Optimization of Size Exclusion Chromatography for the Purification and Quantification of Foot and Mouth Disease Virus Serotype “O”
Optimization of Size Exclusion Chromatography for the Purification and Quantification of Foot and Mouth Disease Virus Serotype “O”
Abdul Razak1, Imran Altaf1*, Aftab Ahmad Anjum1, Ali Raza Awan2 and Farhat Nazir Awan3
ABSTRACT
Foot and mouth disease (FMD) is economically devastating disease of livestock overwhelming the international trading of animal based products. The unpurified inactivated vaccine containing Foot and Mouth Disease Virus a (FMDV) and non-structural proteins (NSPs) used to control the disease stimulated the production of anti-NSP antibodies. Countries striving to establish FMD free status must ensure the absence of circulating FMDV and anti-NSP antibodies in vaccinated animals, which are produced in natural infection. Production of purified FMD vaccines contrary to other animal’s vaccines required extra step of virus purification from its NSPs. Present project was designed to optimize different parameters of size exclusion chromatography (SEC) for the purification of local isolate of FMDV from NSPs. For this purpose Bio-rad Econo-Column 15/50 was packed with SephacrylTM S-300 resin and fitted with BioRad BioLogic LP Chromatographic system. Mixture of blue dextran and bovine serum albumin used as biomarkers for FMDV and NSPs respectively was employed to optimize resin bed height in column based on resolution between curves, while that FMDV was used to optimize flow rate of mobile phase and sample volume based on column efficiency. The chromatographic fraction of FMDV was ensured for purity through protein analysis by SDS-PAGE and serologically by 3ABC-NSP ELISA. It was found that maximum resolution of 1.86 was achieved at 48cm of resin bed height followed by 1.5 and 1.27 at 40cm and 32cm respectively. FMDV was eluted earlier with retention time 138.02+2.02min at flow of 0.7ml/min than 165.76+1.28min at 0.5ml/min along with better column efficiency with sample volume at 4% of column volume. SEC could detect and quantify from 603.75µg/ml in PEG concentrated FMDV up to 3.33µg/ml in 1:64 dilution as lower limit of detection and quantification. Presence of two bands of structural protein with absence of NSPs bands on SDS-PAGE and negative NSP% in hyperimmune sera raised in goats revealed the successful elimination of NSPs contents from FMDV suspension by SEC.
To share on other social networks, click on any share button. What are these?
