纳米磁珠免疫分析技术的建立及其检测人红细胞血型Mi^a(MNS7)抗体的初步实验  

The application of nanomagnetic beads in immunoassays and the research in detecting serum antibodies of Mi^a(MNS7) blood group

作　　者：田晶晶[1] 丁少华[1] 王红梅[1] 段生宝[1] 陈晔洲 李勇[1] 

机构地区：[1]中国科学院苏州生物医学工程技术研究所中科院生物医学检验技术重点实验室,江苏苏州215163

出　　处：《中国输血杂志》2014年第3期244-248,共5页Chinese Journal of Blood Transfusion

基　　金：江苏省自然科学基金青年基金(BK20130343);苏州市医疗器械与新医药专项基金(ZXY2013017)

摘　　要：目的制备偶联人红细胞Miltenberger血型抗原多肽的免疫磁珠,以之为基础建立快速便捷的Miltenberger血型抗体检测技术。方法利用水热合成法合成纳米磁珠,在其表面包被二氧化硅层,并进行氨基化修饰;利用碳酰二亚胺盐(EDC)分别将人免疫球蛋白G(IgG)及牛血清白蛋白(BSA)偶联于磁珠表面,优化偶联条件,并通过流式细胞仪检测荧光标记的抗IgG来评价偶联效果;将人红细胞Miltenberger血型Mia(MNS7)抗原多肽偶联于磁珠表面,检测血清中的抗体。结果纳米磁珠平均粒径为400 nm,包被二氧化硅层后可有效提高磁珠分散性,经氨基化修饰氨基含量约260μmol/g;在提高偶联效率同时保证蛋白与磁珠间的非特异吸附最低,确定了0.01 mol/L PB溶液(pH=7.4)为偶联BSA、0.1 mol/L PB溶液(pH=7.4)为偶连人IgG的最适反应溶剂,非特异性吸附最小;经荧光素标记抗人IgG检测,偶联人IgG磁珠的荧光信号值约为偶联BSA磁珠的荧光信号值的100倍;Mia抗原多肽免疫磁珠对阴性、阳性血清检测的荧光信号差别可达10倍。结论成功制备偶联人红细胞Miltenberger血型系统Mia抗原多肽的免疫磁珠,并确定其在检测人血清抗体中的应用价值。Objective The preparation of immunno-magnetie beads coupled with the pelypeptlde antigen of Mihenberger blood group ,and the foundation of a fast and convenient technique analying the antibodies of Mihenberger blood group based on the immunno-magnetic beads. Methods The nanomagnetic core was synthesized using the hydrothermal method, coated with SiO2 laver,and then modified by amination; Human IgG or BSA was coupled onto the surface of the prepared magnetic beads using EDC. The coupling condition was optimized and the coupling efficiency was evaluated by the fluorescence density of labeled anti-IgG using flow cytometry ; The polypeptide antigen of Mihenberger blood group was coupled onto the immuno-magnetic beads and applied on the analysis of antibodies in the serum. Results The nanomagnetic core has an average parti-cle size of 400 nm and the coating with a layer of SiO2 can greatly improve its dispersity. After amination, the amount of amino group content is 260 mmol/g. The best reaction reagent is 0. 01 mol/L PB （pH =7.4）for the coupling of BSA and 0. 1 mol/ L PB （ pH = 7.4） for the coupling of human IgG, providing the highest coupling efficiency and the lowest nonspecific absorp-tion. The nonspecific absorption is low,judging that the difference of the fluorescent signals is 100-folds between human IgG-coupled and BSA-coupled magnetic beads; the difference of the fluorescent signals can reach as high as 10-folds between the negative and positive serum when detected using the Mi^a-antigen polypeptide coupled immuno-magnetic beads. Conclusion We succecded in the preparation of the immunno-magnetic beads coupled with the Mi^a-antigen polypeptide of Miltenberger blood group, and confirmed its valuable application in the detection of human serum antibodies.

关 键 词：纳米磁珠 免疫分析 Miltenberger血型 Mi^a抗原多肽 抗体检测 

分 类 号：R446.61[医药卫生—诊断学] R457.11[医药卫生—临床医学]