Single Chain Antibody Fragment against Venom from the Snake Daboia russelii formosensis

Lee, Chi Hsin; Lee, Yu-Ching; Lee, Yueh-Lun; Leu, Sy-Jye; Lin, Liang-Tzung; Chen, Chi-Ching; Chiang, Jen-Ron; Mwale, Pharaoh Fellow; Tsai, Bor-Yu; Hung, Ching-Sheng; Yang, Yiyuan

doi:10.3390/toxins9110347

Cited by 26 publications

(21 citation statements)

References 51 publications

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“…This process results in only 10-20% of antibodies being specific to venom toxins, with the remainder directed against environmental antigens, and of those specific antibodies only a proportion will actually be specific to the key pathogenic toxins found in any particular venom (87). Thus, much research effort is now focusing upon increasing the specificity of therapies to specific, pathogenically-important, venom toxins, including the novel application of small molecule inhibitors and monoclonal antibodies (88–93). For both these approaches, screening and identification platforms are important for the selection of molecules exhibiting desirable in vitro neutralizing profiles from large drug or antibodies libraries.…”

Section: Resultsmentioning

confidence: 99%

High throughput screening and identification of coagulopathic snake venom proteins and peptides using nanofractionation and proteomics approaches

Slagboom

Mladic

Xie³

et al. 2019

Preprint

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17Snakebite is a neglected tropical disease that results in a variety of systemic and local pathologies in 18 envenomed victims and is responsible for around 138,000 deaths every year. Many snake venoms cause 19 severe coagulopathy that makes victims vulnerable to suffering life-threating haemorrhage. The 20 mechanisms of action of coagulopathic snake venom toxins are diverse and can result in both anticoagulant 21 and procoagulant effects. However, because snake venoms consist of a mixture of numerous protein and 22 peptide components, high throughput characterizations of specific target bioactives is challenging. In this 23 study, we applied a combination of analytical and pharmacological methods to identify snake venom toxins 24 from a wide diversity of snake species that perturb coagulation. To do so, we used a high-throughput 25 screening approach consisting of a miniaturised plasma coagulation assay in combination with a venom 26 nanofractionation approach. Twenty snake venoms were first separated using reversed-phase liquid 27 chromatography, and a post-column split allowed a small fraction to be analyzed with mass spectrometry, 28 while the larger fraction was collected and dispensed onto 384-well plates before direct analysis using a 29 plasma coagulation assay. Our results demonstrate that many snake venoms simultaneously contain both 30 procoagulant and anticoagulant bioactives that contribute to coagulopathy. In-depth identification analysis 31 from seven medically-important venoms, via mass spectrometry and nanoLC-MS/MS, revealed that 32 phospholipase A 2 toxins are frequently identified in anticoagulant venom fractions, while serine protease 33 and metalloproteinase toxins are often associated with procoagulant bioactivities. The nanofractionation 34 and proteomics approach applied herein seems likely to be a valuable tool for the rational development of 35 next-generation snakebite treatments by facilitating the rapid identification and fractionation of 36 coagulopathic toxins, thereby enabling specific targeting of these toxins by new therapeutics such as 37 monoclonal antibodies and small molecule inhibitors. Classified Personnel Information 39Author summary 40 Snakebite is a neglected tropical disease that results in more than 100,000 deaths every year. 41Haemotoxicity is one of the most common signs of systemic envenoming observed after snakebite, and 42 many snake venoms cause severe impairment of the blood coagulation that makes victims vulnerable to 43 suffering life-threating hemorrhage. In this study, we applied a combination of analytical and 44 pharmacological methods to identify snake venom toxins from a wide diversity of snake species that 45 interfere with blood coagulation. Twenty snake venoms were screened for their effects on the blood 46 coagulation cascade and based on the initial results and the medical relevance of the species, seven 47 venoms were selected for in-depth analysis of the responsible toxins using advanced identification 48 techniques. Our findings reveal a number ...

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Section: Resultsmentioning

confidence: 99%

High throughput screening and identification of coagulopathic snake venom proteins and peptides using nanofractionation and proteomics approaches

Slagboom

Mladic

Xie³

et al. 2019

Preprint

View full text Add to dashboard Cite

show abstract

“…This process results in only 10-20% of antibodies being specific to venom toxins, with the remainder directed against environmental antigens, and of those specific antibodies only a proportion will actually be specific to the key pathogenic toxins found in any particular venom [88]. Thus, much research effort is now focusing upon increasing the specificity of therapies to specific, pathogenically-important, venom toxins, including the novel application of small molecule inhibitors and monoclonal antibodies [89][90][91][92][93][94]. For both these approaches, screening and identification platforms are important for the selection of molecules exhibiting desirable in vitro neutralizing profiles from large drug or antibodies libraries.…”

Section: Plos Neglected Tropical Diseasesmentioning

confidence: 99%

High throughput screening and identification of coagulopathic snake venom proteins and peptides using nanofractionation and proteomics approaches

et al. 2020

View full text Add to dashboard Cite

Snakebite is a neglected tropical disease that results in a variety of systemic and local pathologies in envenomed victims and is responsible for around 138,000 deaths every year. Many snake venoms cause severe coagulopathy that makes victims vulnerable to suffering life-threating haemorrhage. The mechanisms of action of coagulopathic snake venom toxins are diverse and can result in both anticoagulant and procoagulant effects. However, because snake venoms consist of a mixture of numerous protein and peptide components, high throughput characterizations of specific target bioactives is challenging. In this study, we applied a combination of analytical and pharmacological methods to identify snake venom toxins from a wide diversity of snake species that perturb coagulation. To do so, we used a high-throughput screening approach consisting of a miniaturised plasma coagulation assay in combination with a venom nanofractionation approach. Twenty snake venoms were first separated using reversed-phase liquid chromatography, and a post-column split allowed a small fraction to be analyzed with mass spectrometry, while the larger fraction was collected and dispensed onto 384-well plates. After fraction collection, any solvent present in the wells was removed by means of freeze-drying, after which it was possible to perform a plasma coagulation assay in order to detect coagulopathic activity. Our results demonstrate that many snake venoms simultaneously contain both procoagulant and anticoagulant bioactives that contribute to coagulopathy. In-depth identification analysis from seven medically-important venoms, via mass spectrometry and nanoLC-MS/MS, revealed that phospholipase A 2 toxins are frequently identified in anticoagulant venom fractions, while serine protease and metalloproteinase toxins are often associated with procoagulant bioactivities. The nanofractionation and proteomics approach applied herein seems likely to be a valuable tool for the rational development of next-generation snakebite treatments by facilitating the rapid identification and fractionation of coagulopathic toxins, thereby enabling PLOS NEGLECTED TROPICAL DISEASES

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“…However, generation of equine anti-venom is costly and associated with several potential side effects. Chicken IgY-scFv has been generated against glutaraldehyde-attenuated Daboia russelii formosensis (DRF) venom proteins for passive immunization, which can identify and neutralize the toxic activity of the venom components, with only small quantity of antigens required to induce a signi cant antibody response in hens, and can be also used as a rapid diagnostic tool for wound secretions to determine snake types (18).…”

Section: Discussionmentioning

confidence: 99%

Generation and Evaluation of Chicken IgY-scFv for the Purposes of CPV Diagnosis and Therapy

Ge¹,

Xu²,

Li³

et al. 2020

Preprint

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Background: Canine parvovirus (CPV) can cause acute and highly contagious enteritis in dog, antibodies have been used for diagnosis and therapy of CPV disease, generation of functional antibody fragments by using novel antibody engineering platforms is promising in veterinary practice. Results: The IgY single chain fragment variables (scFv) were generated by T7 phage display system and expressed in E. coli system after immunizing hens with virus-like particles (VLP) of CPV-VP2. The titer of the primary scFv library reached to 1.5×10 6 pfu/mL, and 95% of the phages contained the target fragments. The CPV-VLP and CPV-VP2 protein showed similar reaction values to the purified scFv in the ELISA test, and the results of ELISA analysis using IgY-scFv toward CPV clinical samples were consistent with commercial immunochromatographic assay (ICA) and PCR detection, the scFv did not show cross reactivity with canine distemper virus (CDV) and canine coronavirus (CCV). IgY-scFv was successfully expressed in CRFK cells, and in the virus suppression assay, 55% of CPV infections were eliminated within 24 hours. Docking results demonstrated that the number of amino acids of the binding sides between scFv and VP2 were AA37 and AA40, respectively. Conclusions: This study revealed the feasibility of a novel functional antibody fragment development strategy by generating diversified avian IgY-scFv libraries towards the pathogenic target of interest for both detection and therapeutic purposes in veterinary medicine.

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Single Chain Antibody Fragment against Venom from the Snake Daboia russelii formosensis

Cited by 26 publications

References 51 publications

High throughput screening and identification of coagulopathic snake venom proteins and peptides using nanofractionation and proteomics approaches

High throughput screening and identification of coagulopathic snake venom proteins and peptides using nanofractionation and proteomics approaches

High throughput screening and identification of coagulopathic snake venom proteins and peptides using nanofractionation and proteomics approaches

Generation and Evaluation of Chicken IgY-scFv for the Purposes of CPV Diagnosis and Therapy

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