IVIT   27842
INSTITUTO DE VIROLOGIA E INNOVACIONES TECNOLOGICAS
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
VIRAL PARTICLES PREPARED BY SIZE EXCLUSION CHROMATOGRAPHY CAN BE USED AS COATING MATERIAL FOR SEROLOGICAL ASSAYS
Autor/es:
MANSILLA, FLORENCIA CELESTE; TURCO, CECILIA; ANNA LUDI; BURMAN, ALISON; ALEJANDRA CAPOZZO
Lugar:
Bangkok
Reunión:
Congreso; Global Foot-and-Mouth Disease Research Alliance 2019 Scientific Meeting; 2019
Institución organizadora:
Global Foot and Mouth Disease Research Alliance
Resumen:
Anti-foot and mouth disease virus (FMDV) capsid antibodies can by themselves mediate protection against FMDV, and the amount of antibodies, currently assessed by the virus neutralization test (VNT) or Liquid Phase Blocking ELISA (LPBE), is correlated to clinical protection. ELISAs are widely used to quantify and characterize specific antibodies being more reliable and easier to harmonize than the VNT, however, a correlation between VNT and ELISAs have been difficult to achieve, mainly because they measure a different aspect of the antibodyresponse and also due to the fact that the integrity of the viral particle used in the ELISAs is usually not verified. The loss of capsid integrity can hamper protection studies by detecting antibodies against non-exposed epitopes, therefore, the integrity of the capsid is paramount to get accurate results. In our laboratory we developed indirect ELISAs that use purified wholeviral particles (146S) to detect total specific antibodies, their avidity, and subtype. These assays, unlike LPBE, do not require the capture of detector antibodies, making them particularly useful for field viruses and cross-protection studies. The major constrain of these ELISAs is the need ofpurifying the virus, usually performed by sucrose gradient ultracentrifugation (SGU) and pelleting, a cumbersome and time-consuming procedure. In this study we applied size exclusion chromatography (SEC) to purify either vaccine antigen or field virus grown in cell culture and performed the ELISAs by directly coating the plates with the particles eluted from the column (SEC-FMDV), without any further purification step. The integrity of SEC-FMDV was controlled by SGU and antigen ELISA, confirming that the SEC-extracted fraction contained 146S and eventually 75S particles. We validated the use of SEC-FMDV as a coating material for ELISA compared to SGU virus. The use of SEC required a higher concentration of the blocking solution (based on horse serum), with no other modification to the standard protocol. However, when adult bovine serum was used to grow the cells, residual of IgM was co-eluted with the viral particles, thus, the use of this serum should be avoided when preparing ELISA antigen using SEC. We conclude that SEC-FMDV is a suitable reagent for coating ELISA plates ensuring the use of whole particles, paramount to identify antibodies reactive to exposed epitopes.