Construction and functional evaluation of a single-chain antibody fragment that neutralizes toxin AahI from the venom of the scorpion Androctonus australis hector.
9C2 is a murine monoclonal IgG that participates in the neutralization of Androctonus australis hector scorpion venom. It recognizes AahI and AahIII, two of the three main neurotoxins responsible for almost all the toxicity of the venom when injected into mammals. Using PCR we cloned the antibody variable region coding genes from 9C2 hybridoma cells and constructed a gene encoding a single-chain antibody variable fragment molecule (scFv). This scFv was produced in the periplasm of Escherichia coli in a soluble and functional form and purified in a single step using protein L-agarose beads yielding 1-2 mg.L(-1) of bacterial culture. scFv9C2 was predominantly monomeric but also tended to form dimeric and oligomeric structures, all capable of binding toxin AahI. The affinity of scFv and the parental mAb for toxin AahI and homologous toxin AahIII was of the same magnitude, in the nanomolar range. Similarly, purified forms of scFv9C2 completely inhibited the binding of toxin AahI to rat brain synaptosomes. Finally, scFv9C2 was efficient in protecting mice against the toxic effects of AahI after injection of the toxin and scFv to mice by the intracerebroventricular route in a molar ratio as low as 0.36 : 1. Thus, we produced a recombinant scFv that reproduces the recognition properties of the parent antibody and neutralizes the scorpion neurotoxin AahI, thereby opening new prospects for the treatment of envenomation.
Devaux
C
C
CNRS UMR 6560, University of Méditerranée, Marseille, France. dexaux.c@jean-roche.univ-mrs.fr
Moreau
E
E
Goyffon
M
M
Rochat
H
H
Billiald
P
P
D000595:Amino Acid Sequence; D000818:Animals; D000915:Antibody Affinity; D001483:Base Sequence; D001665:Binding Sites; D001921:Brain / Q000378:metabolism; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004306:Dose-Response Relationship, Immunologic; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D005260:Female; D006825:Hybridomas / Q000378:metabolism; D007128:Immunoglobulin Fragments / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D007074:Immunoglobulin G / Q000737:chemistry / Q000378:metabolism; D020128:Inhibitory Concentration 50; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D009498:Neurotoxins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000276:immunology*; D019897:Periplasm / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D015202:Protein Engineering; D011863:Radioimmunoassay; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D012604:Scorpion Venoms / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000276:immunology*; D012605:Scorpions; D013574:Synaptosomes / Q000378:metabolism
D007128:Immunoglobulin Fragments; D007074:Immunoglobulin G; D009498:Neurotoxins; D011994:Recombinant Proteins; D012604:Scorpion Venoms; C065314:scorpion toxin I, Androctonus