Host Defense Clinical and Experimental Immunologic Assays II Domenick Kennedy Dept. Microbiology and...
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Transcript of Host Defense Clinical and Experimental Immunologic Assays II Domenick Kennedy Dept. Microbiology and...
Host Defense
Clinical and Experimental Immunologic Assays II
Domenick KennedyDept. Microbiology and Immunology
Discussion TopicsDiscussion Topics
• Common immunologic assays used for assessment of antibody response:– Production of Monoclonal Antibodies– Immunofluorescence– Flow Cytometry
Monoclonal Antibody – From a single clone of B cells
Clinically Used Monoclonal Abs
InfliximabRheumatoid Arthritis, Crohn’s Disease, Ulcerative ColitisInhibits TNFα
RituximabNon-Hodgkin’s LymphomaTargets CD20
TrastuzumabBreast CancerTargets Her2/Neu (ErbB2) receptor
AdalimumabRheumatoid Arthritis, Crohn’s Disease, Ulcerative ColitisInhibits TNFα
IgIg
Ig
Immunofluorescence
Mouse anti-Human tumor antigen
Patient Serum
Mouse anti-Human Ig
Immunofluorescence Staining
• Circumstances when we would want to identify a given cell population:
Peripheral Blood of HIV Patient
CD4+
CD8+
CD4+
CD8+
CD8+
CD8+
CD8+CD4+
+ anti-CD4, anti-CD8 antibodies
Analyze by Flow Cytometry
CD4+
CD8+CD4+
CD8+
CD8+
CD8+
CD8+
CD4+
Flow CytometryFlow Cytometry
Color
Size
Granularity
http://microvet.arizona.edu/Courses/MIC419/Secure/CaseNoTap/flowfigsize.jpghttp://www.nature.com/nprot/journal/v5/n7/fig_tab/nprot.2010.88_F1.html
SS
C (
Gra
nula
rity)
FSC (Size)
Identification of Immune Cell Populations through Flow Cytometry
Color
CD8- Red
CD4- Green
Measure CD4+ and CD8+ cells
+HIV-HIV
Green
Red
CD8+
CD4+
Questions?