Resumen de Desarrollo de pruebas serológicas para la identificación de ovinos inmunizados con vacunas-GFP
A. Zablaza, B. San Román, P.M. Muñoz, J.M. Blasco, Mª Jesús Grilló Dolset
Serological interference in diagnostic tests is a major problem associated to the use of veterinary vaccines, hampering the differentiation between infected and vaccinated animals (DIVA). To solve this in the ovine brucellosis context, our group is developing new Brucella vaccines tagged with green fluorescent protein (GFP) as a xenogenic marker, to be used in combination with both classical and GFP DIVA-tests. The aim of this work was to develop serological tests (DDG-GFP and ELISA-GFP) to detect GFP antibodies in sheep. The optimal concentration of the proteins in DDG-GFP was that containing 31.25 μg/mL of GFP. In ELISA-GFP, GFP-GST fusion protein was more suitable than GFP as antigen. Moreover, other ELISA-GFP factors (blocking effect, serum and conjugate optimal dilutions, and time of ABTS substrate incubation) were evaluated, setting up an ELISA-GFP detecting all hyperimmune sera, and showing 99 % specificity when 92 negative sheep sera were tested. In conclusion, the serologic tests developed allowed to detect anti-GFP sheep antibodies, and could be useful along with recombinant GFP vaccines, as a DIVA strategy.
