Abstract
In order to develop a recombinant full-length human anti-botulinum neurotoxin A (BoNT/A) antibody, human peripheral blood mononuclear cells (PBMC) were collected from three healthy volunteers and induced for BoNT/A-specific immune response by in vitro immunization. The genes encoding human Fd fragment, consisting of antibody heavy chain variable region and constant region 1 with the genes encoding antibody light chain, were cloned from the immunized PBMC. Afterwards, one combinatory human antigen-binding fragment (Fab) library was constructed using a lambda phage vector system. The size of the constructed library was approximately 105 Escherichia coli transformants. After screening the library by BoNT/A antigen using a plaque lifting with immunostaining approach, 55 clones were identified as positive. The Fab gene of the most reactive clone exhibiting particularly strong BoNT/A binding signal was further subcloned into a full-length human IgG1 antibody gene template in an adenoviral expression vector, in which the heavy and light chains were linked by a foot-and-mouth-disease virus-derived 2A self-cleavage peptide under a single promoter. After the full-length human IgG1 was expressed in mammalian cells and purified with protein L column, sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the heavy and light chains of the antibody were cleaved completely. The affinity expressed as the dissociation constant (K d) for the recombinant human antibody to bind to BoNT/A was determined by indirect enzyme-linked immunosorbent assay and results confirmed that the recombinant full-length human antibody retained BoNT/A-binding specificity with K d value of 10−7 M.
Similar content being viewed by others
References
Hatheway, C. L. (1990). Toxigenic clostridia. Clinical Microbiology Reviews, 3, 66–98.
Valtorta, F., & Arslan, G. (1993). The pharmacology of botulinum toxin. Pharmacological Research, 27, 33–44. doi:10.1006/phrs.1993.1003.
Fujii, N., Kimura, K., Yokosawa, N., Tsuzuki, K., & Oguma, K. (1992). A zinc-protease specific domain in botulinum and tetanus neurotoxins. Toxicon, 30, 1486–1488. doi:10.1016/0041-0101(92)90525-A.
Sugiyama, H. (1980). Clostridium botulinum neurotoxin. Microbiological Reviews, 44, 419–448.
Hayashi, T., McMahon, H., Yamasaki, S., Binz, T., Hata, Y., Sudhof, T. C., et al. (1994). Synaptic vesicle membrane fusion complex: action of clostridial neurotoxins on assembly. The EMBO Journal, 13, 5051–5061.
Arnon, S. S., Schechter, R., Inglesby, T. V., Henderson, D. A., Bartlett, J. G., Ascher, M. S., et al. (2001). Botulinum toxin as a biological weapon: medical and public health management. Journal of the American Medical Association, 285, 1059–1070. doi:10.1001/jama.285.8.1059.
Casadevall, A. (2002). Passive antibody administration (immediate immunity) as a specific defense against biological weapons. Emerging Infectious Diseases, 8, 833–841.
Brown, D. R., Lloyd, J. P., & Schmidt, J. J. (1997). Identification and characterization of a neutralizing monoclonal antibody against botulinum neurotoxin serotype F, following vaccination with active toxin. Hybridoma, 16, 447–456.
Lee, M. S., Lee, J. C., Choi, C. Y., & Chung, J. (2008). Production and characterization of monoclonal antibody to botulinum neurotoxin type B light chain by phage display. Hybridoma, 27, 18–24. doi:10.1089/hyb.2007.0532.
Mah, D. C., Hu, W. G., Pon, J. K., Masri, S. A., Fulton, R. E., Monette, P. L., et al. (2003). Recombinant anti-botulinum neurotoxin A single-chain variable fragment antibody generated using a phage display system. Hybridoma and Hybridomics, 22, 277–283. doi:10.1089/153685903322538791.
Nowakowski, A., Wang, C., Powers, D. B., Amersdorfer, P., Smith, T. J., Montgomery, V. A., et al. (2002). Potent neutralization of botulinum neurotoxin by recombinant oligoclonal antibody. Proceedings of the National Academy of Sciences of the United States of America, 99, 11346–11350. doi:10.1073/pnas.172229899.
Smith, T. J., Lou, J., Geren, I. N., Forsyth, C. M., Tsai, R., Laporte, S. L., et al. (2005). Sequence variation within botulinum neurotoxin serotypes impacts antibody binding and neutralization. Infection and Immunity, 73, 5450–5457. doi:10.1128/IAI.73.9.5450-5457.2005.
Yang, G. H., Kim, K. S., Kim, H. W., Jeong, S. T., Huh, G. H., Kim, J. C., et al. (2004). Isolation and characterization of a neutralizing antibody specific to internalization domain of Clostridium botulinum neurotoxin type B. Toxicon, 44, 19–25. doi:10.1016/j.toxicon.2004.03.016.
Breedveld, F. C. (2000). Therapeutic monoclonal antibodies. Lancet, 355, 735–740. doi:10.1016/S0140-6736(00)01034-5.
Schroff, R. W., Foon, K. A., Beatty, S. M., Oldham, R. K., & Morgan, A. C., Jr. (1985). Human anti-murine immunoglobulin responses in patients receiving monoclonal antibody therapy. Cancer Research, 45, 879–885.
Kuus-Reichel, K., Grauer, L. S., Karavodin, L. M., Knott, C., Krusemeier, M., & Kay, N. E. (1994). Will immunogenicity limit the use, efficacy, and future development of therapeutic monoclonal antibodies? Clinical and Diagnostic Laboratory Immunology, 1, 365–372.
Saleh, M. N., LoBuglio, A. F., Wheeler, R. H., Rogers, K. J., Haynes, A., Lee, J. Y., et al. (1990). A phase II trial of murine monoclonal antibody 17–1A and interferon-gamma: clinical and immunological data. Cancer Immunology, Immunotherapy, 32, 185–190. doi:10.1007/BF01771455.
Adekar, S. P., Al-Saleem, F. H., Elias, M. D., Rybinski, K. A., Simpson, L. L., & Dessain, S. K. (2008). A natural human IgM antibody that neutralizes botulinum neurotoxin in vivo. Hybridoma, 27, 65–69. doi:10.1089/hyb.2007.0549.
Adekar, S. P., Jones, R. M., Elias, M. D., Al-Saleem, F. H., Root, M. J., Simpson, L. L., et al. (2008). A human monoclonal antibody that binds serotype A botulinum neurotoxin. Hybridoma, 27, 11–17. doi:10.1089/hyb.2007.0536.
Adekar, S. P., Takahashi, T., Jones, R. M., Al-Saleem, F. H., Ancharski, D. M., Root, M. J., et al. (2008). Neutralization of botulinum neurotoxin by a human monoclonal antibody specific for the catalytic light chain. PLoS ONE, 3, e3023. doi:10.1371/journal.pone.0003023.
Barbas, C. F., 3rd. (1995). Synthetic human antibodies. Nature Medicine, 1, 837–839. doi:10.1038/nm0895-837.
Burton, D. R., & Barbas, C. F., 3rd. (1994). Human antibodies from combinatorial libraries. Advances in Immunology, 57, 191–280. doi:10.1016/S0065-2776(08)60674-4.
Lonberg, N. (2005). Human antibodies from transgenic animals. Nature Biotechnology, 23, 1117–1125. doi:10.1038/nbt1135.
Burton, D. R., Pyati, J., Koduri, R., Sharp, S. J., Thornton, G. B., Parren, P. W., et al. (1994). Efficient neutralization of primary isolates of HIV-1 by a recombinant human monoclonal antibody. Science, 266, 1024–1027. doi:10.1126/science.7973652.
Crowe, J. E., Jr., Murphy, B. R., Chanock, R. M., Williamson, R. A., Barbas, C. F., 3rd, & Burton, D. R. (1994). Recombinant human respiratory syncytial virus (RSV) monoclonal antibody Fab is effective therapeutically when introduced directly into the lungs of RSV-infected mice. Proceedings of the National Academy of Sciences of the United States of America, 91, 1386–1390. doi:10.1073/pnas.91.4.1386.
Persson, M. A., Caothien, R. H., & Burton, D. R. (1991). Generation of diverse high-affinity human monoclonal antibodies by repertoire cloning. Proceedings of the National Academy of Sciences of the United States of America, 88, 2432–2436. doi:10.1073/pnas.88.6.2432.
Fang, J., Qian, J. J., Yi, S., Harding, T. C., Tu, G. H., VanRoey, M., et al. (2005). Stable antibody expression at therapeutic levels using the 2A peptide. Nature Biotechnology, 23, 584–590. doi:10.1038/nbt1087.
Friguet, B., Chaffotte, A. F., Djavadi-Ohaniance, L., & Goldberg, M. E. (1985). Measurements of the true affinity constant in solution of antigen-antibody complexes by enzyme-linked immunosorbent assay. Journal of Immunological Methods, 77, 305–319. doi:10.1016/0022-1759(85)90044-4.
Yoo, E. M., Chintalacharuvu, K. R., Penichet, M. L., & Morrison, S. L. (2002). Myeloma expression systems. Journal of Immunological Methods, 261, 1–20.
Davies, J. S., Holter, J. L., Knight, D., Beaucourt, S. M., Murphy, D., Carter, D. A., et al. (2004). Manipulating sorting signals to generate co-expression of somatostatin and eGFP in the regulated secretory pathway from a monocistronic construct. Journal of Molecular Endocrinology, 33, 523–532. doi:10.1677/jme.1.01578.
Mizuguchi, H., Xu, Z., Ishii-Watabe, A., Uchida, E., & Hayakawa, T. (2000). IRES-dependent second gene expression is significantly lower than cap-dependent first gene expression in a bicistronic vector. Molecular Therapy, 1, 376–382. doi:10.1006/mthe.2000.0050.
Jerecic, J., Single, F., Kruth, U., Krestel, H., Kolhekar, R., Storck, T., et al. (1999). Studies on conditional gene expression in the brain. Annals of the New York Academy of Sciences, 868, 27–37. doi:10.1111/j.1749-6632.1999.tb11271.x.
Reed, C. D., Rast, H., Hu, W. G., Mah, D., Nagata, L., & Masri, S. A. (2007). Expression of furin-linked Fab fragments against anthrax toxin in a single mammalian expression vector. Protein Expression and Purification, 54, 261–266. doi:10.1016/j.pep.2007.03.007.
Hu, W. G., Chau, D., Wu, J., Jager, S., & Nagata, L. P. (2007). Humanization and mammalian expression of a murine monoclonal antibody against Venezuelan equine encephalitis virus. Vaccine, 25, 3210–3214. doi:10.1016/j.vaccine.2007.01.034.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Hu, WG., Jager, S., Chau, D. et al. Generation of a Recombinant Full-Length Human Antibody Binding to Botulinum Neurotoxin A. Appl Biochem Biotechnol 160, 1206–1216 (2010). https://doi.org/10.1007/s12010-009-8657-1
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s12010-009-8657-1