Mapeamento dos paratopos do anticorpo monoclonal Li mAb(7) anti-proteína dermonecrótica do veneno da aranha Loxosceles intermedia

• Ano de defesa: 2016
• Autor(a) principal: Patrícia Donado Vaz de Melo
• Tipo de documento: Dissertação
• Tipo de acesso: Acesso aberto
• Idioma: por
• Instituição de defesa: Universidade Federal de Minas Gerais; UFMG
• Palavras-chave em Português: Componente dermonecrótico; Neutralização; Loxosceles intermedia; Peptídeos; Li mAb(7); Mapeamento; Paratopo; Esfingomielinase D; Bioquímica e imunologia
• Link de acesso: http://hdl.handle.net/1843/BUBD-AHKP9K

The venom of the spider Loxosceles intermedia contains proteins with sphingomyelinase activity (SMase D), the main dermonecrotic component responsible for the prevalent symptoms of cutaneous and cutaneous visceral loxoscelism like dermonecrosis and acute renal failure. After the immunization of mice with crude venom of Loxosceles intermedia spider, our group obtained the monoclonal antibody Li mAb(7), which has been shown to neutralize the dermonecrotic activity from the venom of L. intermedia in a dose-dependent relationship, 50 ug Li mAb(7) neutralized 10 g of crude venom, and therefore has high potential for use in anti-venom therapy. In addition, the monoclonal antibody Li mAb(7) showed no cross-reactivity with venoms of Loxosceles laeta (Brazil), L. laeta (Perú) and Loxosceles gaucho. In order to obtain peptides with high potential neutralizing the toxic effects of L. intermedia, which are able to overcome difficulties in the production of sera, like the poison obtainment and the maintenance of serum neutralization capacity, this study was dedicated to the mapping of paratope of Li mAb(7). Using on bioinformatics tools and Spot Synthesis methodology the complementarity determining regions were identified, peptides on cellulose membrane were synthesized from structural analyzes of antigen-antibody interactions and the essential amino acids residues for recognition of the protein with sphingomyelinase activity and their locations have been mapped. The soluble peptides were produced and sandwich ELISA assay demonstrated that peptides bind in a specific and dose-dependent manner, the same conformational epitope recognized by Li mAb(7). Competition ELISA assay did not confirm the competition for the same binding site between soluble peptides (607 and 642) and monoclonal, however, it is believed that due to high affinity of antibody for the epitope. Neither the neutralizing capacity of sphingomyelinase activity was demonstrated. Thus, it was demonstrated that peptides derived from the soluble mapping Li mAb(7) specifically recognize the same epitope and have a conformational structure similar to the Fv region of the antibody.