【作者】 佟辉；

【导师】 魏锁成；

【作者基本信息】 西北民族大学 ， 临床兽医学， 2007， 硕士

【摘要】 本试验用血清β2-微球蛋白单克隆抗体酶联免疫吸附的方法建立规范的肿瘤病人血清β2-微球蛋白的检测系统,用于肿瘤病的辅助诊断,从而为动物肿瘤疾病的诊断奠定基础。首先纯化β2-微球蛋白单克隆抗体腹水,其方法是:腹水蛋白质经采用饱和(NH4)2SO4沉淀法和分子筛相结合的方法纯化后得到β2-微球蛋白单克隆抗体。然后制备酶标二抗,具体方法是:用过碘酸钠将辣根过氧化物酶(HRP)的糖基轻微氧化为醛基,再与抗体氨基进行反应,形成辣根过氧化酶(HRP)与抗体的结合物;将β2-微球蛋白单克隆抗体与过氧化物酶的结合物进行纯化,得到酶结合物。最后以建立肿瘤病人血清β2-微球蛋白的检测系统:采用方阵法确定β2-微球蛋白单克隆抗体包被浓度和酶标二抗浓度,最佳抗体包被浓度和最佳酶标二抗浓度分别为1:128000和1:1000,并用ROC曲线法确定血清β2-微球蛋白单克隆抗体ELISA法检测的cut-off值,该值为0.35~0.4。灵敏度和特异性达到相对较高水平,ROC曲线下面积均大于总面积的均大于0.7,显示检测系统具有统计学意义和可用性。将β2-微球蛋白单克隆抗体蛋白检测法的肿瘤病检出率与肿瘤相关物质联合检测试剂盒法和对比,并作统计学分析。用spss11.5软件分析结果:最小期望频数为7.6,N>40,χ2=0.551,df=1, P>0.05,可认为β2-微球蛋白单克隆抗体蛋白检测法的肿瘤病检出率与肿瘤相关物质联合检测试剂盒法检测肿瘤病无显著性差异。该方法灵敏度高、准确性好、稳定性强,而且简便、快速。更多还原

【Abstract】 A standard testing system of serumβ2–microglobulin(β2–m) was found by the method of serumβ2–microglobulin monoclonal antibody enzyme-linked immunosorbent assay(ELISA). Firstly,β2-m monoclonal antibody ascites was purified from the ascites protein, the methods was to acquireβ2-m monoclonal antibody after ascites protein had been purified by using method of saturated ammonium sulfate sedimentation combined with molecule griddle. Secondly,β2-microglobulin monoclonal antibody was signed by HRP: the glycoside base was oxidized to aldehyde group by sodium iodinate and reacted with amino-group joined in theβ2-microglobulin monoclonal antibody, so that the HRP recombined with theβ2-microglobulin monoclonal antibody as enzyme signed secondary antibody. At last, the protein coating concentration and enzyme labeling antibody concentration were determined through square matrix method, the optimal delution concentrations of the first antibody and the enzyme signed secondary antibody were 1:128000 and 1:1000 respectively. The cut-off value ofβ2-microglobulin monoclonal antibody ELISA , which was determined by the ROC method,was 0.35~0.4. After comparing this method with another one, the statistics analysis showed that no significant differences existed( N>40,χ2=0.551, df=1, P>0.05), so this standard ELISA detecting system have been established, which is characterized with high sensitivity, specificity and stability.更多还原