Obtenção de anticorpos e peptídeos recombinantes através e Phage Display para aplicação em imunodiagnóstico

Abstract

Resumo: Neste trabalho a tecnologia de phage display foi empregada para selecionar: 1) anticorpos recombinantes anti-imunoglobulinas G e M humanas; e 2) mimotopos de Treponema pallidum e de virus linfotropico de celulas T humanas (HTLV). 1) Genes correspondentes as cadeias variaveis de imunoglobulinas . obtidos a partir do baco de camundongos imunizados com as fracoes Fc de IgG e Fc5ƒÊ de IgM . foram unidos na forma de segmentos codificantes de anticorpos recombinantes scFv, que foram inseridos no genoma de fagos filamentosos e expressos de modo a serem expostos na superficie viral, fusionados a uma proteina do capsideo. As bibliotecas de anticorpos assim geradas foram entao selecionadas frente ao mesmo antigeno empregado na imunizacao: apesar de terem sido obtidos fagos expondo fragmentos scFv com forte afinidade pela porcao Fc5ƒÊ de IgM, nao foi possivel individualiza-los de maneira bem sucedida, o que impediu a adequada caracterizacao dos mesmos. 2) Bibliotecas de peptideos Ph.D. da New England Biolabs foram submetidas a selecao frente a anticorpos monoclonais anti-T. pallidum e anti-HTLV. Embora nao tenham sido encontrados peptideos com elevada reatividade por este ultimo, ao menos tres sequencias apresentando forte afinidade pelo anticorpo anti-T. pallidum foram selecionadas e isoladas com sucesso. Tais peptideos podem representar excelentes alternativas aos antigenos atualmente empregados no diagnostico de sifilis, e demonstram que a metodologia de phage display pode contribuir para a nacionalizacao da producao de reagentes para imunodiagnostico, tornando mais eficiente a fabricacao dos mesmos e impulsionando a geracao de novos insumos

Abstracte: In this work phage display technology was employed to screen: 1) antibodies against human IgG and IgM immunoglobulins; and 2) mimotopes for Treponema pallidum and human T-lymphotropic virus (HTLV). 1) Genes encoding the variable chains of antibodies – originated from spleen cells of mice immunized with Fc IgG and Fc5ì IgM – were fused to generate segments encoding scFv recombinant antibodies, which were then inserted in the genome of filamentous phage and expressed so as to be displayed at the viral surface, fused to a capsid protein. The antibody libraries thus obtained were then screened against the same antigen employed in the immunization step: Although clones displaying scFv fragments with strong affinity for Fc5ì IgM were obtained, it was not possible to successfully individualize them, preventing their proper characterization. 2) New England Biolabs’ Ph.D. peptide libraries were screened against anti-T. pallidum and anti-HTLV monoclonal antibodies. Although peptides with high reactivity for the latter could not be found, at least three sequences showing strong affinity for the anti-T. pallidum antibody were successfully selected and isolated. These peptides can represent promising alternatives to the antigens currently used in the diagnosis of syphilis, and show that phage display technology can contribute to nationalize the production of diagnostic reagents, making their manufacture more efficient and stimulating the generation of novel products