immunology assays for clinical research n.
Download
Skip this Video
Loading SlideShow in 5 Seconds..
Immunology Assays for Clinical Research PowerPoint Presentation
Download Presentation
Immunology Assays for Clinical Research

Loading in 2 Seconds...

play fullscreen
1 / 27

Immunology Assays for Clinical Research - PowerPoint PPT Presentation


  • 299 Views
  • Uploaded on

Immunology Assays for Clinical Research. Yan Ge, Ph.D. University of Virginia Email: yg9z@virginia.edu. Common Immunology Assays. ELISA ELISPOT Flow cytometry Immunofluorescent staining. ELISA. Enzyme-linked immunosorbent assay

loader
I am the owner, or an agent authorized to act on behalf of the owner, of the copyrighted work described.
capcha
Download Presentation

PowerPoint Slideshow about 'Immunology Assays for Clinical Research' - ovidio


An Image/Link below is provided (as is) to download presentation

Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.


- - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - -
Presentation Transcript
immunology assays for clinical research

Immunology Assays for Clinical Research

Yan Ge, Ph.D.

University of Virginia

Email: yg9z@virginia.edu

common immunology assays
Common Immunology Assays
  • ELISA
  • ELISPOT
  • Flow cytometry
  • Immunofluorescent staining
elisa
ELISA
  • Enzyme-linked immunosorbent assay
  • To detect the level of an antibody or an antigen in a sample
types of elisa
Types of ELISA
  • Indirect ELISA
  • Sandwich ELISA
  • Competitive ELISA
enzymes and substrates
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

enzymes and substrates1
Enzymes and Substrates
  • alkaline phosphatase (AP)
    • PNPP

p-Nitrophenyl Phosphate, Disodium Salt,

maxium absorbance: 405 nm

measurement
Measurement
  • Optical density (OD) at a certain wavelength
  • Standard curve  absolute concentration or arbitrary units
elispot
ELISPOT
  • Enzyme Linked Immunospot
  • Principle: sandwich ELISA
  • To detect secreted proteins (e.g. cytokines, growth factors)
flow cytometry facs flow
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.
applications
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
flow cytometer
Flow Cytometer
  • Components:
    • Fluidics
    • Optics
    • Detectors
    • Electronics
fsc and ssc
FSC and SSC
  • Side-scattered light (SSC): cell granularity or internal complexity
  • Forward-scattered light (FSC): cell-surface area or size
an example
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
data analysis
Data Analysis

Live cells

(7-AAD negative)

lymphocytes

data analysis1
Data Analysis

FL5: CD8-Alexa 750

CD4+ T cells

CD8+ T cells

FL1: CD4-FITC

FL4: CD3-Alexa 750

FL4: CD3-Alexa 750

data analysis2
Data Analysis

Counts

B cells

FL2: CD19-PE

references
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.