新型定性-定量测定血红蛋白氧载体的检测系统Ⅲ.建立抗人源性氧载体类兴奋剂检测系统的初探  

Novel methodologies for detection of specific hemoglobin and-or related derivatives.Ⅲ.initial study for establishment of a detection system for human Hemoglobin-derived HOBCs

作　　者：陈芳[1] 严坤平[1] 王丽莉[1] 褚巍[1] 王凡 陈超[1,2] 但宁[1,2] 

机构地区：[1]西北大学生命科学学院／国家微检测系统工程技术研究中心 [2]陕西西大北美基因股份公司,陕西西安710069

出　　处：《西北大学学报（自然科学版）》2009年第2期255-258,共4页Journal of Northwest University（Natural Science Edition）

基　　金：国家“863计划”基金资助项目(2004AA205030)

摘　　要：目的构建一种高通量蛋白芯片检测人血清中血红蛋白氧载体类兴奋剂的方法。方法用自制SEC微柱快速地将人血清中血红蛋白聚合体(氧载体)和血红蛋白单体(另外加入的内标)分离,逐滴收集洗脱液作为探针,点样结合在蛋白质基片上,与其相对应的荧光标记的多克隆抗体进行杂交,扫描芯片荧光强度检测人血红蛋白氧载体。结果自制SEC微柱能够将戊二醛聚合的人血红蛋白聚合体和单体有效分离,在蛋白芯片的检测中能较好地检测出人血清中1.5%的聚合体人血红蛋白氧载体。结论以SEC技术和蛋白芯片相结合是一种高通量、灵敏、重现性好的检测人血清中的以人血红蛋白为基质氧载体类兴奋剂新方法。Aim To explore and study the basic characteristics and requirements for establishment of an analytic system using size exclusion chromatography （SEC） combined with a protein-chip-based technology for detection of hemoglobin-based oxygen carriers （HBCOs） derived for human hemoglobin for doping analysis. Methods An inhouse-made micro SEC column, Superdex 75 Micro 4/26 column, was used to separate human glutaraldehyde-polymerized hemoglobin （hPolyHb） and unmodified porcine hemoglobin （Hb） ; A protein chip method based on specif- ic antigen-antibody-binding reaction was then used to further confirm the presence or absence of human-derived HBOCs in SEC-separated samples. Results Superdex 75 Micro column effectively separated large-sized components of hPolyHb from unmodified porcine Hb with excellent reproducibility and high sensitivity （0. 015g/L） for detection of hPolyHb in a buffer system. The protein chip method could clearly demonstrate whether hPolyHb existed in the SEC fractions. However, due to the severe dilution of the high-molecular-weight components of hPolyHh in the tested samples by SEC, the overall sensitivity of the SEC-protein chip system for detection of hPolyHh in a buffer system was only 0. 5g/L. Conclusion Detection systems with combination of SEC using Superdex 75-based micro columns and protein-chip-based methodologies are feasible to significantly increase the reliability of the detection results in doping analysis of human-derived HBOCs. However, the system studied had relatively low detection sensitivity. Changing to a more sensitive signal detection mechanism, such as chemil , may gain significant improvement.

关 键 词：SEC-HPLC 血红蛋白氧载体 兴奋剂蛋白质芯片 

分 类 号：Q-3[生物学]