Immuno-affinity chromatography for purification of IgG from hyper-immune sera raised against 146S fraction of Foot and Mouth Disease Virus for diagnostic purposes.

Abstract

Immunoaffinity chromatography (IAC) is a fundamental isolation and purification tool which is incorporated in a substantial range of therapeutic and diagnostic applications. This study has reappraised the usefulness of immunoaffinity chromatography for the purification of polyclonal antibodies. Protein A based IAC is a convenient and reliable method for purification of IgG, from hyperimmunesera (HIS) raised in experimental animals such as rabbits, guinea pigs and mice to be utilized in pharmaceutics and diagnostics. The 146S fraction of Foot and Mouth Disease virus (FMDV) TCID50=10 5.6 was cultured on a baby hamster kidney cell line 21 (BHK-21), concentrated using salt precipitation method using PEG 6000, purified by size exclusion chromatography (SEC) using Sepharose-30 at 254nm absorbance. Purification of 146S FMDV was analyzed using 12% SDS-PAGE which provided two bands of light and heavy chains. The alum-based vaccine, consisting of ≥10μg of 146S FMDV, was applied in 10 male rabbits and 10 male guinea pigs and two animals of each group were taken as a negative control. The titer of serum was calculated using virus neutralization test. A Protein-A kit (Thermo scientific- 44667, 0528.2) was used to purify HIS raised against 146S FMDV and validated using 12% SDS PAGE in reducing condition. The data demonstrate that protein-A affinity chromatography is an efficient tool for the purification of antibodies from hyper-immune sera raised against 146S FMDV and can be used for the production of diagnostic kits e.g. Enzyme linked immuno-sorbent assay (ELISA) and radioimmunoassay.