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.

Correlation between 146S Antigen Content in Foot-and-Mouth Disease Inactivated Vaccines and Immunogenicity Level and Vaccine Potency Alternative Test Methods

“…The quantity of the 146S component in each batch vaccine was tested with the sucrose density gradient centrifugation method [25]. The aqueous phase collected from the demulsified sample was directly applied to the top of a 15-45% sucrose gradient.…”

Calcium Chloride as a Novel Stabilizer for Foot-and-Mouth Disease Virus and Its Application in the Vaccine Formulation

“…FMDV vaccine antigens were quantified by size-exclusion high-performance liquid chromatography (SE-HPLC) on FMDV samples purified by ultracentrifugation as previously described [40]. Briefly, the gel column used in this study was a TSKgel G4000PWXL (300 mm × 7.8 mm I.D.)…”