host defense clinical and experimental immunologic assays ii
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Host Defense Clinical and Experimental Immunologic Assays II. Domenick Kennedy Dept. Microbiology and Immunology [email protected]. Discussion Topics. Common immunologic assays used for assessment of antibody response: Production of Monoclonal Antibodies Immunofluorescence Flow Cytometry. - PowerPoint PPT PresentationTRANSCRIPT
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Host Defense
Clinical and Experimental Immunologic Assays II
Domenick KennedyDept. Microbiology and Immunology
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Discussion TopicsDiscussion Topics
• Common immunologic assays used for assessment of antibody response:– Production of Monoclonal Antibodies– Immunofluorescence– Flow Cytometry
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Monoclonal Antibody – From a single clone of B cells
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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
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IgIg
Ig
Immunofluorescence
Mouse anti-Human tumor antigen
Patient Serum
Mouse anti-Human Ig
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Immunofluorescence Staining
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• Circumstances when we would want to identify a given cell population:
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Peripheral Blood of HIV Patient
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CD4+
CD8+
CD4+
CD8+
CD8+
CD8+
CD8+CD4+
+ anti-CD4, anti-CD8 antibodies
Analyze by Flow Cytometry
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CD4+
CD8+CD4+
CD8+
CD8+
CD8+
CD8+
CD4+
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Flow CytometryFlow Cytometry
Color
Size
Granularity
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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 (G
ranu
larit
y)
FSC (Size)
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Identification of Immune Cell Populations through Flow Cytometry
Color
CD8- Red
CD4- Green
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Measure CD4+ and CD8+ cells
+HIV-HIV
Green
Red
CD8+
CD4+