Araştırma Makalesi
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Usage of Peptide Antigens for Antibody-Based BoNT Detection System

Yıl 2021, , 242 - 249, 01.11.2021
https://doi.org/10.30934/kusbed.935903

Öz

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.

Destekleyen Kurum

TUBITAK

Proje Numarası

117H001

Teşekkür

We are grateful to Prof. Dr. Selçuk Kılıç and Public Health Institution of Turkey for providing BoNT A.

Kaynakça

  • Bigalke H, Rummel A. Medical aspects of toxin weapons. Toxicology. 2005;214(3):210-220. doi:10.1016/j.tox.2005.06.015
  • Middlebrook JL, Franz DR. Botulinum Toxins. Med. Aspects of Chem. and Biol. Warfare. 1997; 603-76.
  • 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
  • Patocka J, Splino M. Botulinum Toxin: From Poison to Medicinal Agent. The ASA Newsletter. 2002; 88: 14-24.
  • 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
  • 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
  • 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
  • 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
  • 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
  • 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
  • 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
  • Schantz EJ, Johnson EA. Properties and use of botulinum toxin and other microbial neurotoxins in medicine. Microbiol Rev. 1992;56(1):80-99.
  • 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.
  • Marks JD. Medical aspects of biologic toxins. Anesthesiol Clin North Am. 2004;22(3):509-vii. doi:10.1016/j.atc.2004.05.010
  • Keller JE. Characterization of new formalin-detoxified botulinum neurotoxin toxoids. Clin Vaccine Immunol. 2008;15(9):1374-1379. doi:10.1128/CVI.00117-08
  • 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
  • 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
  • Diaz-Amigo C. Antibody-Based Detection Methods: From Theory to Practice. Mol. Bio.Imm. Tech. App. For Food Chemists. 2010; 223-45.
  • 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.
  • Van Regenmortel MH. Synthetic peptides versus natural antigens in immunoassay. Annales de Biologie Clinique. 1993; 51(1); 39-41.
  • 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
  • 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.
  • Berman HM, Westbrook J, Feng Z, et al. (2000) The Protein Data Bank Nucleic Acids Res., 28: 235-242.
  • 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.
  • 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
  • Engvall E. Enzyme immunassay ELISA and EMIT. Imm.Tech. Methods in Enzymology. 1980; 70: doi:10.1016/s0076-6879(80)70067-8.
  • Ramana J, Mehla K. Immunoinformatics and Epitope Prediction. Methods Mol Biol. 2020;2131:155-171. doi:10.1007/978-1-0716-0389-5_6
  • Grant GA. Synthetic Peptides for Production of Antibodies that Recognize Intact Proteins. Curr Protoc Mol Biol .2002; 59: 1-11
  • 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
  • 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
  • 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
  • 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
  • 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
  • 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

Antikor Bazlı BoNT Tespit Sistemi için Peptit Antijenlerin Kullanımı

Yıl 2021, , 242 - 249, 01.11.2021
https://doi.org/10.30934/kusbed.935903

Öz

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.

Proje Numarası

117H001

Kaynakça

  • Bigalke H, Rummel A. Medical aspects of toxin weapons. Toxicology. 2005;214(3):210-220. doi:10.1016/j.tox.2005.06.015
  • Middlebrook JL, Franz DR. Botulinum Toxins. Med. Aspects of Chem. and Biol. Warfare. 1997; 603-76.
  • 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
  • Patocka J, Splino M. Botulinum Toxin: From Poison to Medicinal Agent. The ASA Newsletter. 2002; 88: 14-24.
  • 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
  • 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
  • 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
  • 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
  • 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
  • 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
  • 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
  • Schantz EJ, Johnson EA. Properties and use of botulinum toxin and other microbial neurotoxins in medicine. Microbiol Rev. 1992;56(1):80-99.
  • 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.
  • Marks JD. Medical aspects of biologic toxins. Anesthesiol Clin North Am. 2004;22(3):509-vii. doi:10.1016/j.atc.2004.05.010
  • Keller JE. Characterization of new formalin-detoxified botulinum neurotoxin toxoids. Clin Vaccine Immunol. 2008;15(9):1374-1379. doi:10.1128/CVI.00117-08
  • 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
  • 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
  • Diaz-Amigo C. Antibody-Based Detection Methods: From Theory to Practice. Mol. Bio.Imm. Tech. App. For Food Chemists. 2010; 223-45.
  • 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.
  • Van Regenmortel MH. Synthetic peptides versus natural antigens in immunoassay. Annales de Biologie Clinique. 1993; 51(1); 39-41.
  • 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
  • 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.
  • Berman HM, Westbrook J, Feng Z, et al. (2000) The Protein Data Bank Nucleic Acids Res., 28: 235-242.
  • 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.
  • 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
  • Engvall E. Enzyme immunassay ELISA and EMIT. Imm.Tech. Methods in Enzymology. 1980; 70: doi:10.1016/s0076-6879(80)70067-8.
  • Ramana J, Mehla K. Immunoinformatics and Epitope Prediction. Methods Mol Biol. 2020;2131:155-171. doi:10.1007/978-1-0716-0389-5_6
  • Grant GA. Synthetic Peptides for Production of Antibodies that Recognize Intact Proteins. Curr Protoc Mol Biol .2002; 59: 1-11
  • 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
  • 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
  • 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
  • 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
  • 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
  • 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
Toplam 34 adet kaynakça vardır.

Ayrıntılar

Birincil Dil İngilizce
Konular Mikrobiyoloji
Bölüm Özgün Araştırma / Tıp Bilimleri
Yazarlar

Meryem Işık 0000-0001-9990-6571

Zülal Bilici Bu kişi benim 0000-0002-1957-5758

Naci Çine 0000-0001-9063-1073

Selma Öztürk 0000-0002-7949-8993

Proje Numarası 117H001
Yayımlanma Tarihi 1 Kasım 2021
Gönderilme Tarihi 11 Mayıs 2021
Kabul Tarihi 9 Eylül 2021
Yayımlandığı Sayı Yıl 2021

Kaynak Göster

APA Işık, M., Bilici, Z., Çine, N., Öztürk, S. (2021). Usage of Peptide Antigens for Antibody-Based BoNT Detection System. Kocaeli Üniversitesi Sağlık Bilimleri Dergisi, 7(3), 242-249. https://doi.org/10.30934/kusbed.935903
AMA Işık M, Bilici Z, Çine N, Öztürk S. Usage of Peptide Antigens for Antibody-Based BoNT Detection System. KOU Sag Bil Derg. Kasım 2021;7(3):242-249. doi:10.30934/kusbed.935903
Chicago Işık, Meryem, Zülal Bilici, Naci Çine, ve Selma Öztürk. “Usage of Peptide Antigens for Antibody-Based BoNT Detection System”. Kocaeli Üniversitesi Sağlık Bilimleri Dergisi 7, sy. 3 (Kasım 2021): 242-49. https://doi.org/10.30934/kusbed.935903.
EndNote Işık M, Bilici Z, Çine N, Öztürk S (01 Kasım 2021) Usage of Peptide Antigens for Antibody-Based BoNT Detection System. Kocaeli Üniversitesi Sağlık Bilimleri Dergisi 7 3 242–249.
IEEE M. Işık, Z. Bilici, N. Çine, ve S. Öztürk, “Usage of Peptide Antigens for Antibody-Based BoNT Detection System”, KOU Sag Bil Derg, c. 7, sy. 3, ss. 242–249, 2021, doi: 10.30934/kusbed.935903.
ISNAD Işık, Meryem vd. “Usage of Peptide Antigens for Antibody-Based BoNT Detection System”. Kocaeli Üniversitesi Sağlık Bilimleri Dergisi 7/3 (Kasım 2021), 242-249. https://doi.org/10.30934/kusbed.935903.
JAMA Işık M, Bilici Z, Çine N, Öztürk S. Usage of Peptide Antigens for Antibody-Based BoNT Detection System. KOU Sag Bil Derg. 2021;7:242–249.
MLA Işık, Meryem vd. “Usage of Peptide Antigens for Antibody-Based BoNT Detection System”. Kocaeli Üniversitesi Sağlık Bilimleri Dergisi, c. 7, sy. 3, 2021, ss. 242-9, doi:10.30934/kusbed.935903.
Vancouver Işık M, Bilici Z, Çine N, Öztürk S. Usage of Peptide Antigens for Antibody-Based BoNT Detection System. KOU Sag Bil Derg. 2021;7(3):242-9.