IVIT   27842
INSTITUTO DE VIROLOGIA E INNOVACIONES TECNOLOGICAS
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
Development of a fluorescent scFv antibody to improve Rabies diagnosis
Autor/es:
MANSILLA, FLORENCIA CELESTE; ALVAREZ, IRENE; CAPOZZO, ALEJANDRA; TURCO, CECILIA SOLEDAD; SCHAMMAS, JUAN MANUEL; FERRERO, SOL; HELGUERA, GUSTAVO
Lugar:
Seattle
Reunión:
Simposio; International Veterinary Immunology Symposium, 2019; 2019
Resumen:
Background Rabies is the zoonotic infectious disease with the highest case-fatality rate. Its diagnostics relies on laboratory testing and makes use of specific antibodies. Several rabies virus (RABV) neutralizing mouse-derived monoclonal antibodies (mAbs) have been identified. However, mAb production in mammalian cell-culture has a considerable cost, and the use of mice to obtain mAbs from ascitic fluid is increasingly restricted. In this scenario, pursuing novel alternative biotechnological strategies is paramount.MethodWe have developed a specific recombinant mini-antibody against RABV formed by the variable region of the heavy and light chains of a previously described neutralizing mAb (CR57) reactive against epitope-1 of the viral glycoprotein G (RV-GP). Both light and heavy chains variable regions of CR57 were covalently linked by a flexible peptide-linker and fused to the maltose binding protein (MBP) to improve its solubility. MBP-scFv57 expression was optimized using an E.coli expression system. Affinity-purified antibody was then covalently conjugated to fluorescein isothiocyanate (FITC) and its ability to recognize transfected and/or infected cells was determined by direct immunofluorescence test (DIF) and FACS.ResultsMBP-scFv57 was recovered, yielding 7.5g/l of initial culture. The integrity and stability was verified by SDS-PAGE/western blot, confirming that MBP fusion improves its stability. The scFv57 recognized RV-GP in the membrane of transiently-transfected HEK-293 cells. After conjugation, scFv57-FITC was able to detect 99% of the transfected cells by FACS. The presence of RV-GP was also determined by IFD on transfected HEK293 cells and on infected VERO and BHK cells with similar performance compared to standard reagents. ConclusionThe scFv57 is efficiently produced as a soluble and stable recombinant fusion-protein in bacteria and can be affinity-purified in one step, preserving its binding activity. The fusion-protein has been FITC-conjugated, resulting in an inexpensive reagent capable of detecting the presence of virus in tissue samples or infected cells by DIF.