[Rapid immunotitration of individual toxins from Androctonus australis venom].
Passive immunotherapy against scorpion envenomations is facilitated by the preliminary titration of circulating toxins in envenomed patients. Currently, routinely used ELISA tests allow only the titration of the whole venom, without reference to the toxins which compose the venom and spread variably within the tissue. Taking as a model one of the three toxins responsible for the lethal effects of Androctonus australis hector (Aahl) venom, we developed an ELISA sandwich test based on a fragment of recombining antibody (scFv) consisting of the variable chains of the monoclonal IgG 9C2 coupled to a decapeptide showing high affinity for streptavidine. Conjugate scFvlStrep-tag was prepared by genetic engineering. It was produced in the periplasm of recombining bacteria, in a reproducible way, in a soluble form, at low cost and with an output, after purification, of 0.8 mg/L of bacterial culture. The recombinant protein, of small size (28 kDa), is bifunctional. It preserves a very high affinity for the toxin Aah I (Kd of 2.3 10(-10) M, very close to that of IgG 9C2), yet recognises streptavidine and its conjugate (streptavidine-peroxidase). The titration of the Aahl toxin used an ELISA sandwich test in which the toxin was captured in a specific way by a monoclonal antibody; the immunocomplexes were then detected by recombinant immunoconjugate, thus conferring a high specificity on titration. The test is quick (90 mn), reproducible and sensitive, with a limit of detection of 0.6 toxin (ng.ml-1). This method could be extended to two other lethal toxins of the venom of the scorpion Androctonus australis hector and to those of other species. New perspectives are thus possible for the diagnosis of the envenomations.
Aubrey
N
N
Laboratoire d'études et de recherches sur les arthropodes irradiés(LERAI), Muséum national d'histoire naturelle, 57 rue Cuvier, 75231 Paris, France.
Devaux
C
C
Billiald
P
P
D000818:Animals; D000997:Antivenins / Q000008:administration & dosage*; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay / Q000191:economics / Q000379:methods* / Q000592:standards; D006801:Humans; D007116:Immunization, Passive / Q000379:methods; D007074:Immunoglobulin G / Q000276:immunology; D007135:Immunoglobulin Variable Region / Q000276:immunology; D013058:Mass Spectrometry; D009498:Neurotoxins / Q000032:analysis* / Q000235:genetics / Q000276:immunology; D009842:Oligopeptides / Q000276:immunology; D065008:Scorpion Stings / Q000097:blood / Q000175:diagnosis* / Q000276:immunology / Q000628:therapy; D012604:Scorpion Venoms / Q000032:analysis* / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D012605:Scorpions; D012680:Sensitivity and Specificity; D013997:Time Factors; D017186:Titrimetry / Q000191:economics / Q000379:methods / Q000592:standards
C099517:Ala-Trp-Arg-His-Pro-Gln-Phe-Gly-Gly; D000997:Antivenins; D007074:Immunoglobulin G; D007135:Immunoglobulin Variable Region; D009498:Neurotoxins; D009842:Oligopeptides; D012604:Scorpion Venoms; C065314:scorpion toxin I, Androctonus