¹University of Veterinary and Animal Sciences, Faculty of Veterinary Sciences, Institute of Microbiology, 54000 Lahore, PAKISTAN
²University of Veterinary and Animal Sciences, Faculty of Biosciences, Institute of Biochemistry and Biotechnology, 54000 Lahore, PAKISTAN DOI : 10.9775/kvfd.2026.36325 Foot and Mouth Disease (FMD) is an economically significant livestock disease in developing countries such as Pakistan, where serotypes A, Asia1, and O of Foot and Mouth Disease Virus (FMDV) are prevalent. Effective disease control relies on immunization using potent vaccines, and evaluation of vaccine potency in terms of protective dose 50 (PD₅₀) is a critical step during purified vaccine formulation. The present study aimed to prepare chromatographically purified vaccines of the three prevalent serotypes in different formulations and to determine PD₅₀ values. Viruses were propagated in cell culture, biologically titrated, and chemically inactivated. To obtain highly purified antigens with reduced non-structural protein content and improved quantification compared with conventional methods, viral antigens were purified, and quantified using Size Exclusion Chromatography (SEC) with Sephacryl S-300 followed by purity check through SDS-PAGE. Five formulations of each serotype (16, 8, 4, 2, and 1 μg/dose) were prepared and inoculated into five animals per concentration. Serum samples were analyzed by SPCE to identify protected animals. PD₅₀ values were calculated for each antigen and used to formulate a trivalent vaccine. The trivalent vaccine was evaluated for structural and non-structural proteins using SPCE and 3ABC ELISA, respectively. Results demonstrated that SEC efficiently removed NSPs, as confirmed by SDS-PAGE. PD₅₀ values for serotypes A, Asia1, and O were estimated as 2.14, 1.90, and 1.63 μg/dose, respectively. Accordingly, trivalent vaccine concentrations of 6.42, 5.70, and 4.90 μg/dose (3PD₅₀) were formulated. The trivalent purified vaccine induced protective immune responses with less than 20% NSP content in SPCE and 3ABC ELISA. Keywords : Size exclusion chromatography, Sephacryal S-300, SDS-PAGE, Nonstructural protein, PD50, Vaccine