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

A Munir, A A Anjum, I Altaf, A R Awan
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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.

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用于从针对口蹄疫病毒146S级分的高免疫血清中纯化IgG的免疫亲和层析用于诊断目的。
免疫亲和层析(IAC)是一种基本的分离和纯化工具,广泛应用于治疗和诊断。本研究重新评价了免疫亲和层析在纯化多克隆抗体方面的作用。基于蛋白A的IAC是一种从实验动物(如兔、豚鼠和小鼠)中饲养的高免疫系统(HIS)中纯化IgG的方便可靠的方法,可用于药物和诊断。将口蹄疫病毒(FMDV)TCID50=10 5.6的146S级分在幼仓鼠肾细胞系21(BHK-21)上培养,使用PEG 6000使用盐沉淀法浓缩,使用Sepharose-30在254nm吸光度下通过尺寸排阻色谱(SEC)纯化。用提供轻链和重链两条带的12%SDS-PAGE分析了146S FMDV的纯化。以明矾为基础的疫苗,由≥10μg的146S FMDV组成,应用于10只雄兔和10只雄豚鼠,每组2只动物作为阴性对照。用病毒中和试验计算血清滴度。使用Protein-A试剂盒(Thermo-scientific-44667052.2)纯化针对146S FMDV的HIS,并在还原条件下使用12%SDS-PAGE进行验证。数据表明,蛋白-A亲和层析是从针对146S FMDV的高免疫血清中纯化抗体的有效工具,并且可用于生产诊断试剂盒,例如酶联免疫吸附测定(ELISA)和放射免疫测定。
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