Four major immunologic methods

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Four major immunologic methods

  1. 1. Four Major Immunologic Methods
  2. 2. • ELISA (Enzyme-linked immunosorbant Assay • Immunofluorescence- Flow cytometry (FACS) Lecture by Dr. Witte • Western blot • Immunoprecipitation
  3. 3. Structure of Antibody Antigen binding site Five classes of antibody: IgM, IgG, IgA, IgE and
  4. 4. Ag + Ab Ag-Ab k 1 -1 k Ka = [AgAb] [Ab] [Ag] Association of antigen and antibody • Ag-Ab reactions are reversible • Affinity of Ab for Ag can be measured
  5. 5. EPITOPE = ANTIGEN-BINDING SITE POLYCLONAL vs MONOCLONAL ANTIBODIES • Protein Antigens have many epitopes • B cells make Ab to a single epitope • Different clones of B cells make Ab to different epitopes
  6. 6. Polyclonal and Monoclonal Antibodies
  7. 7. Antigen Excess Antibody Excess Antigen-Antibody Interactions
  8. 8. Haemagglutination
  9. 9. ELISA- qualitative method (a) Indirect ELIZA Detection of Ab Detection of Ag
  10. 10. Structure of Antibody
  11. 11. Competitive ELISA (quantitative method)
  12. 12. Radioimmunoassay (quantitative method)
  13. 13. Radioimmunoassay- Standard curve
  14. 14. Immunoprecipitation - qualitative
  15. 15. Figure A-18 part 1 of 2 Immunoprecipitation of Cell Lysates
  16. 16. Figure A-18 part 2 of 2 Immunoprecipitation of Cell Lysates
  17. 17. Figure A-20 Western Blot (qualitative and quantitative)
  18. 18. 1. Describe the difference between monoclonal and polyclonal antibodies 2. List 2 advantages of indirect reagents for antibody assays. 3. State the specificity of an indirect reagent. 4. Describe how to develop a "dip-stick" test for pregnancy using antibody to human chorionic gonadotropin, a peptide hormone found in elevated levels in urine of pregnant women. 55. Design an ELISA for detection of antibody to HIV. 66. Design an immunologic assay to quantitate levels of insulin in a patient’s blood. Problems

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