药物设计和筛选.ppt

A. Heterogeneous Assays 多步筛选方法 multiple additions/incubations/washings/ transfers/filtrations/readings of the signal 特点 Labor intensive, complicated step, hard to automate （1）Nonradioactive Heterogeneous Assays Enzyme immunoassays (widely used in vitro assays) ELISA （Enzyme Linked Immunosorbent Assay） 酶联免疫吸附试验 An ELISA plate An HIV ELISA, sometimes called an HIV enzyme immunoassay (EIA) is the first and most basic test to determine if an individual is positive for a selected pathogen, such as HIV. The test is performed in a 8 cm x 12 cm plastic plate which contains an 8 x 12 matrix of 96 wells, each of which is about 1 cm high and 0.7 cm in diameter. Positive ELISA Test Negative ELISA Test HIV antigens pre-coated onto an ELISA plate Patient serum containing antibodies. If the patient is HIV+, then this serum will contain antibodies to HIV, and those antibodies will bind to the HIV antigens on the plate. Anti-human immunoglobulin coupled to an enzyme. This is the second antibody, and it binds to human antibodies. Chromogen or substrate which changes color when cleaved by the enzyme attached to the second antibody. S E P Antibody Antibody-2 colour, fluorescence, chemiluminescence Inhibitor Screening S Substrate E Enzyme P Product ELISA 方法药物筛选原理 Read ! EGFR 抑制剂筛选 ELISA试剂盒 （2）Radioactive Heterogeneous Assays Very commonly used, highly sensitive and robust despite handling hazards and radioactive waste generation 分离放射性产物的一般方法 Radioactive Product Radioactive Substract Glass-fiber filters (filtration) Washing Drying at rt. Transferred into a vial add scintillant Counted in a scintillation counter a. Filtration Assays b. Adsorption Assays In protein kinase reactions the phosphorylated product (acidic) by ionic interaction is captured on phosphocell- ulose （磷酸纤维素）filters; filter washed, air-dried and transferred into a vial; scintillant is added, and the vial is counted in a scintillation counter. c. Precipitation Assays In the traditional enzyme ass