Host Defense

Clinical and Experimental Immunologic Assays II

Domenick KennedyDept. Microbiology and Immunology

dokennedy@lumc.edu

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?

dokennedy@lumc.edu