(精编)【医学课件】 Molecular Diagnostics教学课件.pptVIP

Chapter 9-Molecular Diagnostics Enzyme-Linked Immunosorbent Assay (ELISA) Monoclonal Antibodies DNA Diagnostic Systems (DNA fingerprinting) Molecular Diagnosis of Genetic Disease (RFLPs, SNP genotyping, genotyping by sequencing) Old vs. New Molecular Diagnostics Old: grow cells/pathogen-test Such growth can be a problem as it is sometimes slow, costly, and specific New: direct test (either immunological or DNA based) Detection must be: specific, sensitive, and simple (fast and automatable are also nice) Fig. 9.1 Enzyme-Linked Immunosorbent Assay (ELISA): immunological detection Target molecule antigenic site i i i i i i i i i i i i i i i Support A. Bind sample to the support (commonly plastic or a membrane) B. Add primary antibody; wash C. Add secondary antibody-enzyme conjugate; wash D. Add substrate Y Y Y Y bound primary antibody Y E Y E Y E Y E enzyme linked secondary antibody colorless substrate colored product Fig. 9.2 Target antigens and polyclonal versus monoclonal antibodies Polyclonal antibodies are made against and react with multiple antigenic sites (epitopes) on a target antigen. Monoclonal antibodies are directed against a particular antigenic site. Target antigen with various antigenic determinants (epitopes) 1 2 3 4 5 6 7 Fig. 9.6 DNA diagnostic systems Bind ssDNA (target) to membrane Hybridize to labeled ssDNA or RNA (probe) Wash membrane to remove unbound probe Detect hybrid sequences formed between the probe and target DNA (concern: false +s -s) membrane DNA based diagnosis of Malaria and Typanosoma cruzi A DNA probe from a highly repeated DNA sequence of Plasmodium falciparum, the parasite that causes malaria, is used to screen blood samples via hybridization assays DNA primers are made against the ends of a 188 bp repeated sequence contained in the protozoan parasite Typanosoma cruzi, the causative agent of Chagas disease and used in a PCR/polyacrylamide gel electrophoresis detection method Other examples of DNA-based detection: Salmonella t