上海交通大学
硕士学位论文
基于纳米磁性微球的免疫层析定量检测技术研究
姓名：刘辉荣
申请学位级别：硕士
专业：生物化学与分子生物学
指导教师：古宏晨；徐宏
20080201
基于纳米磁性微球的免疫层析定量检测技
术研究
摘要纳米技术在现实生活中的应用
免疫层析技术是20世纪90年代初发展起来的一种新型免疫检测技术。具有快速、简便、单人份检测、经济的优点，已经广泛应用于医学检测、食品质量监测、环境监测、农业和畜牧业、出入境检验检疫、法医定案等领域。目前，免疫层析技术朝着定量、提高灵敏度、多元检测、层析系统集成化等方向发展，然而以胶体金或者乳胶微球为标记的光学定量免疫层析检测技术是通过检测硝化纤维膜表面标记物的光反射信号的密度，其定量检测的灵敏度较低；因此，目前基于磁信号的检测成为近两年定量免疫层析技术的研究热点。
本文采用EDC/NHS活化本实验室专利技术制备得到的高饱和磁化强度、分散稳定性好的羧基磁珠，在此基础上偶联抗β-hCG抗体，制备得到直径259 nm、分散良好的免疫磁珠。以hCG为模式检测蛋白，用免疫磁珠代替免疫胶体金构建免疫层析试纸条，实现了对hCG的定量检测。在试纸条构建过程中，考察了试纸条的特异性和灵敏度；以及一些效用物质（表面活性剂、盐离子、高聚物）对特异性与灵敏度的影响；最终实现了试纸条的较高特异性和较高灵敏度。通过用磁信号阅读仪检测不同浓度hCG试纸条的T线和C线上
的磁信号，得到hCG浓度和对应磁信号线性曲线，检测线性范围从0~5000mIU/ml，检测时间小于20min。另外，老化试验证明免疫磁珠试纸条的免疫活性保存期高于免疫胶体金试纸条。
关键词：磁珠，免疫磁珠，免疫层析，定量，检测
RESEARCH OF QUANTITATIVE LATERAL FLOW
TEST BASED ON IMMUNOMAGNETIC BEADS
ABSTRACT
Lateral flow test is a new kind of immunoassay developed from 1990s.With the advantages of rapidness、simplicity、single-test、economy, lateral flow has been used in the following field : medical test、quality monitor of food、supervision of environment、agriculture and stockbreeding、test and quarantine in custom、legal medical verdict. Nowadays lateral flow test  was developed towards for quantitative test、sensitivity improvement、multiplex detection、change of lateral flow system. Optical quantitative lateral flow test with the colloidal gold and colored latex as signal material was based on the detection of light intensity reflected from the signal material on the surface of the nitromembrane. The sensitivity of the quantitative lateral flow test based on colliodal was low. So the quantitative lateral flow test based on immunomagnetic nanobeads has become the research hot recently.
In this paper , the well dispersed immunomagnetic beads whose diameter was 259nm were prepared by the reaction
between the aminos of the anti-β-hCG antibodies and the carboxyl on the surface of magnetic beads whose carboxyl groups were catalyzed by EDC/NHS. Immunomagnetic beads were used in lateral flow strips as substitute for colloidal gold. HCG was quantitative tested as model protein by lateral flow strips of immunomagnetic beads. During of the construction of lateral flow strips ,effect of some material(surfactant、ion、polymer) on the specificity and sensitivity was observed. Magnetic signal on the test line and control line of hCG in different concentration were measured by magnetic signal reader. So the hCG concentration can be quantitative tested by measure the magnetic signal of the lateral flow strip. Diameter of immunomagnetic nanobeads(250nm) are far bigger than that of the colloidal gold(40nm), lateral flow strips with immunomagnetic nanobeads substituted for colloidal gold can test quantitatively and improve sensitivity and accuracy.
KEY WORDS: magnetic nanobeads, immunomagnetic nanobe- ads, lateral flow, quantitative, test