Development of an indirect ELISA based on purified, inactivated Rabies Virus as a first step to evaluate immune response induced by recombinant anti-rabies vaccines

DEL MÉDICO ZAJAC MARÍA PAULA ; GARANZINI DÉBORA ; J ; CALAMANTE GABRIELA ; URADO ROSANA ; PÉREZ OSCAR

CABA

Congreso; Rabies in the Americas (RITA) 2018; 2018

Fundación Cassará

Preexposure rabies vaccination is recommended for susceptible animals in areas where the disease is endemic. In our laboratory we develop pox and adenovirus-vectored vaccines to prevent paralytic rabies in bovines. In order to rapidly evaluate the induction of specific antibodies by our recombinant vectors prior to test them in efficacy trials, a simple method is required. Thus, the aim of this work was the development of an indirect ELISA to detect anti-rabies antibodies in animals inoculated with the recombinant immunogens.ELISA was performed by immobilization of the antigen in microplates of 96 wells. Several parameters were evaluated in order to improve the specific signal with minimal background: 1) amount and type of antigen bound on the plates; 2) period of time for antigen binding; 3) blocking solutions; 4) dilution of the positive sera.Various antigens were evaluated at different quantities per well: Rabipur vaccine (1-3 ug), concentrated inactivated rabies virus (RVi) (50-800 ng), sucrose-gradient purified RVi (0,4-3,2 ug) or extracts from cells infected with recombinant baculovirus (Bv) expressing rabies glycoprotein (50-600 ng of RG). The antigen binding was accomplished for 1 h at 37ºC or 16 h at 4ºC. The blocking solutions used were: 3 or 5 % skimmed-milk and 1% PVP solution. The negative and positive mice sera were diluted 1/25; 1/50 or 1/100 in blocking solution. As a second antibody a HRP-anti mouse antibody was used. After 30 min of incubation with ABTS substrate solution, the optical density was measured at 405 nm. Best results were obtained using 2 ug/well of purified RVi bound 16 h at 4ºC, PVP solution for blocking and sera diluted 1/50. Finally, we confirmed by ELISA that MVA vector expressing rabies glycoprotein induces in mice a specific immune response significantly different from those animals inoculated with non-recombinant MVA.