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Antibodies against Venom of the Snake Deinagkistrodon acutus.

Posted by on in 2015
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Lee CH1Lee YC2Liang MH3Leu SJ4Lin LT4Chiang JR5Yang YY6. 2015. Appl Environ Microbiol. 82(1):71-80. doi: 10.1128/AEM.02608-15.

  • 1Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, Taipei, Taiwan.
  • 2The Center of Translational Medicine, Taipei Medical University, Taipei, Taiwan Ph.D. Program for Biotechnology in Medicine, College of Medical Science and Technology, Taipei Medical University, Taipei, Taiwan Core Facility for Antibody Generation and Research, Taipei Medical University, Taipei, Taiwan.
  • 3Core Facility for Antibody Generation and Research, Taipei Medical University, Taipei, Taiwan.
  • 4Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, Taipei, Taiwan Department of Microbiology and Immunology, School of Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan.
  • 5Center for Research, Diagnostics and Vaccine Development, Centers for Disease Control, Ministry of Health and Welfare, Taipei, Taiwan.
  • 6Core Facility for Antibody Generation and Research, Taipei Medical University, Taipei, Taiwan School of Medical Laboratory Science and Biotechnology, College of Medical Science and Technology, Taipei Medical University, Taipei, Taiwan Department of Laboratory Medicine, Wan Fang Hospital, Taipei Medical University, Taipei, Taiwan yangyuan@tmu.edu.tw.

Abstract

Snake venom protein from Deinagkistrodon acutus (DA protein), one of the major venomous species in Taiwan, causes hemorrhagic symptoms that can lead to death. Although horse-derived antivenin is a major treatment, relatively strong and detrimental side effects are seen occasionally. In our study, yolk immunoglobulin (IgY) was purified from eggs, and DA protein was recognized using Western blotting and an enzyme-linked immunosorbent assay (ELISA), similar to therapeutic horse antivenin. The ELISA also indicated that specific IgY antibodies were elicited after the fifth booster, plateaued, and lasted for at least 3 months. To generate monoclonal single-chain variable fragment (scFv) antibodies, we used phage display technology to construct two libraries with short or long linkers, containing 6.24 × 10(8) and 5.28 × 10(8) transformants, respectively. After four rounds of biopanning, the eluted phage titer increased, and the phage-based ELISA indicated that the specific clones were enriched. Nucleotide sequences of 30 individual clones expressing scFv were analyzed and classified into four groups that all specifically recognized the DA venom protein. Furthermore, based on mass spectrometry, the scFv-bound protein was deduced to be snake venom metalloproteinase proteins. Most importantly, both IgY and mixed scFv inhibited the lethal effect in mice injected with the minimum lethal dosage of the DA protein. We suggest that together, these antibodies could be applied to the development of diagnostic agents or treatments for snakebite envenomation in the future.

Copyright © 2015, American Society for Microbiology. All Rights Reserved.

PMID:
 
26475102
 
[PubMed - in process]
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