Development of new Raw Material for the Serologic diagnosis of dengue: Antigens, Viral turn recombinant natives.
Dengue is the major tropical arbovirosis, in terms of morbidity and mortality. It?s transmitted by hematophoges mosquito?s bite of the genera Aedes and it is responsible for over one hundred million cases of dengue (DF) per year of which fivehundred thousand are hemorrhagic (DHF). In Brazil, the epidemiological situation is alarming, because the country represents 80% of all cases recorded in the Americas.Dengue virus belongs to the family Flaviviridae and has four serotypes (DEN-1, DEN- 2, DEN-3 and DEN-4) and at the moment no vaccine is available. A fast and efficientdiagnostic can confirm cases of dengue and guide future treatment and prognosis. Several diagnostic tests exist, but ELISA (enzyme-linked-immunosorbent-assay) is the most widely used one, because it is quick, easy to perform and can used with several samples at the same time. The antigens used are inactivated viral particles produced in cell culture or crude extract, such as virally infected mouse brain, whichare limited by several factors, such as, lot variability, the growing problem of bioethics and laboratory animal manipulations and finally the risks from manipulatingpathogenic material. A good alternative is to use recombinant antigens that can replace the ones presently used, give a more reproducible result, with lower cost and safer. In order to improve serological diagnostic in Brazil, the major objectives of this work were; (I) to obtain high quality antigens from virally infected cell cultures, (II) todevelop a panel of recombinant antigens with the four viral serotypes, (III) to test the applicability of these recombinant antigens in serologic diagnostic tests for dengue.The IBMP native antigens were obtained by infection of C6/36 cells and subsequent precipitation, concentration and inativation. The chosen recombinant antigens were,the B domain of protein E, because it is an antigenic domain exposed on the molecule?s surface and is very reactive against patient?s serum, and the prM and Eproteins, which were expressed in a chimerical way and deleted of their hydrophobic anchorage regions (prM/EDH). All the proteins were expressed in high amounts withthe prokaryotic system Qiaexpressionist. Two test formats were used; i.e. ELISA and imunoblotting, using 44 serum -positive and serum-negative patients, and also serafrom patients vaccinated against yellow fever. The IBMP native antigens were tested in parallel with the infected mouse brain antigens (Bio-Manginhos kit) and PanBioantigens, a commercially available test (in this case used to validate the test), using the same sera panel. The best results were obtained with the IBMP native antigens,which shows 95,4% reactivity with the positive sera when compared to the 90,9% reactivity of the Bio-Manguinhos antigens, showing more sensitivity and specificity.The results of DomB used in ELISA tests aren?t satisfactory, because inespecific reaction occurs with the negative sera. The prM/EDH recombinant antigens were used in tests and imunoblotting for detection of IgM and IgG antibodies. Results are very promising and protocol standardization is under way.
Advisor:Claudia Nunes Duarte dos Santos
School:Faculdades Oswaldo Cruz
Source Type:Master's Thesis
Date of Publication:12/17/2004