现代医学实验学_实验技术新.pptVIP

Types of ELISA Methods: Direct ELISA:?Antigens are immobilized and enzyme-conjugated primary antibodies are used to detect or quantify antigen concentration. The specificity of the primary antibody is very important. PROS: minimum procedure; avoids cross-reactivity from secondary antibody. CONS: requires labeling of all primary antibodies - high cost; not every antibody is suitable for labeling.  Indirect ELISA:?Primary antibodies are not labeled, but detected instead with enzyme-conjugated secondary antibodies that recognize the primary antibodies. PROS: secondary antibodies are capable of signal amplification; many available secondary antibodies can be used for different assays; unlabeled primary antibodies retain maximum immunoreactivity. CONS: cross-reactivity may occur.  Sandwich ELISA:?The antigen to be measured is bound between a layer of capture antibodies and a layer of detection antibodies. The two antibodies must be very critically chosen to prevent cross-reactivity or competition of binding sites. PROS: sensitive, high specificity, antigen does not need to be purified prior to use. CONS: antigens must contain at least two antibody binding sites.  Competitive ELISA:?The antigen of interest from the sample and purified immobilized antigen compete for binding to the capture antibody. A decrease in signal when compared to assay wells with purified antigen alone indicates the presence of antigens in the sample. PROS: crude or impure samples may be used, high reproducibility. CONS: lower overall sensitivity and specificity. Northern 的发明人不叫Northern: ?James Alwine, David Kemp, and George Stark 正常细胞细胞核发出均匀的荧光，凋亡细胞细胞核致密浓染,发出亮蓝色荧光。红色箭头所示为凋亡细胞。对照组偶见少量自然凋亡的细胞，模型组凋亡细胞数量明显增多。预先加入PCF可剂量依赖性减少凋亡细胞数,凋亡率结果如下 四环素控制的条件基因表达/沉默：转录的激活子（Transactivator）rTA，在无四环素存在的前提下，能与一个特别定制的操纵子tet-O结合，激活下游目标基因的表达，而四环素（如多西环素）的应用将使此激活子与四环素结合，迅速中止目标基因的表达。 RNA干扰 1 原理： 近年来的研究表明，将与mRNA对应的正义RNA和反义RNA组成的双链RNA(dsRNA)导入细胞，可以使mRNA发生特异性的降解，导致其相应的基因沉默。这种转录后基因沉默机制(post-transcriptional gene silencing,