Citation

Lee CH, Lee YC, Liang MH, Leu SJ, Lin LT, Chiang JR, Yang YY. Appl. Environ. Microbiol. 2015; 82(1): 71-80.

Affiliation

Core Facility for antibody Generation and Research, Taipei Medical University, Taipei, Taiwan. School of Medical Laboratory Sciences and Biotechnology, Taipei Medical University, Taipei, Taiwan. Department of Laboratory Medicine, Wan Fang Hospital, Taipei Medical University, Taipei, Taiwan. yangyuan@tmu.edu.tw.

Copyright

(Copyright © 2015, American Society for Microbiology)

DOI

10.1128/AEM.02608-15

PMID

26475102

Abstract

Snake venom protein from Deinagkistrodon acutus (DA), one of the major venomous species in Taiwan, causes hemorrhagic symptoms leading to death. Although horse-derived antivenin is a major treatment, relatively high 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 three 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 bio-panning, 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.

Language: en