title: Generation and molecular characterization of a monoclonal antibody reactive with conserved epitope in sphingomyelinases D from Loxosceles spider venoms.
abstract: We report the production of a neutralizing monoclonal antibody able to recognize the venoms of three major medically important species of Loxosceles spiders in Brazil. The mAb was produced by immunization of mice with a toxic recombinant L. intermedia sphingomyelinase D {SMases D isoform (rLiD1)} [1] and screened by enzyme-linked immunosorbent assay (ELISA) using L. intermedia, L. laeta and L. gaucho venoms as antigens. One clone (LiD1mAb16) out of seventeen anti-rLiD1 hybridomas was cross-reactive with the three whole Loxosceles venoms. 2D Western blot analysis indicated that LiD1mAb16 was capable of interacting with 34 proteins of 29-36kDa in L. intermedia, 33 in L. gaucho and 27 in L. laeta venoms. The results of immunoassays with cellulose-bound peptides revealed that the LiD1mAb16 recognizes a highly conserved linear epitope localized in the catalytic region of SMases D toxins. The selected mAb displayed in vivo protective activity in rabbits after challenge with rLiD1. These results show the potential usefulness of monoclonal antibodies for future therapeutic approaches and also opens up the perspective of utilization of these antibodies for immunodiagnostic assays in loxoscelism.
authors.affiliation: Departamento de Bioquímica e Imunologia, Instituto Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil.
authors.forename: C
authors.initials: C
authors.lastname: Dias-Lopes
authors.affiliation: Departamento de Bioquímica e Imunologia, Instituto Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil.
authors.forename: L
authors.initials: L
authors.lastname: Felicori
authors.affiliation: SysDiag, UMR3145,CNRS/Biorad, Montpellier, France.
authors.forename: L
authors.initials: L
authors.lastname: Rubrecht
authors.affiliation: SysDiag, UMR3145,CNRS/Biorad, Montpellier, France.
authors.forename: S
authors.initials: S
authors.lastname: Cobo
authors.affiliation: SysDiag, UMR3145,CNRS/Biorad, Montpellier, France.
authors.forename: L
authors.initials: L
authors.lastname: Molina
authors.affiliation: SysDiag, UMR3145,CNRS/Biorad, Montpellier, France.
authors.forename: C
authors.initials: C
authors.lastname: Nguyen
authors.affiliation: SysDiag, UMR3145,CNRS/Biorad, Montpellier, France.
authors.forename: P
authors.initials: P
authors.lastname: Galéa
authors.affiliation: SysDiag, UMR3145,CNRS/Biorad, Montpellier, France.
authors.forename: C
authors.initials: C
authors.lastname: Granier
authors.affiliation: SysDiag, UMR3145,CNRS/Biorad, Montpellier, France.
authors.forename: F
authors.initials: F
authors.lastname: Molina
authors.affiliation: Departamento de Bioquímica e Imunologia, Instituto Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil. Electronic address: olortegi@icb.ufmg.br.
authors.forename: C
authors.initials: C
authors.lastname: Chávez-Olortegui
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D057134:Antibodies, Neutralizing / Q000276:immunology; D003429:Cross Reactions; D018604:Epitope Mapping; D000939:Epitopes / Q000276:immunology*; D006825:Hybridomas; D051379:Mice; D008807:Mice, Inbred BALB C; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009500:Neutralization Tests; D010727:Phosphoric Diester Hydrolases / Q000276:immunology*; D011817:Rabbits; D011994:Recombinant Proteins / Q000276:immunology; D013111:Spider Venoms / Q000201:enzymology* / Q000276:immunology; D013112:Spiders / Q000201:enzymology
chemical_list: D000911:Antibodies, Monoclonal; D057134:Antibodies, Neutralizing; D000939:Epitopes; D011994:Recombinant Proteins; D013111:Spider Venoms; C055161:loxosceles venom; D010727:Phosphoric Diester Hydrolases; C055160:sphingomyelin phosphodiesterase D
keywords: Epitopes; Loxosceles spider venoms; Monoclonal antibody; Sphingomyelinases D