistry, 286(11), 8961-8976.
Figure 8 Pre-bound VHHs do not impair trisaccharide binding to TcdA.
SPR sensorgrams illustrate the injection of A20.1 (a, solid and dashed lines), followed by injection of A20.1(b,dashed line), or A20.1CD-grease(b,solid line), and finally injection of buffer (c, solid and dashed lines).
Hussack, G., Arbabi-Ghahroudi, M., van Faassen, H., Songer, J. G., Ng, K. K. S., MacKenzie, R., & Tanha, J. (2011). Neutralization of Clostridium difficile toxin A with single-domain antibodies targeting the cell receptor binding domain. Journal of Biological Chemistry, 286(11), 8961-8976.
Figure 9 In vitro neutralizing activity and in vivo expression of A20.1 and A26.8 VHH antibodies.
Dose-effect curve of TcdA toxicity on CHO-K1 cell line in the presence or absence of CM from B. longum 44B [pESH100/VHH-A20.1] (BL-A20.1) and B. longum 44B [pESH100/VHH-A26.8] (BL-A20.1). CM were applied on the CHO-K1 cells prior to the treatment with TcdA toxin (5.5–1400 ng/ml). The percentage of rounded cells was manually calculated using phase contrast microscopy 24 h after treatment.
Shkoporov, A. N., Khokhlova, E. V., Savochkin, K. A., Kafarskaia, L. I., & Efimov, B. A. (2015). Production of biologically active scFv and VHH antibody fragments in Bifidobacterium longum. FEMS microbiology letters, 362(12).
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CAT | Product Name | Application | Type |
---|---|---|---|
PNBL-010 | Recombinant Anti-C. difficile TcdA VHH Single Domain Antibody (PNBL-010) | ELISA, SPR, Inhib, WB | Llama VHH |
PNBL-011 | Recombinant Anti-C. difficile TcdA VHH Single Domain Antibody (PNBL-011) | Neut, FuncS | Llama VHH |
CAT | Product Name | Application | Type |
---|---|---|---|
MRO-141CT-F(E) | Human Anti-TcdA Recombinant Antibody; Fab Fragment (MRO-141CT-F(E)) | WB, ELISA, Neut | Human Fab |
MRO-142CT-F(E) | Human Anti-TcdA Recombinant Antibody; Fab Fragment (MRO-142CT-F(E)) | WB, ELISA, Neut | Human Fab |
MRO-143CT-F(E) | Human Anti-TcdA Recombinant Antibody; Fab Fragment (MRO-143CT-F(E)) | WB, ELISA, Neut | Human Fab |
MRO-144CT-F(E) | Human Anti-TcdA Recombinant Antibody; Fab Fragment (MRO-144CT-F(E)) | WB, ELISA, Neut | Human Fab |
MRO-145CT-F(E) | Human Anti-TcdA Recombinant Antibody; Fab Fragment (MRO-145CT-F(E)) | WB, ELISA, Neut | Human Fab |
CAT | Product Name | Application | Type |
---|---|---|---|
HPAB-0854LY-S(P) | Human Anti-TcdA Recombinant Antibody; scFv Fragment (HPAB-0854LY-S(P)) | Neut, ELISA | Human scFv |
HPAB-0855LY-S(P) | Human Anti-TcdA Recombinant Antibody; scFv Fragment (HPAB-0855LY-S(P)) | Neut, ELISA | Human scFv |
HPAB-AP192-YC-S(P) | Human Anti-TcdA Recombinant Antibody; scFv Fragment (HPAB-AP192-YC-S(P)) | WB, Neut | Human scFv |
CAT | Product Name | Application | Type |
---|---|---|---|
HPAB-0854LY | Human Anti-TcdA Recombinant Antibody (HPAB-0854LY) | Neut, ELISA | Human IgG1 |
HPAB-0855LY | Human Anti-TcdA Recombinant Antibody (HPAB-0855LY) | Neut, ELISA | Human IgG1 |
HPAB-AP192-YC | Human Anti-TcdA Recombinant Antibody (HPAB-AP192-YC) | WB, Neut | Human IgG |
CAT | Product Name | Application | Type |
---|---|---|---|
HPAB-0854LY-F(E) | Human Anti-TcdA Recombinant Antibody; Fab Fragment (HPAB-0854LY-F(E)) | Neut, ELISA | Human Fab |
HPAB-0855LY-F(E) | Human Anti-TcdA Recombinant Antibody; Fab Fragment (HPAB-0855LY-F(E)) | Neut, ELISA | Human Fab |
HPAB-AP192-YC-F(E) | Human Anti-TcdA Recombinant Antibody; Fab Fragment (HPAB-AP192-YC-F(E)) | WB, Neut | Human Fab |
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