title: Immunodetection of the "brown" spider (Loxosceles intermedia) dermonecrotoxin with an scFv-alkaline phosphatase fusion protein.
abstract: Bites by spiders from Loxosceles genus often lead to a wide variance in envenomation profile of patients and diagnosis is difficult due to the number of diseases that mimic loxoscelism. In such a context, it is of interest to consider the design of standardized recombinant colorimetric antibodies for diagnosis and specific detection of individual circulating toxins in biological fluids of envenomed patients. We have previously prepared a monoclonal murine IgG (LiMab7) that reacts with Loxosceles intermedia venom components of 32-35kDa and neutralizes the dermonecrotic activity of the venom. Here, we re-engineered LiMab7 into a colorimetric bifunctional protein consisting in the corresponding single-chain antibody fragment (scFv) fused to alkaline phosphatase (AP) of Escherichia coli. The immune tracer was tested in two different types of immunoassays and it proved to be efficient in both. Thus, this recombinant fusion protein (scFv-LiMab7/AP) can be used for rapid and specific immunotitration of L. intermedia venom with a linear range of 39-20000ng/mL and a detection limit of 39ng/mL without any cross-reaction.
authors.affiliation: Laboratório de Imunoquímica, Departamento de Patologia Básica, Universidade Federal do Paraná, Curitiba CEP 81531-980, PR, Brazil.
authors.forename: Isabella
authors.initials: I
authors.lastname: Jiacomini
authors.affiliation: Laboratório de Imunoquímica, Departamento de Patologia Básica, Universidade Federal do Paraná, Curitiba CEP 81531-980, PR, Brazil.
authors.forename: Sabrina K
authors.initials: SK
authors.lastname: Silva
authors.affiliation: Université de Tours-INRA UMR 1282, Infectiologie et Santé Publique, 37200 Tours, France.
authors.forename: Nicolas
authors.initials: N
authors.lastname: Aubrey
authors.affiliation: Muséum national d'Histoire naturelle, UMR MNHN-CNRS 7245, 12 rue Buffon, Paris 75231, France.
authors.forename: Julien
authors.initials: J
authors.lastname: Muzard
authors.affiliation: Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil.
authors.forename: Carlos
authors.initials: C
authors.lastname: Chavez-Olortegui
authors.affiliation: Laboratório de Imunoquímica, Departamento de Patologia Básica, Universidade Federal do Paraná, Curitiba CEP 81531-980, PR, Brazil.
authors.forename: Juliana
authors.initials: J
authors.lastname: De Moura
authors.affiliation: Muséum national d'Histoire naturelle, UMR MNHN-CNRS 7245, 12 rue Buffon, Paris 75231, France; Université Paris-Sud, Faculté de Pharmacie, 5 rue J.-B. Clément, 92296 Châtenay-Malabry, France. Electronic address: billiald@mnhn.fr.
authors.forename: Philippe
authors.initials: P
authors.lastname: Billiald
authors.affiliation: Laboratório de Imunoquímica, Departamento de Patologia Básica, Universidade Federal do Paraná, Curitiba CEP 81531-980, PR, Brazil. Electronic address: lmalvarenga@ufpr.br.
authors.forename: Larissa M
authors.initials: LM
authors.lastname: Alvarenga
mesh_terms: D000469:Alkaline Phosphatase / Q000235:genetics; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D057134:Antibodies, Neutralizing / Q000378:metabolism; D063287:Brown Recluse Spider / Q000502:physiology*; D003937:Diagnosis, Differential; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D051379:Mice; D009498:Neurotoxins / Q000032:analysis* / Q000276:immunology; D010727:Phosphoric Diester Hydrolases / Q000032:analysis* / Q000276:immunology; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012015:Reference Standards; D012680:Sensitivity and Specificity; D057127:Single-Chain Antibodies / Q000235:genetics / Q000378:metabolism; D012867:Skin / Q000378:metabolism* / Q000473:pathology; D001098:Spider Bites / Q000175:diagnosis* / Q000276:immunology; D013111:Spider Venoms / Q000032:analysis* / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; D057134:Antibodies, Neutralizing; D009498:Neurotoxins; D011993:Recombinant Fusion Proteins; D057127:Single-Chain Antibodies; D013111:Spider Venoms; C055161:loxosceles venom; D000469:Alkaline Phosphatase; D010727:Phosphoric Diester Hydrolases
keywords: Detection; Immunoassay; Immunotracer; Loxosceles sp.; Quantification; scFv