Preliminary assays to specific serotype monoclonal antibodies production against the dengue virus types 1 and 2 using synthetic peptides designed after structural analysis of the E protein
The dengue virus E protein is the major target of immunity humoral response. E protein presents differences in primary structure (four structural isoforms) that reflects the possibility of four differents serotypes against this protein, in an immune response. Monoclonal antibodies against these specific regions of each isoform could be used to develop a rapid and efficient serotype-specific dengue diagnosis for disease monitoring. The nucleotide and amino acid sequences of E protein were previously published, available in data base and systematically analyzed through bioinformatics programs which allowed to be designed B epitopes (Zanchi, Master Degree work, 2003, Universidade Federal de Rondônia). Three of these peptides were used for the assays of the monoclonal antibodies production. Isogenics BALB/c mice were immunized with definite sequence peptides: 1) peptide 1 (Glu64 Ala Lys Ile Ser Asn Thr Thr Thr Asp Ser Arg75 ? DENV-1); 2) peptide 2 (Ser224 Gly Ala Ser Thr Ser Gln Glu Thr Trp Asn Arg Gln Asp237 ? DENV-1); and 3) peptide 4 (Lys47 Thr Glu Ala Lys Gln Pro Ala Thr Leu Arg Lys Tyr59 ? DENV-2). The next step was the serocirculating polyclonal antibodies analyses which only anti-peptide 4 recognized the DENV2 in C6/36 infected cells. The corresponding mice were used for monoclonal antibodies production. Monoclonal antibodies anti-peptide 4 (mAbP4/1) and 2 (mAbP4/2) of IgG1 and IgG2b subclasses, respectively. The mAbsP4/1 and 2 were purified by affinity chromatografy in sepharose-proteinA column. These monoclonal antibodies showed low affinity for the respective peptide. Unfortunately, mAbsP4 1 and 2 cross-reacted with human serum proteins and they didn?t react with DENV2. On the other side view, this present work has been contributed to monoclonal antibodies technique implantation using the synthetic peptide immunization in mice, a completely new technique in the Region North of the Country.
Advisor:Rodrigo Guerino Stabeli
School:Fundação Universidade Federal de Rondônia
Source Type:Master's Thesis
Date of Publication:12/01/2005