Resumo

The cellular prion protein (PrPc) is a membrane associated protein with two glycosilating sites occurring in a wide range of eukaryotic cells and encountered especially in the nervous system. Since it is highly conserved and therefore must play a role of general importance, many assumptions have already been made about its function, which is not yet completely understood. The aim of this work is to produce monoclonal antibodies with high specificity that could target PrPc in different environments and conditions. PrP-knockout mice were immunized with a recombinant form of PrPc and had their sera tested both by ELISA and Western-blot, achieving titers of up to 1:13000 in the former. The mice spleen cells were fused with myeloma cells (SP2/0) in a 5:1 ratio, generating hybridoma cells. Those cells were screened by ELISA and we found 22 clones which secreted antibodies in high titers. However, only six of them proved to be stable. One of them, however, is able to recognize only the recombinant form of the protein, which is deglycosilated. To further investigate this, extracts of HEK cells transfected with PrP and treated with tunicamycin, a drug that blocks the glycosilation pathway of the cell, were incubated with the hybridoma supernatant and we found that this hybridoma, denominated 7F10, recognizes only the deglycosilated form of the protein. This result is a strong indicative of what might be the epitope recognized by this mAb, given that PrPc has only two glycosilation sites. Limiting dilution was made with every clone that has shown ELISA positivity and that has proved to be stable. In general, clones obtained from the limiting dilution had higher titers. These clones secrete monoclonal antibodies which recognize both recombinant (WB and ELISA) and brain expressed PrPc (WB). Supported by CNPq, CAPES, FAPESP, Fundação Araucária, SETI-UGF.