Recombinant Human Antibody (AR2) is capable of binding to C. botulinum botA, expressed in HEK 293 cells. Expressed as the combination of a heavy chain (HC) containing VH from anti-C. botulinum botA mAb and CH1-3 region of human IgG1 and a light chain (LC) encoding VL from anti-C. botulinum botA mAb and CL of human kappa light chain. Exists as a disulfide linked dimer of the HC and LC hetero-dimer under non-reducing condition.
Figure 1 Selection of yeast-displayed BoNT/A HC with mutations that knocked out mAb binding.
BoNT/A HC clones with mutations knocking out binding to mAb 3D12 or mAb AR2 were selected from a library of yeast-displayed BoNT/A HC mutants using FACS. (a) The first round of sorting was designed to merely enrich for yeast displaying BoNT/A HC capable of binding 3D12 and/or AR2 in preparation for subsequent rounds of sorting. This was accomplished by simultaneously staining yeast with mAbs 3D12 and AR2 labeled with Alexa-647 and SV5-Alexa-488. Yeast-displayed BoNT/A HC that were bound by mAbs 3D12 and/or AR2 (APC channel) were gated for selection (gate P2), regardless of the display level (SV5-Alexa-488 (FITC) staining). (b) The second round of sorting was designed to isolate yeast-displayed BoNT/A HC containing mutations that resulted in loss of binding to one of the two mAbs but retained binding to the other BoNT/A mAb. Requiring binding to the second BoNT/A mAb helped ensure that the selected domains were folded properly. This was accomplished by staining yeast with 3D12-Alexa-647 and AR2-Alexa-488. Yeast that lost binding to AR2 (gate P3), or that had lost binding to 3D12 (gate P2) were gated and sorted separately. (c) The final round of sorting was designed to complete the isolation of yeast-displayed BoNT/A HC with mutations that resulted in the loss of binding to one of the two BoNT/A mAbs but retained binding to the other BoNT/A mAb. For the final round of sorting, yeast from second round gates P2 and P3 were stained in separate reactions with 3D12-Alexa-647 and AR2-Alexa-488 mAbs and anti-SV5-epitope tag mAb followed by anti-mouse PE. For sorting of yeast from the second round gate P3 selected for loss of binding to AR2, yeast without binding to AR2 (gate P3, blue dots) but with retained binding to 3D12 (gate P4, purple dots) were selected by intersecting the P3 and P4 gates (yellow dots) and collecting only those yeast in both gates ((c) top panels). Yeast with the same phenotype, but without SV5 binding (truncation mutants, gate P2, green dots) were also sorted. For the final round of sorting of yeast from the second round gate P2 selected for loss of binding to 3D12, yeast without binding to 3D12 (gate P5, yellow dots) but with retained binding to AR2 (gate P4, purple dots) were selected by intersecting the P5 and P4 gates (dark green dots) and collecting only those yeast in both gates ((c) bottom panels). Yeast with the same phenotype, but without SV5 binding (truncation mutants, gate P2, green dots) were also sorted.
Levy, R., Forsyth, C. M., LaPorte, S. L., Geren, I. N., Smith, L. A., & Marks, J. D. (2007). Fine and domain-level epitope mapping of botulinum neurotoxin type A neutralizing antibodies by yeast surface display. Journal of molecular biology, 365(1), 196-210.
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CAT | Product Name | Application | Type |
---|---|---|---|
PNBL-061 | Recombinant Anti-C. botulinum BoNT/A VHH Single Domain Antibody (F12) | WB, FuncS | Llama VHH |
CAT | Product Name | Application | Type |
---|---|---|---|
PABW-016 | Recombinant Human Anti-C. botulinum botA Antibody (CR1) | WB, IHC, IF, FuncS | IgG |
CAT | Product Name | Application | Type |
---|---|---|---|
PFBZ-016 | Recombinant Human Anti-C. botulinum botA Antibody Fab Fragment (AR2) | WB, ELISA | |
PFBW-016 | Recombinant Human Anti-C. botulinum botA Antibody Fab Fragment (CR1) | WB, IHC, IF, FuncS |
CAT | Product Name | Application | Type |
---|---|---|---|
PSBZ-016 | Recombinant Human Anti-C. botulinum botA Antibody scFv Fragment (AR2) | WB, ELISA | scFv |
PSBW-016 | Recombinant Human Anti-C. botulinum botA Antibody scFv Fragment (CR1) | WB, IHC, IF, FuncS | scFv |
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