特异性免疫磁珠联合毛细管电泳技术检测蛋白质的方法探讨

摘要

Objective The detection of urinary proteins plays vital role for disease diagnosis,prognosis and therapy evaluation.The conventional proteins assays cannot detect the specific low concentration proteins in urine.To detect low concentration proteins in urine and extend its application in disease diagnosis,our laboratory attempted to use the method of immunomagnetic beads coupled with capillary electrophoresis to detect specific low concentration proteins.We chose β-actin monoclonal antibody to verify the method.Methods Immunomagnetic beads were prepared by coating β-actin monoclonal antibody onto the surfaces of carboxylate-modified magnetic beads in covalent binding form.1％ BSA was used to block the β-actin immunomagnefic beads.Then borax buffer (pH10.0,0.02mol/L)was used to wash the β-actin immunomagnefic beads and the eluate part was collected.To prove β-actin immunomagnetic beads were prepared successfully,capillary electrophoresis was used to analyze the eluate solution.Results The electrophoretogram of the eluate solution contains β-actin antibody and BSA,proving carboxylate-modified magnetic beads were coated by β-actin monoclonal antibody and blocked by BSA successfully.Conclusion β-actin immunomagnetic beads were prepared and verified by capillary electrophoresis successfully,which has the vital significance for the detection of low concentration proteins in urine by immunomagnetic beads coupled with capillary electrophoresis.%目的 尿液蛋白的检测在疾病的诊断、监测和疗效评估中发挥着重要作用.目前常用的蛋白检测方法无法检测尿液低浓度蛋白.为了实现对尿液低浓度蛋白的检测,使其应用到临床诊断等方面,本实验室尝试应用特异性免疫磁珠联合毛细管电泳方法检测尿液低浓度蛋白.本实选择β-actin单克隆抗体来制备特异性免疫磁珠对该方法进行可行性探讨和方法学研究.方法 将β-actin单克隆抗体以共价偶联的方式包被到羧基磁珠表面,用含1％牛血清白蛋白(BSA)的PBS溶液封闭磁珠,获得特异性β-actin免疫磁珠.使用pH 10.0的0.02mol/L硼砂溶液对β-actin免疫磁珠进行洗脱,将洗脱液进行毛细管电泳,验证免疫磁珠是否制备成功.结果 β-actin免疫磁珠洗脱液的毛细管电泳图中可见到β-actin抗体峰和BSA峰,结果表明β-actin抗体与羧基磁珠偶联成功以及BSA封闭羧基磁珠成功.结论 本实验成功的制备了特异性β-actin免疫磁珠,并应用毛细管电泳方法进行相关验证,此实验为特异性免疫磁珠联合毛细管电泳方法检测尿液低浓度蛋白奠定了基础.