塞内卡病毒多克隆抗体的制备及其在免疫组织化学中的初步应用

Preparation of Polyclonal Antibody against Senecavirus A and Its Perliminary Application in Immunohistochemistry

为了制备纯度高、特异性强的塞内卡病毒(SVA)多克隆抗体并研究其在免疫组织化学(IHC)中的应用,本试验用甲醛溶液灭活SVA,将其与矿物油佐剂充分混合乳化后,接种兔制备多克隆抗体并测定中和抗体效价,经Protein A亲和层析介质纯化,用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)鉴定抗体纯度,用斑点杂交法(Dot blot)鉴定抗体特异性;以该抗体作为一抗,应用IHC技术检测SVA感染猪的蹄部组织切片中抗原的表达情况。结果显示,制备的SVA多克隆抗体中和抗体效价为1∶1024,纯化后的抗体经SDS-PAGE鉴定仅有分子量约55 kDa和25 kDa两条目的蛋白条带;Dot blot结果显示,该抗体特异性强,最低检测浓度为0.15μg/mL;IHC结果显示,该抗体在1∶500稀释时能够检测到组织切片中抗原的表达。结果表明,本试验制备的SVA多克隆抗体符合IHC技术要求,可为下一步SVA致病机理研究提供重要的试验材料。

In order to prepare highly pure and specific polyclonal antibody against Senecavirus A(SVA)and investigate its preliminary application in immunohistochemistry(IHC),formalin-inactivated SVA was fully mixed and emulsified with mineral oil adjuvant,followed by immunization of rabbits for polyclonal antibody production in this study.The neutralizing antibody titer was determined,and the purified antibody was obtained using Protein A-sepharose affinity chromatography.The purity and specificity of the polyclonal antibody were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE)and Dot blot,respectively.The purified antibody was used as primary antibody for IHC to detect the antigen expression in tissue sections of SVA-infected pigs'hooves.The results showed that the neutralizing antibody titer of the prepared SVA polyclonal antibody was 1:1024.After purification,SDS-PAGE analysis revealed two protein bands with molecular weights of approximately 55 kDa and 25 kDa.The Dot blot assay confirmed the strong specificity of the antibody,with a minimum detection concentration of 0.15μg/mL.The IHC results demonstrated that the antibody could detect antigen expression in tissue sections at 1:500 dilution.These findings indicate that the prepared SVA polyclonal antibody meets the requirements of IHC technology,and can provide important experimental materials for the further research on the pathogenesis of SVA.