Transcript Immunology Assays for Clinical Research: ELISA ELISPOT

Immunology Assays for
Clinical Research
Yan Ge, Ph.D.
University of Virginia
Email: [email protected]
Common Immunology Assays
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ELISA
ELISPOT
Flow cytometry
Immunofluorescent staining
ELISA
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Enzyme-linked
immunosorbent
assay
To detect the level
of an antibody or an
antigen in a sample
Types of ELISA
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Indirect ELISA
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Sandwich ELISA
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Competitive ELISA
Indirect ELISA
– Antibody Detection
Sandwich ELISA
– Antigen Detection
Enzymes and Substrates
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horse radish peroxidase (HRP)
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OPD
o-phenylenediamine dihydrochloride,
maximum absorbance: 492 nm
TMB
3,3',5,5'-tetramethylbenzidine,
maximum absorbance: 450 nm
ABTS
2,2'-Azinobis [3-ethylbenzothiazoline-6sulfonic acid]-diammonium salt,
maximum absorbance: 410 nm
Enzymes and Substrates
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alkaline phosphatase (AP)
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PNPP
p-Nitrophenyl Phosphate, Disodium
Salt,
maxium absorbance: 405 nm
Measurement
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Optical density (OD) at a certain
wavelength
Standard curve  absolute
concentration or arbitrary units
ELISPOT
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Enzyme Linked Immunospot
Principle: sandwich ELISA
To detect secreted proteins (e.g.
cytokines, growth factors)
Flow Cytometry (FACS, Flow)
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A technique used to measure the
physical and chemical properties of
cells or cellular components
Cells are measured individually, but
in large numbers.
Applications
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Immunophenotyping
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Intra-cellular proteins (e.g. cytokines,
chemokines)
Kinetics
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E.g. Ca2+ mobilization using Indo-1
DNA and cell cycle
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Identification of cells using cell-surface
markers
DAPI, propidium iodide, 7-AAD
Cell sorting
Cell Staining with Fluorochromes
Flow Cytometer
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Components:
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Fluidics
Optics
Detectors
Electronics
Fluidics
FSC and SSC
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Side-scattered light
(SSC): cell
granularity or
internal complexity
Forward-scattered
light (FSC): cellsurface area or size
Compensation
An example
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Stain cells with:
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FL1:
FL2:
FL3:
FL4:
FL5:
CD4-FITC
CD19-PE
7-AAD (live/dead cells)
CD3-Alexa Fluor 647
CD8-Alexa Fluor 750
Data Analysis
Live cells
lymphocytes
(7-AAD
negative)
CD4+
T cells
FL4: CD3-Alexa 750
FL5: CD8-Alexa 750
FL1: CD4-FITC
Data Analysis
CD8+
T cells
FL4: CD3-Alexa 750
Data Analysis
Counts
B cells
FL2: CD19-PE
References
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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.