BiotechnologyandGeneticEngineering-PBIO450550.ppt

Molecular Diagnosis of Genetic Disease Cystic fibrosis Sickle-cell anemia (see Fig. 9.28) Chapter 9-Molecular Diagnostics Enzyme-Linked Immunosorbent Assay (ELISA) Monoclonal Antibodies DNA Diagnostic Systems (DNA fingerprinting) Molecular Diagnosis of Genetic Disease 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. 9.4 Procedure for producing a monoclonal antibody to protein XNote: B lymphocytes or B cells produce antibodies but do not reproduce in culture. Some B cells can become cancerous and are known as myelomas which can reproduce in culture.See Chapter 9 animation/lodish7e/#800911__811944__ Fig. 9.3 Explanation of how HAT medium works Myeloma cells are HGPRT- and will die on HAT media having hypoxanthine, aminopterin (an antifolate), and thymidine. Spleen cells are HGPRT+ , so spleen-myeloma (hybridoma) cells can grow on HAT. (Note: spleen cells by themselves cannot grow in culture.) Table 9.2 Targets for diagnostic monoclonal antibodies Polypeptide hormones (chorionic gonadotropin, g