variations in the elisa technique
DESCRIPTION
Lab 6. Preparation of rabbit IgG Enzyme linked Immunosorbent Assay. Variations in the ELISA technique. Used for testing the amount of antibody to an antigen in serum. Add the solution containing antigen to be measured. The sensitivity of the Sandwich ELISA is dependent on four factors: - PowerPoint PPT PresentationTRANSCRIPT
Variations in the ELISA technique
Used for testing the amount of antibody to an antigen in serum
Lab 6. Preparation of rabbit IgG
Enzyme linked Immunosorbent Assay
Add the solution containing antigen to be measured
The sensitivity of the Sandwich ELISA is dependent on four factors:
1. The number of molecules of the first antibody that are bound to the solid phase.
2. The avidity of the first antibody for the antigen.
3. The avidity of the second antibody for the antigen.
4. The specific activity of the enzyme attached to the second antibody.
http://www.chemicon.com/resource/ANT101/a2C.asp
Antibody titres are worked out empirically.
Dilutions ofknown Standard
Try several dilutions of antibody1:250/ 1:500/ 1:1000/ 1:10,000
Cannot just take the Ab concentration from a different protocol
Always do duplicates of each test
Ideally the enzyme substrates should be stable, safe and inexpensive.
Popular enzymes are those which convert a colorless substrate to a colored product,
e.g. p-nitrophenylphosphate (pNPP) which is converted to the yellow p-nitrophenol by alkaline phosphatase.
Kuby 6-11
E=enzyme
CS-chromogenic substrate
CP-coloured pdt
ELISPOT Assay
http://www.elispot-analyzers.de
The specified number of IFN--producing T cellswas mixed into one million antigen presenting cells (APC).
The number of IFN--producing cells was measured by ELISPOT (upper panel) and intracytoplasmic staining (middle panel); results of ELISA measurements are shown in the lower panel.
Sensitivity of the ELISPOT assay.
•High-throughput. •Fewer cells are required compared to other cellular assays. •Lymphocytes survive the testing in ELISPOT assays.
Spot size is proportional to secretion
SDS covers proteins in a net negative chargeAddition of 2-mercaptoethanol reduces disulphide bonds and Boiling is used to further denature proteins.
Migrate in gel according to mass
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Before SDS
Charged R groups
H
H
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Hydrophobic areas
Sodium dodecyl sulfate polyacrylamide gel electrophoresis
Wikipedia
Range of separation of proteins depends on the percentage of polyacrylamide used (typical range 12-16%).
Proteins are separated in a ‘discontinuous’ system.
Stacking gel has looser pores to allow proteins to line up first.
How does an SDS-PAGE gel really work?
http://mullinslab.ucsf.edu/Protocols%20HTML/
SDS_PAGE_protocol.htm
pH 8.8Cl-
Proteins
Glycine
Leading ions
Glycine –ve charge
pH 6.8
How the discontinuous gel works.
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Coomassie Blue stained gel silver staining
Western blots- Ab used to identify Ag immobilized on nylon
SDS PAGE gel separates proteins present in a sample
All proteins are covered with
negatively charged SDS and
migrate according to mass
Native PAGE gels run under non-denaturing conditions-
SDS and 2-mercaptoethanol are omitted from the gel and sample
Proteins separate according to charge, size, shape
Silver stain
IgM serum
Western blot
mAb detects light chain
Ig serum
Bromage, E. Comp Biochem Physiol B Biochem Mol Biol. 2006 Jan;143(1):61-9. Epub 2005 Dec 1.
What does a Western blot
tell you that a protein
gel does not?
Protein blotting
Two major factors affect the efficiency
1. The elution from the gel
-use the lowest percentage of acrylamide that will
allow resolution
-high molecular weight proteins blot poorly
2. Efficiency of binding to the membrane
- nitrocellulose (not covalently bound)
- Polyvinylidene fluoride (PVDF)
- Activated nylon
Transfer of proteins to the membrane
Western blotting-wet transfer apparatus
Western blot-semi-dry transfer of proteins
Detection
Primary antibody followed by:
Radioactive-labelled
125I staphlococcal protein A or streptococcal protein G
Enzyme-linked secondary antibodies
-horseradish peroxidase (HRP)
-alkaline phosphatase-BCIP/NBT
BCIP (5-Bromo-4-Chloro-3'-Indolyphosphate p-Toluidine Salt) and
NBT (Nitro-Blue Tetrazolium Chloride).
Chemiluminescent detection-
HRP catalyzes the oxidation of luminol in hydrogen peroxide.
Luminol decays by light emission.
AP catalyzes the dephosphyorylation of adamantyl-1-2-dioxetane
phosphate, resulting in emission of light.
Can see proteins that are not normally visible
Far western technique
Detection of protein-protein interactions using a labelled bait protein
Figure 7 Distribution of the 52 kDa protein in various mouse tissues as analysed by Southwestern blot analysisFigure 7 Distribution of the 52 kDa protein in various mouse tissues as analysed by Southwestern blot analysis
Biochemical Journal (1998) 329, 623-629 - Biochemical Journal (1998) 329, 623-629 -
www.biochemj.orgwww.biochemj.org
Southwestern blot