title: Synthetic peptides for in vitro evaluation of the neutralizing potency of Loxosceles antivenoms.
abstract: An important step in the development of therapeutic antivenoms is the pre-clinical testing using in vivo methods to assess their neutralizing potency. For spider antivenoms (Loxosceles species), horse serum potency against the necrotizing activities of Loxosceles intermedia crude venom is currently tested in rabbits. These procedures are time consuming and involve a large number of animals. The aim of this study was to develop an in vitro method to assess the neutralizing potency of anti-Loxosceles sera. We first demonstrated that it was not possible to establish a correlation between the ELISA antibody reactivity of horse anti-Loxosceles serum and their neutralizing potency. We then showed that the antivenoms recognized several peptide epitopes from different regions of SMase-D proteins, which are toxic antigens from Loxosceles venoms. The recognition of some peptides was observed only when high neutralizing potency sera was used. Based on these results, three peptides (peptide 1, DNRRPIWNLAHMVNA and peptide 3, DFSGPYLPSLPTLDA corresponding to residues 2-16 and 164-178, respectively, of SMase-1 protein from Loxosceles laeta, and peptide 2, EFVNLGANSIETDVS corresponding to residues 22-36 of A1H - LoxGa protein from Loxosceles gaucho and LiD1 protein from L. intermedia) were selected. The peptides were synthesized, coupled to bovine serum albumin (BSA), and used as antigens in indirect ELISA to test their reactivity with horse anti-Loxosceles serum of varying neutralizing potencies. We found certain assay conditions that discriminated between the high and low neutralizing potency sera. This study introduced an in vitro and peptide-based neutralization assay for anti-Loxosceles antivenoms.
authors.affiliation: Limq, Laboratório de Imunoquímica, Departamento de Patologia Básica, SCB, UFPR, Curitiba, PR, Brazil.
authors.forename: Juliani Salvini
authors.initials: JS
authors.lastname: Ramada
authors.forename: Alessandra
authors.initials: A
authors.lastname: Becker-Finco
authors.forename: João Carlos
authors.initials: JC
authors.lastname: Minozzo
authors.forename: Liza Figueiredo
authors.initials: LF
authors.lastname: Felicori
authors.forename: Ricardo Andrez
authors.initials: RA
authors.lastname: Machado de Avila
authors.forename: Franck
authors.initials: F
authors.lastname: Molina
authors.forename: Christophe
authors.initials: C
authors.lastname: Nguyen
authors.forename: Juliana
authors.initials: J
authors.lastname: de Moura
authors.forename: Carlos
authors.initials: C
authors.lastname: Chávez-Olórtegui
authors.forename: Larissa Magalhães
authors.initials: LM
authors.lastname: Alvarenga
mesh_terms: D000595:Amino Acid Sequence; D000704:Analysis of Variance; D000818:Animals; D000997:Antivenins / Q000096:biosynthesis* / Q000494:pharmacology*; D019295:Computational Biology; D015195:Drug Design; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000235:genetics / Q000378:metabolism; D006736:Horses / Q000097:blood; D007106:Immune Sera / Q000378:metabolism; D008969:Molecular Sequence Data; D009500:Neutralization Tests / Q000379:methods*; D010455:Peptides / Q000235:genetics / Q000378:metabolism; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism; D012710:Serum Albumin, Bovine; D013111:Spider Venoms / Q000037:antagonists & inhibitors*; D013112:Spiders / Q000737:chemistry* / Q000201:enzymology
chemical_list: D000997:Antivenins; D000939:Epitopes; D007106:Immune Sera; D010455:Peptides; D013111:Spider Venoms; D012710:Serum Albumin, Bovine; D010727:Phosphoric Diester Hydrolases; C055160:sphingomyelin phosphodiesterase D
keywords: Antivenom; Epitope mapping; In vitro neutralization assay; Loxosceles sp.; Synthetic peptides