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Effect of Adjuvants on Antibody Titer of Synthetic Recombinant Light Chain of Botulinum Neurotoxin Type B and its Diagnostic Potential for Botulism

  • Jain, Swati (Biotechnology Division, Defence Research and Development Establishment) ;
  • Ponmariappan, S. (Biotechnology Division, Defence Research and Development Establishment) ;
  • Kumar, Om (Biotechnology Division, Defence Research and Development Establishment) ;
  • Singh, Lokendra (Biotechnology Division, Defence Research and Development Establishment)
  • 투고 : 2010.10.21
  • 심사 : 2011.03.30
  • 발행 : 2011.07.28

초록

Botulism is a neuroparalytic disease caused by Clostridium botulinum, which produces seven (A-G) antigenically diverse neurotoxins (BoNTs). BoNTs are the most poisonous substances known to humans, with a median lethal dose ($LD_{50}$) of approximately 1 ng/kg of body weight. Owing to their extreme potency and lethality, they have the potential to be used as a bioterrorism agent. The mouse bioassay is the gold standard for the detection of botulinum neurotoxins; however, it requires at least 3-4 days for completion. Attempts have been made to develop an ELISA-based detection system, which is potentially an easier and more rapid method of botulinum neurotoxin detection. The present study was designed using a synthetic gene approach. The synthetic gene encoding the catalytic domain of BoNT serotype B from amino acids 1-450 was constructed with PCR overlapping primers (BoNT/B LC), cloned in a pQE30 UA vector, and expressed in an E. coli M15 host system. Recombinant protein production was optimized at 0.5 mM IPTG final concentration, 4 h post induction, resulting in a maximum yield of recombinant proteins. The immunogenic nature of the recombinant BoNT/B LC protein was evaluated by ELISA. Antibodies were raised in BALB/c mice using various adjuvants. A significant rise in antibody titer (p<0.05) was observed in the Alum group, followed by the Titermax Classic group, Freund's adjuvant, and the Titermax Gold group. These developed high-titer antibodies may prove useful for the detection of botulinum neurotoxins in food and clinical samples.

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참고문헌

  1. Agarwal, A. K., A. Goel, A. Kohli, A. Rohtagi, and R. Kumar. 2004. Food-borne botulism. J. Assoc. Physicians India 52: 677-678.
  2. Akdeniz, H., T. Buzgan, M. Tekin, H. Karsen, and M. K. Karahocagil. 2007. An outbreak of botulism in a family in Eastern Anatolia associated with eating suzme yoghurt buried under soil. Scand. J. Infect. Dis. 39: 108-114. https://doi.org/10.1080/00365540600951317
  3. Arnon, S. S., R. Schechter, T. V. Inglesby, D. A. Henderson, J. G. Bartlett, M. S. Ascher, et al. 2001. Botulinum toxin as a biological weapon: Medical and public health management. J. Am. Med. Assoc. 285: 1059-1070. https://doi.org/10.1001/jama.285.8.1059
  4. Barash, J. R., T. W. Tang, and S. S. Arnon. 2005. First case of infant botulism caused by Clostridium baratii type F in California. J. Clin. Microbiol. 43: 4280-4282. https://doi.org/10.1128/JCM.43.8.4280-4282.2005
  5. Binz, T., J. Blasi, S. Yamasaki, A. Baumeister, E. Link, T. C. Sudhof, R. Jahn, and H. Niemann. 1994. Proteolysis of SNAP- 25 by types E and A botulinal neurotoxins. J. Biol. Chem. 269: 1617-1620.
  6. Blasi, J., E. R. Chapman, E. Link, T. Binz, S. Yamasaki, P. De Camilli, T. C. Sudhof, H. Niemann, and R. Jahn. 1993. Botulinum neurotoxin A selectively cleaves the synaptic protein SNAP-25. Nature 365: 160-163. https://doi.org/10.1038/365160a0
  7. Blasi, J., E. R. Chapman, S. Yamasaki, T. Binz, H. Niemann, and R. Jahn. 1993. Botulinum neurotoxin C1 blocks neurotransmitter release by means of cleaving HPC-1/syntaxin. EMBO J. 12: 4821-4828.
  8. Bomford, R. 1980. The comparative selectivity of adjuvants for humoral and cell-mediated immunity. I. Effect on the antibody response to bovine serum albumin and sheep red blood cells of Freund's incomplete and complete adjuvants, alhydrogel, Corynebacterium parvum, Bordetella pertussis, muramyl dipeptide and saponin. Clin. Exp. Immunol. 39: 426-434.
  9. Cai, S., B. R. Singh, and S. Sharma. 2007. Botulism diagnostics: From clinical symptoms to in vitro assays. Crit. Rev. Microbiol. 33: 109-125. https://doi.org/10.1080/10408410701364562
  10. Dillon, P. J. and C. A. Rosen. 1990. A rapid method for the construction of synthetic genes using the polymerase chain reaction. Biotechniques 9: 298, 300.
  11. Doellgast, G. J., M. X. Triscott, G. A. Beard, J. D. Bottoms, T. Cheng, B. H. Roh, M. G. Roman, P. A. Hall, and J. E. Brown. 1993. Sensitive enzyme-linked immunosorbent assay for detection of Clostridium botulinum neurotoxins A, B, and E using signal amplification via enzyme-linked coagulation assay. J. Clin. Microbiol. 31: 2402-2409.
  12. Foran, P., G. W. Lawrence, C. C. Shone, K. A. Foster, and J. O. Dolly. 1996. Botulinum neurotoxin C1 cleaves both syntaxin and SNAP-25 in intact and permeabilized chromaffin cells: Correlation with its blockade of catecholamine release. Biochemistry 35: 2630-2636. https://doi.org/10.1021/bi9519009
  13. Franz, D. R., P. B. Jahrling, A. M. Friedlander, D. J. McClain, D. L. Hoover, W. R. Bryne, J. A. Pavlin, G. W. Christopher, and E. M. Eitzen Jr. 1997. Clinical recognition and management of patients exposed to biological warfare agents. J. Am. Med. Assoc. 278: 399-411. https://doi.org/10.1001/jama.1997.03550050061035
  14. Frean, J., L. Arntzen, J. van den Heever, and O. Perovic. 2004. Fatal type A botulism in South Africa, 2002. Trans. R. Soc. Trop. Med. Hyg. 98: 290-295. https://doi.org/10.1016/S0035-9203(03)00069-5
  15. Gill, D. M. 1982. Bacterial toxins: A table of lethal amounts. Microbiol. Rev. 46: 86-94.
  16. Gilsdorf, J., N. Gul, and L. A. Smith. 2006. Expression, purification, and characterization of Clostridium botulinum type B light chain. Protein Expr. Purif. 46: 256-267. https://doi.org/10.1016/j.pep.2005.09.024
  17. Harandi, A. M., D. Medaglini, and R. J. Shattock. 2010. Vaccine adjuvants: A priority for vaccine research. Vaccine 28: 2363-2366. https://doi.org/10.1016/j.vaccine.2009.12.084
  18. King, L. A., T. Niskanen, M. Junnikkala, E. Moilanen, M. Lindstrom, H. Korkeala, et al. 2009. Botulism and hot-smoked whitefish: A family cluster of type E botulism in France, September 2009. Euro. Surveill. 14(45): pii=19394.
  19. Lacy, D. B. and R. C. Stevens. 1999. Sequence homology and structural analysis of the clostridial neurotoxins. J. Mol. Biol. 291: 1091-1104. https://doi.org/10.1006/jmbi.1999.2945
  20. Montecucco, C. and G. Schiavo. 1994. Mechanism of action of tetanus and botulinum neurotoxins. Molec. Microbiol. 13: 1-8. https://doi.org/10.1111/j.1365-2958.1994.tb00396.x
  21. Montecucco, C. and G. Schiavo. 1995. Structure and function of tetanus and botulinum neurotoxins. Q. Rev. Biophys. 28: 423-472. https://doi.org/10.1017/S0033583500003292
  22. Oguma, K., Y. Fujinaga, and K. Inoue. 1995. Structure and function of Clostridium botulinum toxins. Microbiol. Immunol. 39: 161-168. https://doi.org/10.1111/j.1348-0421.1995.tb02184.x
  23. Rivera, V. R., F. J. Gamez, W. K. Keener, J. A. White, and M. A. Poli. 2006. Rapid detection of Clostridium botulinum toxins A, B, E, and F in clinical samples, selected food matrices, and buffer using paramagnetic bead-based electrochemiluminescence detection. Anal. Biochem. 353: 248-256. https://doi.org/10.1016/j.ab.2006.02.030
  24. Rossetto, O., F. Deloye, B. Poulain, R. Pellizzari, G. Schiavo, and C. Montecucco. 1995. The metallo-proteinase activity of tetanus and botulism neurotoxins. J. Physiol. Paris 89: 43-50. https://doi.org/10.1016/0928-4257(96)80550-X
  25. Sakaguchi, G. 1982. Clostridium botulinum toxins. Pharmacol. Ther. 19: 165-194. https://doi.org/10.1016/0163-7258(82)90061-4
  26. Schiavo, G., F. Benfenati, B. Poulain, O. Rossetto, P. Polverino de Laureto, B. R. DasGupta, and C. Montecucco. 1992. Tetanus and botulinum-B neurotoxins block neurotransmitter release by proteolytic cleavage of synaptobrevin. Nature 359: 832-835. https://doi.org/10.1038/359832a0
  27. Schiavo, G., O. Rossetto, S. Catsicas, P. Polverino de Laureto, B. R. DasGupta, F. Benfenati, and C. Montecucco. 1993. Identification of the nerve terminal targets of botulinum neurotoxin serotypes A, D, and E. J. Biol. Chem. 268: 23784-23787.
  28. Schiavo, G., C. C. Shone, O. Rossetto, F. C. Alexander, and C. Montecucco. 1993. Botulinum neurotoxin serotype F is a zinc endopeptidase specific for VAMP/synaptobrevin. J. Biol. Chem. 268: 11516-11519.
  29. Schmidt, J. J. and K. A. Bostian. 1995. Proteolysis of synthetic peptides by type A botulinum neurotoxin. J. Protein Chem. 14: 703-708. https://doi.org/10.1007/BF01886909
  30. Sharma, S. K., B. S. Eblen, R. L. Bull, D. H. Burr, and R. C. Whiting. 2005. Evaluation of lateral-flow Clostridium botulinum neurotoxin detection kits for food analysis. Appl. Environ. Microbiol. 71: 3935-3941. https://doi.org/10.1128/AEM.71.7.3935-3941.2005
  31. Sheth, A. N., P. Wiersma, D. Atrubin, V. Dubey, D. Zink, G. Skinner, et al. 2008. International outbreak of severe botulism with prolonged toxemia caused by commercial carrot juice. Clin. Infect. Dis. 47: 1245-1251. https://doi.org/10.1086/592574
  32. Singh, B. R. 2000. Intimate details of the most poisonous poison. Nat. Struct. Biol. 7: 617-619. https://doi.org/10.1038/77900
  33. Sobel, J., M. Malavet, and S. John. 2007. Outbreak of clinically mild botulism type E illness from home-salted fish in patients presenting with predominantly gastrointestinal symptoms. Clin. Infect. Dis. 45: e14-e16. https://doi.org/10.1086/518993
  34. Szilagyi, M., V. R. Rivera, D. Neal, G. A. Merrill, and M. A. Poli. 2000. Development of sensitive colorimetric capture ELISAs for Clostridium botulinum neurotoxin serotypes A and B. Toxicon 38: 381-389. https://doi.org/10.1016/S0041-0101(99)00168-3
  35. Vaidyanathan, V. V., K. Yoshino, M. Jahnz, C. Dorries, S. Bade, S. Nauenburg, H. Niemann, and T. Binz. 1999. Proteolysis of SNAP-25 isoforms by botulinum neurotoxin types A, C, and E: Domains and amino acid residues controlling the formation of enzyme-substrate complexes and cleavage. J. Neurochem. 72: 327-337. https://doi.org/10.1046/j.1471-4159.1999.0720327.x
  36. Vugia, D. J., S. R. Mase, B. Cole, J. Stiles, J. Rosenberg, L. Velasquez, A. Radner, and G. Inami. 2009. Botulism from drinking pruno. Emerg. Infect. Dis. 15: 69-71. https://doi.org/10.3201/eid1501.081024

피인용 문헌

  1. Production of recombinant botulism antigens: A review of expression systems vol.28, pp.None, 2011, https://doi.org/10.1016/j.anaerobe.2014.06.003
  2. Development of immunodetection system for botulinum neurotoxin serotype E vol.147, pp.6, 2011, https://doi.org/10.4103/ijmr.ijmr_1375_16