第11章_生物素-亲合素放大技术-课件.pptVIP

Ag-Ab1-Ab2-B + A-E Ag-Ab1-Ab2-B-A-E +A-E BAS实验方法及反应层次 方法 反应层次 直接法 BA Ag-(Ab-B)-A* BAB Ag- (Ab-B)-A-B* ABC Ag- (Ab-B)-AB*C 间接法 BA Ag-Ab1-(Ab2-B)-A* BAB Ag-Ab1- (Ab2-B)-A-B* ABC Ag- Ab1- (Ab2-B)-AB*C 二、生物素-亲合素系统在酶免疫测定中的应用 BAS在ELISA中的应用 生物素-亲合素系统在均相酶免疫测定 A B 固相化抗原或抗体 B 电化学发光免疫测定示意图 用于终反应的放大 三、生物素-亲合素系统在荧光免疫技术中的应用 BAS用于荧光抗体技术，通常采用BA法，即用荧光素直接标记亲合素（或链酶亲合素）；也可采用游离亲合素（或链酶亲合素）搭桥，两端分别连接生物素化抗体和荧光素标记的生物素（BAB法）或荧光标记的抗亲合素（或链酶亲合素）抗体的夹心法。 四、生物素-亲合素系统在放射免疫测定中的应用 BAS主要与免疫放射分析(IRMA)检测体系偶联，用于对终反应的放大（BA法） BAS也可用于IRMA反应后B、F成分的分离 五、生物素-亲合素系统在分子生物学中的应用 以生物素标记核酸探针进行的定位检测 用BAS制备的亲和吸附剂进行基因的分离纯化 将免疫测定技术与PCR结合建立免疫-PCR(immuno-PCR)用于抗原的检测 Anti-HCV Sandwich principle.Total duration:18minutes. 1st incubation:40μL of sample,60 μ L of reagent containing biotinylated HCV antigens and 60 μ L of a reagent containing HCV antigens labeled with a ruthenium complex react to form a sandwich complex. 2nd incubation:After addition of strepetavidin-coated microparticles,the complex becomes bound to the solid phase via interaction of biontin and streptavidin. The reaction mixture is aspirated into the measuring cell where the microparticles are magnetically captured onto the surface of the electrode.Unbound substances are then removed with ProCell.Application of a voltage to the electrode then induces chemiluminescent emission which is measured by a photomultiplier. Results are determined automatically by the Elecsys software by comparing the electrochemiluminescence signal obtained from the sample with the cutoff value obtained by anti-HCV calibration. HBsAgII Sandwich principle.Total duration of assay:18 minutes. 1st incubation:50 μL of sample,two biotinylated monoclonal anti-HBsAg antibodies, and a mixture of monoclonal anti-HBsAg antibody and polyclonal anti-HBsAg antibodies labeled with a ruthenium complex form a sandwich complex. 2nd:incubation:After addition of streptavidin-coated microparticles,the complex becomes bound to the solid phase via interaction of biotin and streptavidin.