title: Human ScFv that block sodium ion channel activity of tetrodotoxin.
abstract: Tetrodotoxin (TTX) is a heterocyclic guanidinium alkaloid (C11H17N3O8) with molecular mass of ∼320 Da. The TTX and toxic analogs block sodium ion activity of mammalian nerve cells resulting in failure to conduct nerve impulse which manifested clinically in host as variable degrees of organ paralysis. Human intoxication occurs after consuming food containing the toxins. Current treatment of the poisoning is supportive and symptomatic. There has been no specific drug or antidote for the TTX mediated malady. In this study, phage clones displaying human single chain antibody fragments (HuScFv) were selected from a human ScFv phage display library. HuScFv derived from phagemid transformed Escherichia coli clones (clones s16 and s35) bound to the TTX as tested by indirect ELISA and band shift assay. Homology modeling and molecular docking revealed that VL domain of the s16-HuScFv interacted with the hydroxyl groups of C6, C9, C10 and C11 of the TTX by means of Tyr 223, Ser226 and Tyr228, while the Asp53 and Asp55 of the VH domain of s35-HuScFv interacted with the positions 1 and 2 of the guanidinium group and the hydroxyl groups at C9 and C10 of the TTX. The s16- and s35-HuScFv neutralized the TTX bioactivity in nerve cell based- and mouse bio-assays. Moreover, the HuScFv could rescue the intoxicated mice from the TTX mediated lethality. Thus, the HuScFv derived from the transformed E. coli clones have high potential as a safe, effective and specific therapeutic remedy for TTX intoxication in humans and warrant further trials.
authors: Graduate Program in Immunology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand.
authors: Monrat
authors: M
authors: Chulanetra
authors: Kunan
authors: K
authors: Bangphoomi
authors: Nitat
authors: N
authors: Sookrung
authors: Jeeraphong
authors: J
authors: Thanongsaksrikul
authors: Potjanee
authors: P
authors: Srimanote
authors: Yuwaporn
authors: Y
authors: Sakolvarvaree
authors: Kiattawee
authors: K
authors: Choowongkomon
authors: Wanpen
authors: W
authors: Chaicumpa
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D001681:Biological Assay; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000494:pharmacology; D008297:Male; D051379:Mice; D008813:Mice, Inbred ICR; D008969:Molecular Sequence Data; D012150:Polymorphism, Restriction Fragment Length; D017422:Sequence Analysis, DNA; D057127:Single-Chain Antibodies / Q000494:pharmacology*; D015222:Sodium Channels / Q000187:drug effects* / Q000378:metabolism; D053719:Tandem Mass Spectrometry; D023721:Tetraodontiformes; D013779:Tetrodotoxin / Q000037:antagonists & inhibitors* / Q000633:toxicity
chemical_list: D000911:Antibodies, Monoclonal; D007135:Immunoglobulin Variable Region; D057127:Single-Chain Antibodies; D015222:Sodium Channels; D013779:Tetrodotoxin