Viral particles prepared by size exclusion chromatography can be used as coating material for serological assays

LUDI, ANNA; TURCO CECILIA SOLEDAD; CAPOZZO, ALEJANDRA; BURMAN, ALISON

Bangkok

Encuentro; Global Foot-and-Mouth Disease Research Alliance, 2019 Scientific Meeting; 2019

Anti-foot and mouth disease virus (FMDV) capsid antibodies can by themselves mediate protection against FMDV. The amount of antibodies, currently assessed by the virus neutralization test (VNT) or ELISAs, is correlated to clinical protection. ELISA is widely used to quantify and characterize specific antibodies being more reliable and easier to harmonize than the VNT, however, correlation between VNT and ELISAs have been difficult to achieve, mainly because they measure a different aspect of the antibody response 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 whole viral particles (146S) to detect total specific antibodies, the avidity of those antibodies and their IgG subtype. These assays, unlike Liquid phase blocking ELISA (LPBE), do not require capture of detector antibodies, making them particularly useful for field viruses and cross-protection studies. The major constrain of these ELISAs is actually the need of purifying the virus, usually performed by sucrose gradient ultracentrifugation (SGU) and pelleting, a cumbersome expensive procedure, difficult to standardise. 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 coating the plated with the particles directly eluted from the column (SEC-FMDV), after only one purification step. The integrity of SEC-FMDV was controlled by SGU and antigen ELISA of each fraction, confirming that the SEC-extracted fraction contains only 146S and eventually 75S particles. We validated the use of SEC-FMDV as coating material for in 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. 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 virus-surface epitopes.