The Novel Clostridial Neurotoxin Produced by Strain IBCA10-7060 Is Immunologically Equivalent to BoNT/HA
Abstract
:1. Introduction
2. Results
2.1. BoNT/H LC-HN Fragment Expression and Mouse Immunization
2.2. Isolation and Initial Characterization of mAbs from Mice Immunized with BoNT/H LC-HN
2.3. Isolation and Initial Characterization of mAbs from Mice Immunized with BoNT/F LC-HN
2.4. The BoNT/H LC-HN scFv mAbs Bind Nine Non-Overlapping Epitopes
2.5. mAbs Binding BoNT/H LC-HN do not Bind BoNT Serotypes A–G
2.6. Some mAbs can Bind both BoNT/A HC and BoNT/H
3. Discussion
4. Conclusions
5. Materials and Methods
5.1. Materials
5.2. Protein Expression and Purification
5.3. Ethics Statement
5.4. Mouse Immunization, Spleen Harvest, and Antiserum Titer
5.5. scFv library Construction and FACS Sorting
5.6. scFv binding Confirmation and Affinity Determination
5.7. Epitope Overlap Determination
5.8. MAb Affinity Maturation
5.9. Measurement of Solution Phase Affinity at Equilibrium
6. Patents
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Library Name | Immunogens | Library Size |
---|---|---|
BoNT/F libraries | BoNT/F1HC and F1 holotoxin BoNT/F3 LC-HN BoNT/F5 LC-HN BoNT/F6 LC-HN BoNT/F7 LC-HN | 2 × 108 |
BoNT/H library | BoNT/H LC-HN | 5 × 107 |
Domain Specificity | BoNT/F Binding1 | KD on Yeast (nM) | Epitope Group | ||
---|---|---|---|---|---|
H LC-HN | BoNT/Hi | ||||
MAbs from BoNT/H Library | |||||
72A1 | H LC | No | 3.08 | 2.41 | II |
72A7 | H LC-HN | No | 0.78 | 1.79 | IX |
72B4 | H LC-HN | No | 12.5 | >200 | VI |
72B7 | H LC-HN | No | 25.1 | 32.85 | V/IX |
72B8 | H LC-HN | No | 14.2 | 52.4 | IX |
72B12 | H LC-HN | No | 29.4 | 19.5 | IX/IV |
72C4 | H LC-HN | No | 17.1 | 43.5 | VII |
72D10 | H LC-HN | No | 40.8 | No Binding | IX |
72E6 | H LC-HN | No | 0.99 | 0.88 | IX |
72G8 | H LC-HN | No | 5.00 | 2.4 | VIII |
72G9 | H LC | No | 6.62 | 53.9 | I/II |
73C4 | H LC-HN | No | 182 | >100 | V |
73D9 | H LC-HN | No | 30.0 | No Binding | VII/IX |
74F1 | H HN | No | 1.77 | 2.18 | II |
74G6 | H HN | No | 4.05 | 5.50 | III |
MAbs from BoNT/F Libraries | |||||
71B8 | H LC-HN | No | 1490 | No Binding | VIII/IX |
71C7 | H LC-HN | No | 38.8 | No Binding | IV |
71D4 | H LC-HN | No | 184.8 | No Binding | III |
71E4 | H LC | F5 LC-HN | 70.65 (3984) | 68.8 | I |
6H12 | 2.89 (13.24) | 3.07 | |||
71F4 | H LC | No | 124.4 | No Binding | II |
71F4B3 | 34.6 | >200 | |||
71H6 | H LC-HN | No | 20.5 | No Binding | VII/IX |
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Fan, Y.; Barash, J.R.; Conrad, F.; Lou, J.; Tam, C.; Cheng, L.W.; Arnon, S.S.; Marks, J.D. The Novel Clostridial Neurotoxin Produced by Strain IBCA10-7060 Is Immunologically Equivalent to BoNT/HA. Toxins 2020, 12, 9. https://doi.org/10.3390/toxins12010009
Fan Y, Barash JR, Conrad F, Lou J, Tam C, Cheng LW, Arnon SS, Marks JD. The Novel Clostridial Neurotoxin Produced by Strain IBCA10-7060 Is Immunologically Equivalent to BoNT/HA. Toxins. 2020; 12(1):9. https://doi.org/10.3390/toxins12010009
Chicago/Turabian StyleFan, Yongfeng, Jason R. Barash, Fraser Conrad, Jianlong Lou, Christina Tam, Luisa W. Cheng, Stephen S. Arnon, and James D. Marks. 2020. "The Novel Clostridial Neurotoxin Produced by Strain IBCA10-7060 Is Immunologically Equivalent to BoNT/HA" Toxins 12, no. 1: 9. https://doi.org/10.3390/toxins12010009
APA StyleFan, Y., Barash, J. R., Conrad, F., Lou, J., Tam, C., Cheng, L. W., Arnon, S. S., & Marks, J. D. (2020). The Novel Clostridial Neurotoxin Produced by Strain IBCA10-7060 Is Immunologically Equivalent to BoNT/HA. Toxins, 12(1), 9. https://doi.org/10.3390/toxins12010009