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

+

+

+

+

+

+

--

-

-

-

-

Before SDS

Charged R groups

H

H

-

-

--

--- -

-- -

--

--

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