NOVEL ENZYME LINKED IMMUNOSORBENT ASSAY TO DETECT ANTI-RABIES VIRUS ANTIBODIES IN SERUM SAMPLES DERIVED FROM VACCINATED ANIMALS

JUAN IGNACIO MARFÍA; SILVINA SONIA BOMBICINO; ALDANA TRABUCCHI; RUBEN FRANCISCO IACONO; ALEXANDRA MARISA TARGOVNIK; LEONARDO GABRIEL ALONSO; MARÍA VICTORIA MIRANDA; SILVINA NOEMÍ VALDEZ1

San Luis

Congreso; Reunion anual de la SAI; 2023

SAI

Rabies is a zoonotic disease, which means it can be transmitted from animals to humans. Rabies in bulls is a rare but potentially serious disease. Therefore, any case of rabies in animals, including bulls, is a cause for concern and should be managed seriously to prevent the spread of the disease to other animals and people. There is no test to monitor the effectiveness of the anti-rabies vaccination in our Country. For this reason, the aim of this work was to develop a novel immunoassay to detect antibodies to rabies employing a rabies antigen produced in baculovirus expression system.Materials and methods: The ectodomain of the rabies virus glycoprotein G (GE) was synthesized using the baculovirus expression system in Sf9 insect cells. This approach involved the fusion of the polh promoter with a 120 nt segment derived from the promoter of the orf46 gene from Spodoptera exigua multiple nucleopolyhedrovirus, resulting in a chimeric promoter (polh-pSeL). The protein was recovered from the cell lysates and purified in one step by metal ion affinity chromatography. Thirty-two serum samples of bulls vaccinated against rabies and 30 sera from non-vaccinated bulls kindly donated by ProinVet were used for the development of the immunoassay. An indirect ELISA was performed, based on the interaction of anti-rabies virus antibodies present in samples with purified GE immobilized in the solid phase. Bound antibodies were detected with anti-bovine IgG antibodies conjugated to peroxidase. The formation of immunocomplexes was revealed by the addition of TMB, the reaction was stopped with H2SO4 and the oxidized product was measured using a spectrophotometer. The data obtained for each sample was analyzed with the GraphPad Software and the results were expressed as specific absorbance / OD (OD = the mean of each sample minus the mean of the blank control). The cut-off value of the assay was set at OD = 0.706.