大豆球蛋白兔源多克隆抗血清的制备及其免疫学特性鉴定

为建立大豆球蛋白致敏原检测方法和蛋白致敏性亚基的表位定位,从脱脂豆粉中分离纯化大豆球蛋白,制备多克隆抗血清,并进行免疫学特性分析。大豆球蛋白等电点pH值为6.4,采用碱溶酸沉法对大豆球蛋白进行粗提,用Sepharose CL-6B层析纯化大豆球蛋白粗提物,用SDS-PAGE检测蛋白纯度,免疫新西兰兔制备多抗血清,并对血清进行免疫学特性鉴定。结果表明:1号兔效价达到1∶6×10^4,由间接竞争ELISA抑制曲线可知,半数抑制率IC50为527 ng/mL,1号兔的多抗血清敏感性最高。特异性测定结果表明,该多抗血清除了与β-伴大豆球蛋白和花生蛋白的交叉反应率为8.6%和4.6%,与其他蛋白交叉反应率均小于0.2%。制备的兔源多抗血清效价高、敏感性强、特异性好,为大豆球蛋白单克隆抗体的制备及大豆蛋白致敏性亚分子结构的定位奠定了基础。

Preparation and immunological characteristics identification of rabbit polyclonal antiserum derived from soybean globulin

In order to establish soybean globulin allergen detection method and epitope mapping of protein allergenic subunit,soybean globulin was isolated and purified from defatted soybean powder to prepare polyclonal antiserum,and its immunological characteristics were analyzed.Soybean globulin isoelectric point pH is 6.4.Soybean globulin was crudely extracted by alkali extraction and acid precipitation,followed by purified by Sepharose CL-6B chromatography and its purity was detected by SDS-PAGE.New Zealand rabbits were immunized with soybean globulin to prepare polyclonal antiserum,and its immunological characteristics were analyzed.The results showed that the titer of rabbit No.1 was 1∶6×10^4,and the half inhibition rate IC50 was 527 ng/mL,which showed the highest sensitivity.The specificity results demonstrated that the cross-reaction rate of the serum with protein was less than 0.2%,except forβ-conglycinin and peanut protein(8.6%and 4.6%.)The prepared rabbit polyclonal antiserum had high titer,sensitivity and specificity,which layed a foundation for the preparation of soybean globulin monoclonal antibody and the mapping of soybean protein allergenic submolecular structure.