title: Humanized-single domain antibodies (VH/VHH) that bound specifically to Naja kaouthia phospholipase A2 and neutralized the enzymatic activity.
abstract: Naja kaouthia (monocled cobra) venom contains many isoforms of secreted phospholipase A2 (sPLA(2)). The PLA(2) exerts several pharmacologic and toxic effects in the snake bitten subject, dependent or independent on the enzymatic activity. N. kaouthia venom appeared in two protein profiles, P3 and P5, after fractionating the venom by ion exchange column chromatography. In this study, phage clones displaying humanized-camel single domain antibodies (VH/V(H)H) that bound specifically to the P3 and P5 were selected from a humanized-camel VH/V(H)H phage display library. Two phagemid transfected E. coli clones (P3-1 and P3-3) produced humanized-V(H)H, while another clone (P3-7) produced humanized-VH. At the optimal venom:antibody ratio, the VH/V(H)H purified from the E. coli homogenates neutralized PLA(2) enzyme activity comparable to the horse immune serum against the N. kaouthia holo-venom. Homology modeling and molecular docking revealed that the VH/V(H)H covered the areas around the PLA(2) catalytic groove and inserted their Complementarity Determining Regions (CDRs) into the enzymatic cleft. It is envisaged that the VH/V(H)H would ameliorate/abrogate the principal toxicity of the venom PLA(2) (membrane phospholipid catabolism leading to cellular and subcellular membrane damage which consequently causes hemolysis, hemorrhage, and dermo-/myo-necrosis), if they were used for passive immunotherapy of the cobra bitten victim. The speculation needs further investigations.
authors.affiliation: Graduate Program in Immunology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand. sandwashed@hotmail.com
authors.forename: Charnwit
authors.initials: C
authors.lastname: Chavanayarn
authors.forename: Jeeraphong
authors.initials: J
authors.lastname: Thanongsaksrikul
authors.forename: Kanyarat
authors.initials: K
authors.lastname: Thueng-In
authors.forename: Kunan
authors.initials: K
authors.lastname: Bangphoomi
authors.forename: Nitat
authors.initials: N
authors.lastname: Sookrung
authors.forename: Wanpen
authors.initials: W
authors.lastname: Chaicumpa
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D061067:Antibodies, Monoclonal, Humanized / Q000737:chemistry*; D002162:Camelus; D003001:Cloning, Molecular; D022801:Complementarity Determining Regions / Q000378:metabolism; D004546:Elapid Venoms / Q000737:chemistry / Q000201:enzymology*; D017815:Elapidae; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006461:Hemolysis / Q000187:drug effects; D006470:Hemorrhage / Q000139:chemically induced; D006736:Horses / Q000276:immunology; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000378:metabolism; D008969:Molecular Sequence Data; D054467:Phospholipases A2 / Q000378:metabolism*; D010743:Phospholipids / Q000378:metabolism; D012150:Polymorphism, Restriction Fragment Length; D011487:Protein Conformation; D061905:Single-Domain Antibodies / Q000737:chemistry*
chemical_list: D061067:Antibodies, Monoclonal, Humanized; D022801:Complementarity Determining Regions; D004546:Elapid Venoms; D007135:Immunoglobulin Variable Region; D010743:Phospholipids; D061905:Single-Domain Antibodies; D054467:Phospholipases A2