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Host Defense Clinical and Experimental Immunologic Assays II

Host Defense Clinical and Experimental Immunologic Assays II. Domenick Kennedy Dept. Microbiology and Immunology dokennedy@lumc.edu. Discussion Topics. Common immunologic assays used for assessment of antibody response: Production of Monoclonal Antibodies Immunofluorescence Flow Cytometry.

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Host Defense Clinical and Experimental Immunologic Assays II

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  1. Host DefenseClinical and Experimental Immunologic Assays II Domenick Kennedy Dept. Microbiology and Immunology dokennedy@lumc.edu

  2. Discussion Topics • Common immunologic assays used for assessment of antibody response: • Production of Monoclonal Antibodies • Immunofluorescence • Flow Cytometry

  3. Monoclonal Antibody – From a single clone of B cells

  4. Clinically Used Monoclonal Abs • Infliximab • Rheumatoid Arthritis, Crohn’s Disease, Ulcerative Colitis • Inhibits TNFα • Rituximab • Non-Hodgkin’s Lymphoma • Targets CD20 • Trastuzumab • Breast Cancer • Targets Her2/Neu (ErbB2) receptor • Adalimumab • Rheumatoid Arthritis, Crohn’s Disease, Ulcerative Colitis • Inhibits TNFα

  5. Immunofluorescence Mouse anti-Human Ig Ig Mouse anti-Human tumor antigen Patient Serum Ig Ig

  6. Immunofluorescence Staining

  7. Circumstances when we would want to identify a given cell population:

  8. Peripheral Blood of HIV Patient

  9. CD4+ CD4+ CD8+ CD8+ CD8+ CD8+ CD8+ CD4+ + anti-CD4, anti-CD8 antibodies Analyze by Flow Cytometry

  10. CD4+ CD4+ CD8+ CD8+ CD8+ CD8+ CD8+ CD4+

  11. Flow Cytometry Color Granularity Size

  12. SSC (Granularity) FSC (Size) http://www.nature.com/nprot/journal/v5/n7/fig_tab/nprot.2010.88_F1.html http://microvet.arizona.edu/Courses/MIC419/Secure/CaseNoTap/flowfigsize.jpg

  13. Identification of Immune Cell Populations through Flow Cytometry CD8- Red CD4- Green Color

  14. CD8+ Measure CD4+ and CD8+ cells CD4+ Green Red -HIV +HIV

  15. Questions? dokennedy@lumc.edu

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