Obtención de un anticuerpo monoclonal anti α--enolasa humana y caracterización de su interacción con el antígeno

Abstract

Enolase is a glycolytic enzyme that catalyzes the dehydration of 2-phospho-D-glycerate to phosphoenolpyruvate. In humans there are three isoforms: α, β and γ. One of them, α-enolase has been found to play different roles in a variety of cellular compartments and tissues. Monoclonal Antibodies against several proteins have been generated showing interesting results about the interaction between antigen-antibody that might have important implications in biomedicine. In this work we produced a monoclonal antibody using recombinant human α-enolase as antigen. Five female Balb/C mice were immunized with this antigen for seven weeks and the immunologic response was evaluated by indirect ELISA assay. Subsequently, the mouse that showed a better immune response to antigen recombinant human α-enolase was summited to splenectomy. Hybridomas were obtained by fusing the mouse spleen cells with myeloma cells X63Ag8.653. In order to select either the hybridoma or its clones, cell supernatants were screened by indirect ELISA using as antigens human α-enolase and a peptide that was designed and synthesized to control the selection of clones. This peptide was designed by in silico analysis of the sequence and the three-dimensional structure of human α-enolase and corresponds to a region of the sequence of human α-enolase that is poorly conserved among human enolases, is immunogenic and is exposed to the solvent in the three-dimensional structure. Finally monoclonal antibody specificity was assessed by indirect ELISA using also other enolases like Saccharomyces cerevisiae and Trichomonas vaginalis.