Development of a fluorescents cFv antibody to improve Rabies diagnosis

MANSILLA FLORENCIA; ALVAREZ IRENE; CAPOZZO ALEJANDRA; FERRERO S; HELGUERA G; TURCO CECILIA; JUAN MANUEL SCHAMMAS

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Congreso; 12th International Veterinary Immunology Symposium (IVIS); 2019

Background: Rabies is the zoonotic infectiousdisease with the highest case-fatality rate. Itsdiagnostics relies on laboratory testing andmakes use of specific antibodies. Severalrabies virus (RABV) neutralizing mousederivedmonoclonal antibodies (mAbs) havebeen identified. However, mAb production inmammalian cell-culture has a considerablecost, and the use of mice to obtain mAbsfrom ascitic fluid is increasingly restricted.In this scenario, pursuing novel alternativebiotechnological strategies is paramount.Method: We have developed a specificrecombinant mini-antibody against RABVformed by the variable region of the heavyand light chains of a previously describedneutralizing mAb (CR57) reactive againstepitope-1 of the viral glycoprotein G (RV-GP).Both light and heavy chains variable regionsof CR57 were covalently linked by a flexiblepeptide-linker and fused to the maltose bindingprotein (MBP) to improve its solubility. MBPscFv57expression was optimized using anE.coli expression system. Affinity-purifiedantibody was then covalently conjugated tofluorescein isothiocyanate (FITC) and its abilityto recognize transfected and/or infected cellswas determined by direct immunofluorescencetest (DIF) and FACS.Results: MBP-scFv57 was recovered, yielding7.5g/l of initial culture. The integrity andstability was verified by SDS-PAGE/westernblot, confirming that MBP fusion improves itsstability. The scFv57 recognized RV-GP in themembrane of transiently-transfected HEK-293cells. After conjugation, scFv57-FITC was ableto detect 99% of the transfected cells by FACS.The presence of RV-GP was also determinedby IFD on transfected HEK293 cells and oninfected VERO and BHK cells with similarperformance compared to standard reagents.Conclussion: The scFv57 is efficientlyproduced as a soluble and stable recombinantfusion-protein in bacteria and can be affinitypurifiedin one step, preserving its bindingactivity. The fusion-protein has been FITCconjugated,resulting in an inexpensive reagentcapable of detecting the presence of virus intissue samples or infected cells by DIF.