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Immunology Assays for Clinical Research

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Immunology Assays for Clinical Research Yan Ge, Ph.D. University of Virginia Email: yg9z_at_virginia.edu Common Immunology Assays ELISA ELISPOT Flow cytometry ... – PowerPoint PPT presentation

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Title: Immunology Assays for Clinical Research


1
Immunology Assays for Clinical Research
  • Yan Ge, Ph.D.
  • University of Virginia
  • Email yg9z_at_virginia.edu

2
Common Immunology Assays
  • ELISA
  • ELISPOT
  • Flow cytometry
  • Immunofluorescent staining

3
ELISA
  • Enzyme-linked immunosorbent assay
  • To detect the level of an antibody or an antigen
    in a sample

4
Types of ELISA
  • Indirect ELISA
  • Sandwich ELISA
  • Competitive ELISA

5
Indirect ELISA Antibody Detection
6
Sandwich ELISA Antigen Detection
7
Enzymes and Substrates
  • horse radish peroxidase (HRP)
  • OPD
  • o-phenylenediamine dihydrochloride, maximum
    absorbance 492 nm
  • TMB
  • 3,3',5,5'-tetramethylbenzidine,
  • maximum absorbance 450 nm
  • ABTS
  • 2,2'-Azinobis 3-ethylbenzothiazoline-6-sulfonic
    acid-diammonium salt,
  • maximum absorbance 410 nm

8
Enzymes and Substrates
  • alkaline phosphatase (AP)
  • PNPP
  • p-Nitrophenyl Phosphate, Disodium Salt,
  • maxium absorbance 405 nm

9
Measurement
  • Optical density (OD) at a certain wavelength
  • Standard curve ? absolute concentration or
    arbitrary units

10
ELISPOT
  • Enzyme Linked Immunospot
  • Principle sandwich ELISA
  • To detect secreted proteins (e.g. cytokines,
    growth factors)

11
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12
Flow Cytometry (FACS, Flow)
  • A technique used to measure the physical and
    chemical properties of cells or cellular
    components
  • Cells are measured individually, but in large
    numbers.

13
Applications
  • Immunophenotyping
  • Identification of cells using cell-surface
    markers
  • Intra-cellular proteins (e.g. cytokines,
    chemokines)
  • Kinetics
  • E.g. Ca2 mobilization using Indo-1
  • DNA and cell cycle
  • DAPI, propidium iodide, 7-AAD
  • Cell sorting

14
Cell Staining with Fluorochromes
15
Flow Cytometer
  • Components
  • Fluidics
  • Optics
  • Detectors
  • Electronics

16
Fluidics
17
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18
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19
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20
FSC and SSC
  • Side-scattered light (SSC) cell granularity or
    internal complexity
  • Forward-scattered light (FSC) cell-surface area
    or size

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22
Compensation
23
An example
  • Stain cells with
  • FL1 CD4-FITC
  • FL2 CD19-PE
  • FL3 7-AAD (live/dead cells)
  • FL4 CD3-Alexa Fluor 647
  • FL5 CD8-Alexa Fluor 750

24
Data Analysis
Live cells (7-AAD negative)
lymphocytes
25
Data Analysis
FL5 CD8-Alexa 750
CD4 T cells
CD8 T cells
FL1 CD4-FITC
FL4 CD3-Alexa 750
FL4 CD3-Alexa 750
26
Data Analysis
Counts
B cells
FL2 CD19-PE
27
References
  • Introduction to Flow Cytometry A learning Guide.
    Manual Part Number 11-11032-01, April 2000, BD
    Sciences.
  • Introduction to Flow Cytometry. Flow cytometry
    core facility. Biomedical Instrumentation Center,
    Unifored Services University.
  • Introduction to Flow Cytometry. By Misha Rahman,
    AbD Serotec.
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