1. Host Defense Clinical 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. Ig Immunofluorescence Mouse anti-Human tumor antigen Patient Serum Mouse anti-Human Ig

6. Immunofluorescence Staining

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

8. Peripheral Blood of HIV Patient

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

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

11. Flow Cytometry Color Size Granularity

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

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

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

15. Questions? dokennedy@lumc.edu