Chicken antibodies against venom proteins of Trimeresurus stejnegeri in 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 × 10 7 and 6.8 × 10 7 antibody clones, respectively. An increase in the titers of eluted phage indicated anti-TS clones remarkably enriched after 2 nd 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.

Development of a Monoclonal scFv against Cytotoxin to Neutralize Cytolytic Activity Induced by Naja atra Venom on Myoblast C2C12 Cells

“…The drawbacks of conventional antivenoms have led to attempts to develop alternative therapeutic reagents for snakebite treatment, including egg IgY, monoclonal antibodies, small molecule drugs, and synthetic nanoparticles [ 30 , 31 , 32 , 33 , 34 , 35 ]. Egg IgY-based antivenom is easier and less costly to manufacture than conventional antivenom.…”

“…The generation of the scFv library and the selection of anti-CTX scFv was performed according to the procedures as described in previous studies [ 33 , 50 , 51 ]. Briefly, hens were sacrificed after the final immunization.…”

“…Phage pellets were transferred into PBS containing 1% bovine serum albumin (BSA) and 20% glycerol and stored at −20 °C for further biopanning. The procedure of scFv biopanning was performed as described in previous reports [ 33 , 50 , 51 ]. In brief, CTX protein (400 ng/well) was coated on a 96-well microplate and blocked with 1% BSA in a PBS buffer.…”

Advances in Chicken IgY-Based Immunoassays for the Detection of Chemical and Biological Hazards in Food Samples

“…Compared with hybridoma technology, phage display, which does not require cell fusion and has the advantages of convenience, rapidity, inexpensiveness, etc., is more popular for producing chicken mAbs. , The detailed preparation of a chicken single-chain variable fragment (scFv) by phage display is shown in Figure . Compared with mice and rabbits, chickens can produce high-affinity scFvs against various highly conserved mammalian proteins owing to phylogenetic differences. , In recent years, some chicken scFvs have been used for developing immunoassays. − However, it should be noted that the performance of immunoassays based on not only chicken scFvs but also marine scFvs were usually shown to be inferior to their corresponding counterparts based on the intact Ig molecule due to the instability of scFvs Despite this, scFvs possess one strong advantage: user customizability. Over recent decades, advances in protein engineering, which received the Nobel Prize in Chemistry in 2018, have greatly improved the ability to manipulate antibody performance.…”