Antikor Bazlı BoNT Tespit Sistemi için Peptit Antijenlerin Kullanımı

Abstract

Amaç: Bu çalışmada, doğal yapılı BoNT A toksininin kendisi yerine biyoinformatik araçlar kullanılarak tasarlanan peptitleri kullanarak BoNT'ye özgü bir bağışıklık tepkisi ortaya çıkarmayı ve bu anti-peptit antikorlarının bir toksin tespit sisteminde kullanımını göstermeyi amaçladık. Yöntem: BoNT A'nın LC, HN ve HC bölgelerine özgü sentetik epitopik bölgeler, IEDB'den B hücresi "Epitop Tahmin Araçları" kullanılarak antijen olarak seçildi ve bir yazılımla (Discovery Studio 4.0) BoNT A yüzeyinde olduğu gösterildi. Seçilen peptidler, fare bağışıklamalarında kullanıldı ve geliştirilen anti-peptid antikorları ile doğal yapılı BoNT A arasındaki ilişki incelendi. Bulgular: Farelerde üç farklı peptide (P1, P2 ve P3) karşı geliştirilen anti-peptid antikorları ile mililitrede pikogram seviyelerinde doğal yapılı BoNT A'nın saptanması gerçekleştirildi. Sonuç: Bu çalışma, sentetik peptitlerin, toksinlere karşı yüksek afiniteli antikorları geliştirmek için en az doğal toksin veya toksoidin kendisi kadar etkili olduğunu göstermektedir. Ayrıca botulizmin hızlı teşhisine duyulan ihtiyaç ve halihazırda kullanılan test sistemlerinde çok sayıda deney hayvanının kurban edildiği göz önüne alındığında, bu sonuçlar hem hayvan sayısını hem de toksin kullanım miktarını azaltmak için sentetik peptit immünojenlerinin kullanılmasının gerekliliğini ortaya koymaktadır.

Keywords

• Botulinum Nörotoksin A
• Toksin Tespiti
• Sentetik Peptitler
• Botulizm
• antikor üretimi

Usage of Peptide Antigens for Antibody-Based BoNT Detection System

Abstract

Objective: In the current study, we aimed to elicit a BoNT-specific immune response via using peptides designed by using bioinformatics tools instead of using intact native BoNT A toxin itself and to demonstrate the usage of these anti-peptide antibodies in a toxin detection system. Methods: Synthetic epitopic regions specific to LC, HN and HC regions of BoNT A were selected as antigen using B cell “Epitope Prediction Tools” form IEDB and shown to be on the surface of BoNT A with a software (Discovery Studio 4.0). Selected peptides were used in mice immunizations and the interaction between developed anti-peptide antibodies and the native intact BoNT A was examined. Results: The detection of native intact BoNT A at picogram levels per milliliter was performed with anti-peptide antibodies developed against three different peptides (P1, P2, and P3) in mice. Conclusion: The current study shows that synthetic peptides are at least as effective as the native toxin or the toxoid itself for raising high-affinity antibodies against toxins. In addition, considering the need for a quick diagnosis of botulism and, already used test systems in which many experimental animals are sacrificed, these results demonstrate the necessity of synthetic peptide immunogens usage to reduce both the number of animals and the amount of toxin usage.

Keywords

• Botulinum Neurotoxin A
• Toxin Detection
• Synthetic Peptides
• Antibody Generation
• Botulism

References

1. Bigalke H, Rummel A. Medical aspects of toxin weapons. Toxicology. 2005;214(3):210-220. doi:10.1016/j.tox.2005.06.015
2. Middlebrook JL, Franz DR. Botulinum Toxins. Med. Aspects of Chem. and Biol. Warfare. 1997; 603-76.
3. White SM. Chemical and biological weapons. Implications for anaesthesia and intensive care. Br J Anaesth. 2002;89(2):306-324. doi:10.1093/bja/aef168
4. Patocka J, Splino M. Botulinum Toxin: From Poison to Medicinal Agent. The ASA Newsletter. 2002; 88: 14-24.
5. Gu S, Rumpel S, Zhou J, et al. Botulinum neurotoxin is shielded by NTNHA in an interlocked complex. Science. 2012;335(6071):977-981. doi:10.1126/science.1214270
6. Schiavo G, Benfenati F, Poulain B, et al. Tetanus and botulinum-B neurotoxins block neurotransmitter release by proteolytic cleavage of synaptobrevin. Nature. 1992;359(6398):832-835. doi:10.1038/359832a0
7. Blasi J, Chapman ER, Link E, et al. Botulinum neurotoxin A selectively cleaves the synaptic protein SNAP-25. Nature. 1993;365(6442):160-163. doi:10.1038/365160a0
8. Simpson LL. Clinically relevant aspects of the mechanism of action of botulinum neurotoxin. Journal of Voice. 1992; 6(4); 358-364. doi:10.1016/s0892-1997(05)80034-7

9. Hong W. SNAREs and traffic [published correction appears in Biochim Biophys Acta. 2005 Jul 10;1744(3):465]. Biochem Biophys Acta. 2005;1744(2):120-144. doi:10.1016/j.bbamcr.2005.03.014
10. Elias M, Al-Saleem F, Ancharski DM, et al. Evidence that botulinum toxin receptors on epithelial cells and neuronal cells are not identical: implications for development of a non-neurotropic vaccine. J Pharmacol Exp Ther. 2011;336(3):605-612. doi:10.1124/jpet.110.175018
11. Arnon SS, Schechter R, Inglesby TV, et al. Botulinum toxin as a biological weapon: medical and public health management [published correction appears in JAMA 2001 Apr 25;285(16):2081]. JAMA. 2001;285(8):1059-1070. doi:10.1001/jama.285.8.1059
12. Schantz EJ, Johnson EA. Properties and use of botulinum toxin and other microbial neurotoxins in medicine. Microbiol Rev. 1992;56(1):80-99.
13. Solomon HM, Lilly TJ. Clostridium botulinum. Bacteriological analytical manual. FDA. 2001. Available from: https://www.fda.gov/food/laboratory-methods-food/bam-chapter-17-clostridium-botulinum.
14. Marks JD. Medical aspects of biologic toxins. Anesthesiol Clin North Am. 2004;22(3):509-vii. doi:10.1016/j.atc.2004.05.010
15. Keller JE. Characterization of new formalin-detoxified botulinum neurotoxin toxoids. Clin Vaccine Immunol. 2008;15(9):1374-1379. doi:10.1128/CVI.00117-08
16. Cai H, Reinisch K, Ferro-Novick S. Coats, tethers, Rabs, and SNAREs work together to mediate the intracellular destination of a transport vesicle. Dev Cell. 2007;12(5):671-682. doi:10.1016/j.devcel.2007.04.005
17. Čapek P, J. Dickerson T. Correction: Čapek, P., et al. Sensing the Deadliest Toxin: Technologies for Botulinum Neurotoxin Detection.Toxins. 2010; 2(1):93-94. https://doi.org/10.3390/toxins2010093
18. Diaz-Amigo C. Antibody-Based Detection Methods: From Theory to Practice. Mol. Bio.Imm. Tech. App. For Food Chemists. 2010; 223-45.
19. Wang YF, Kobayashi M. Antibody Detection: Principles and Applications. Adv. Tech. Diag. Microbiology. 2012:53-73. doi:10.1007/978-1-4614-3970-7_4.
20. Van Regenmortel MH. Synthetic peptides versus natural antigens in immunoassay. Annales de Biologie Clinique. 1993; 51(1); 39-41.
21. Niman HL, Houghten RA, Walker LE, et al. Generation of protein-reactive antibodies by short peptides is an event of high frequency: implications for the structural basis of immune recognition. Proc Natl Acad Sci U S A. 1983;80(16):4949-4953. doi:10.1073/pnas.80.16.4949
22. Hancock DC, OReilly NJ. Synthetic Peptides as Antigens for Antibody Production. Methods Mol Biol. 2005; 295:13–25. doi:10.1385/1-59259-873-0:013.
23. Berman HM, Westbrook J, Feng Z, et al. (2000) The Protein Data Bank Nucleic Acids Res., 28: 235-242.
24. Vita R, Mahajan S, Overton JA, et al. The Immune Epitope Database (IEDB): 2018 update. Nucleic Acids Res. 2018 Oct 24. doi: 10.1093/nar/gky1006.
25. Zarebski LM, Vaughan K, Sidney J, et al. Analysis of epitope information related to Bacillus anthracis and Clostridium botulinum. Expert Rev Vaccines. 2008;7(1):55-74. doi:10.1586/14760584.7.1.55
26. Engvall E. Enzyme immunassay ELISA and EMIT. Imm.Tech. Methods in Enzymology. 1980; 70: doi:10.1016/s0076-6879(80)70067-8.
27. Ramana J, Mehla K. Immunoinformatics and Epitope Prediction. Methods Mol Biol. 2020;2131:155-171. doi:10.1007/978-1-0716-0389-5_6
28. Grant GA. Synthetic Peptides for Production of Antibodies that Recognize Intact Proteins. Curr Protoc Mol Biol .2002; 59: 1-11
29. Chiao DJ, Wey JJ, Shyu RH,et al. Monoclonal antibody-based lateral flow assay for detection of botulinum neurotoxin type A. Hybridoma (Larchmt). 2008;27(1):31-35. doi:10.1089/hyb.2007.0550
30. Sharma SK, Ferreira JL, Eblen BS,et al. Detection of type A, B, E, and F Clostridium botulinum neurotoxins in foods by using an amplified enzyme-linked immunosorbent assay with digoxigenin-labeled antibodies. Appl Environ Microbiol. 2006;72(2):1231-1238. doi:10.1128/AEM.72.2.1231-1238.2006
31. Stanker LH, Merrill P, Scotcher MC, et al. Development and partial characterization of high-affinity monoclonal antibodies for botulinum toxin type A and their use in analysis of milk by sandwich ELISA. J Immunol Methods. 2008;336(1):1-8. doi:10.1016/j.jim.2008.03.003
32. Ayyar BV, Tajhya RB, Beeton C, et al. Antigenic sites on the HN domain of botulinum neurotoxin A stimulate protective antibody responses against active toxin. Sci Rep. 2015;5:15776. Published 2015 Oct 28. doi:10.1038/srep15776
33. Lee BS, Huang JS, Jayathilaka LP, et al. Antibody Production with Synthetic Peptides. Methods Mol Biol. 2016;1474:25-47. doi:10.1007/978-1-4939-6352-2_2
34. Lipman NS, Jackson LR, Trudel LJ, et al. Monoclonal Versus Polyclonal Antibodies: Distinguishing Characteristics, Applications, and Information Resources, ILAR Journal. 2005;46:258–268. https://doi.org/10.1093/ilar.46.3.258