Chicken antibodies against venom proteins of Trimeresurus stejnegeri in Taiwan

 

Chi-Hsin Lee1,2, Chia-I Liu1,2, Sy-Jye Leu3,4, Yu-Ching Lee5, Jen-Ron Chiang6, Liao-Chun Chiang7, Yan-Chiao Mao8, Bor-Yu Tsai9, Ching-Sheng Hung10, Chi-Ching Chen11, Yi-Yuan Yang1,2,12

 

1 School of Medical Laboratory Science and Biotechnology, College of Medical Science and Technology, Taipei Medical University, Taipei, Taiwan.

2 Graduate Program in Medical Biotechnology, College of Medical Science and Technology, Taipei Medical University, Taipei, Taiwan.

3 Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, Taipei, Taiwan.

4 Department of Microbiology and Immunology, College of Medicine, Taipei Medical University, Taipei, Taiwan.

5 The Center of Translational Medicine, Taipei Medical University, Taipei, Taiwan.

6 Center for Research, Diagnostics and Vaccine Development, Centers for Disease Control, Ministry of Health and Welfare, Taiwan.

7 College of Life Sciences, National Tsing Hua University, Hsinchu, Taiwan.

8 Division of Clinical Toxicology, Department of Emergency Medicine, Taichung Veterans General Hospital, Taichung, Taiwan.

9 Navi Bio-Therapeutics Inc., Taipei, Taiwan.

10 Department of Laboratory Medicine, Wan Fang Hospital, Taipei Medical University, Taipei, Taiwan.

11 Department of Pathology and Laboratory Medicine, Landseed Hospital, Taoyuan, Taiwan.

12 Core Laboratory of Antibody Generation and Research, Taipei Medical University, Taipei, Taiwan.

 

Abstract

Background: The venom of bamboo vipers (Trimeresurus stejnegeri – TS), commonly found in Taiwan, contains deadly hemotoxins that cause severe envenomation. Equinederived antivenom is a specific treatment against snakebites, but its production costs are high and there are some inevitable side effects. The aim of the present work is to help in the development of an affordable and more endurable therapeutic strategy for snakebites.

 

Methods: T. stejnegeri venom proteins were inactivated by glutaraldehyde in order to immunize hens for polyclonal immunoglobulin (IgY) antibodies production. After IgY binding assays, two antibody libraries were constructed expressing single-chain variable fragment (scFv) antibodies joined by the short or long linker for use in phage display antibody technology. Four rounds of biopanning were carried out. The selected scFv antibodies were then further tested for their binding activities and neutralization assays to TS proteins.

 

Results: Purified IgY from egg yolk showed the specific binding ability to TS proteins. The dimensions of these two libraries contain 2.4 × 107 and 6.8 × 107 antibody clones, respectively. An increase in the titers of eluted phage indicated anti-TS clones remarkably enriched after 2nd panning. The analysis based on the nucleotide sequences of selected scFv clones indicated that seven groups of short linkers and four groups of long linkers were identified. The recombinant scFvs showed significant reactivity to TS venom proteins and a cross-reaction to Trimeresurus mucrosquamatus venom proteins. In in vivo studies, the data demonstrated that anti-TS IgY provided 100% protective effects while combined scFvs augmented partial survival time of mice injected with a lethal amount of TS proteins.

 

Conclusion: Chickens were excellent hosts for the production of neutralization antibodies at low cost. Phage display technology is available for generation of monoclonal antibodies against snake venom proteins. These antibodies could be applied in the development of diagnostic kits or as an alternative for snakebite envenomation treatment in the near future.

 

Keywords: Trimeresurus stejnegeri IgY antibody Phage display technology Single-chain variable fragment antibody

 

Correspondence: yangyuan@tmu.edu.tw

 

Received: 15 April 2020; Accepted: 06 October 2020; Published online: 20 November 2020