host defense clinical and experimental immunologic assays ii domenick kennedy dept. microbiology and...
TRANSCRIPT
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+
