葡聚糖抗体的制备及其Elisa检测方法的建立

Preparation of Dextran Antibody and Establishment of It's Elisa Immunoassay

制备并筛选葡聚糖拟糖蛋白抗原,通过动物免疫获得葡聚糖多克隆抗体并建立葡聚糖的Elisa检测方法,应用于实际样品检测。以还原胺化法制备大分子葡聚糖-牛血清蛋白(BSA)拟糖蛋白质,通过SDS-PAGE与自由氨基测定,优化葡聚糖拟糖蛋白抗原制备反应,建立直接竞争Elisa筛选抗原活性。以制得的葡聚糖T40-BSA拟糖蛋白抗原为免疫原,经过免疫白兔后获得高效价多抗;建立检测葡聚糖多抗的间接非竞争Elisa方法。确定拟糖蛋白抗原的最佳制备条件(n(葡聚糖)/n(氧化剂NaIO4)=1/120,偶联时间24 h);筛选获得不同制备条件下与标准抗体结合最强的抗原产物T40-BSA,动物免疫获得多抗,优化实验条件:以质量浓度10μg/mL的T40-BSA为包被抗原,37℃时血清多抗和酶标多抗的反应时间均为45 min,质量分数5%小牛血清为封闭液后洗板;建立了基于T40-BSA拟糖蛋白抗原的不同相对分子质量葡聚糖间接竞争Elisa检测方法,并应用于对市售白糖中葡聚糖T40的含量检测。研究结果表明,葡聚糖-BSA拟糖蛋白抗原制备条件不同,对其免疫活性有影响。建立的检测方法有良好的线性,能用于实际样品中葡聚糖含量的检测。

In this study,dextran neoglycoprotein antigens were prepared evaluated anti finally obtained dextran polyehonal antibodies,and an enzyme-linked immunosorbent assay （Elisa） detection method was developed according to Elisa theory. The method was put to use for quantitative analysis of dextran in practical samples. Dextran neoglycoprotein antigens were prepared by Reduetive Amination method,and were confirmed by SDS-PAGE and free amino detection. The impact factors such as different oxidation degree of dextran,the conjugate reaction time to BSA were investigated. The antigens interacted with standard antibody and were evaluated through Elisa. The immunogen was immunized with white rabbits to obtained antibody respectively. A general and broad class-specific Elisa immunoassay was developed for dextran detection. The best preparation conditions were obtained （ndextran/noxidant, of NaIO4=1/120,the reaction time of 24 11）,and the antigen with best combination with standard was selected. The optimized conditions of assay used coating antigen at 10 μg/mL,reaction time of antibody and rabbit-anti-bovine IgG in 45 min,blocking reagents with 5% calf serum. The developed indirect competitive Elisa method was put to use for quantitative analysis of dextran T40 in commercial sugar. The different preparation condition of dextran neoglycoprotein antigen has effect on its immunocompetence. The developed Elisa detection method with good linear and accuracy was practical for dextran determination.