Chicken antibodies against venom proteins of Trimeresurus stejnegeri in Taiwan.

BACKGROUND: The venom of bamboo vipers (Trimeresurus stejnegeri - TS), commonly found in Taiwan, contains deadly hemotoxins that cause severe envenomation. Equine-derived 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.

Chicken antibodies against venom proteins of Trimeresurus stejnegeri in Taiwan

The venom of bamboo vipers (Trimeresurus stejnegeri - TS), commonly found in Taiwan, contains deadly hemotoxins that cause severe envenomation. Equine-derived 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.(AU)

Chicken antibodies against venom proteins of Trimeresurus stejnegeri in Taiwan

The venom of bamboo vipers (Trimeresurus stejnegeri - TS), commonly found in Taiwan, contains deadly hemotoxins that cause severe envenomation. Equine-derived 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.(AU)

Snake venomics and antivenomics of Protobothrops mucrosquamatus and Viridovipera stejnegeri from Taiwan: keys to understand the variable immune response in horses.

The proteomes of the venoms of the snakes Viridovipera stejnegeri and Protobothrops mucrosquamatus from Taiwan were characterized by N-terminal sequencing, MALDI-TOF mass fingerprinting, and collision-induced dissociation tandem mass spectrometry of in-gel generated tryptic peptides. Proteins belonging to the following toxin classes were identified: metalloproteinase, phospholipase A(2) (PLA(2)), serine proteinase, C-type lectin-like, CRISP, l-amino acid oxidase, disintegrin, and peptides (vasoactive and inhibitors of SVMPs). Nine horses were immunized with a mixture of these venoms. All horses developed a satisfactory immune response against lethality of the venom of V. stejnegeri, whereas only three horses reached the accepted neutralizing potency against the venom of P. mucrosquamatus. Antivenoms were prepared from pools of 'good responder' (GR) and 'poor responder' (PR) horses and compared by antivenomics and neutralization tests. A similar neutralizing response was observed between the GR and PR antivenoms against the venom of V. stejnegeri, whereas antivenom from PR had a lower neutralizing activity against effects of P. mucrosquamatus venom than antivenom from GR. The low potency of the plasma of some horses against this venom is a consequence of the low immunogenicity of the neurotoxic PLA(2) trimucrotoxin. Our results provide clues for innovating the immunization scheme to generate improved antivenoms.