Comparison of High-Performance Liquid Chromatography with Sucrose Density Gradient Ultracentrifugation for the Quantification of Foot-and-Mouth Disease Vaccine Antigens.

Abstract

Foot-and-mouth disease (FMD) causes substantial economic losses in the livestock industry. The protective immunizing component of the FMD virus (FMDV) is a ribonucleoprotein particle with a sedimentation coefficient of 146S. Size-exclusion high-performance liquid chromatography (SE-HPLC) was introduced to replace sucrose density gradient ultracentrifugation (SDG), which is the gold standard for the quantification of FMDV 146S particles. SE-HPLC showed a pattern similar to that of SDG; however, the two methods resulted in different quantities for the same amount of 146S particles. This study aimed to identify the reason for this disparity and adjust the difference between the two methods by employing a standard material. While SE-HPLC displayed all the virus particles in the peak fraction by SDS-PAGE and Western blotting, the virus particles were widely dispersed in multiple fractions, including peak fractions in the SDG. To adjust the difference between the two methods, a stable surrogate virus, bovine enterovirus, was devised to draw a standard curve, and the gap was reduced to <10%. To our knowledge, this is the first report to provide experimental evidence on the difference between SDG and SE-HPLC for the quantification of FMDV particles.