Abstract
Traditional, horse-derived antivenin is currently the most efficient treatment against snake bites. However, it is costly and has unpredictable side effects. Thus, alternative, cost-effective strategies for producing antivenin are needed. In this study, we immunized hens with inactivated NNA venom proteins from the cobra Naja naja atra (NNA). Purified yolk IgY antibodies showed specific anti-NNA binding activity comparable to that of the equine-derived antivenin. We used phage display technology to generate two antibody libraries containing 9.0 × 108 and 8.4 × 108 clones with a short or long linker, respectively. The phage ELISA indicated that anti-NNA clones displaying single-chain variable fragments (scFv) were significantly enriched after biopanning. The nucleotide sequences of the light and heavy chain genes of 30 monoclonal scFv antibodies were determined and classified into six groups with the short linker and nine groups with the long linker. These scFv clones specifically bound to NNA proteins but not to venom proteins from other snakes. Their binding affinities were further determined by competitive ELISA. Animal model studies showed that anti-NNA IgY antibodies exhibited complete protective effects, while a combination of scFv antibodies raised the survival rates and times of mice challenged with lethal doses of NNA venom proteins.
Highlights
Snake envenomation is considered a neglected tropical disease and is an often-discussed medical issue, especially in tropical regions [1,2]
In an attempt to evaluate an alternative strategy for neutralizing the toxicity of venom proteins, and to develop reagents for the rapid diagnosis of snakebites, we used attenuated Naja naja atra (NNA) venom proteins administered to chickens to produce specific polyclonal immunoglobulin Y (IgY), while monoclonal scFv was obtained from the antibody libraries constructed by phage display technology
These IgY and scFv antibodies were assessed for their binding activities to six snake venom proteins and for their neutralization activities to determine their protective efficacy by injecting mice with a minimum lethal dose (MLD)
Summary
Snake envenomation is considered a neglected tropical disease and is an often-discussed medical issue, especially in tropical regions [1,2]. In an attempt to evaluate an alternative strategy for neutralizing the toxicity of venom proteins, and to develop reagents for the rapid diagnosis of snakebites, we used attenuated NNA venom proteins administered to chickens to produce specific polyclonal IgY, while monoclonal scFv was obtained from the antibody libraries constructed by phage display technology. These IgY and scFv antibodies were assessed for their binding activities to six snake venom proteins and for their neutralization activities to determine their protective efficacy by injecting mice with a minimum lethal dose (MLD). These model IgY and scFv antibodies were created with the hope that they could potentially be applied to the future development of alternative rapid diagnostic and therapeutic reagents for snake envenomation
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