What is ELISA?

What is ELISA?

Enzyme-linked Immunosorbent Assay

The first ELISA tests

Originally developed for antibody measurement

Tests have since been adapted to detect antigens

What is the current use of an ELISA test?

ELISA tests are now widely utilized to detect substances that have antigenic properties such as:

Hormones

Microbial antigens

Antibodies

How does an ELISA test work? pt 2

Partially purified, inactivated [HIV as example} antigen is added to the wells and allowed to incubate

Allows attachment of antigen to walls of the well

Unbound Ag washed away

A blocking agent is added to the wells

Prevents nonspecific binding of antibody

How does an ELISA test work? pt 2

Enzyme Substrate added and incubated

A color change indicates a positive result

Enzyme conjugated to second antibody acts on substrate

Intensity of color may be quantified where darker color indicates more serum bound to antigen

Intensity of colors at one wavelength may be quantified using a spectrophotometer

Reports absorbance in optical density units

>0.500 OD = positive test

0.300-0.499 OD are indeterminate and need to be retested

< 0.300 OD = negative test

In most cases, a patient will be retested if the serum gives a positive result.

western blotting analysis.

What are the advantages of ELISA?

Highly sensitive and specific

Results are quickly obtained

No need for radioisotopes or a costly radiation counter (as needed for radioimmune assays)

Why is ELISA so sensitive?

Enzyme conjugate has a high catalytic turnover rate

One conjugate can generate millions of product molecules

Why is it necessary to block unoccupied binding sites in the wells?

Smooth surfaces

No false positive readings

Why is a positive and negative control always run?

to make sure the test works properly

Why is anti-HIV-1 screened instead of the virus itself?

HIV-1 has a proviral stage where it lies dormant in T cells

During the early disease stages there is only traces of circulating virus present

The viral mutation rate is too high to be dependably monitored by any given preparation of antibodies

Why is secondary Ab used and where does it come from?

comes from non human animals

Indirect assay

What we have covered

One of the most common types of ELISAs

Sandwich ELISA

The other most common type of ELISA

The analyte is bound between two antibodies

Capture antibody

Detection antibody

Direct or indirect

Both must look for non-overlapping epitopes!

Immunodiffusion Techniques

Based on the most basic Ag-Ab principles

Binding may involve >1 Ab or Ag and large macromolecular complexes can form

These complexes form precipitates which can be cleared from the body

These precipitates are also useful for laboratory and diagnostic tests.

When Abs and Ags are inserted into different areas of an agarose gel, they diffuse toward each other

They form opaque bands of precipitate at the interface of their diffusion fronts

The precipitates are Ag-Ab complexes

Double Immunodiffusion – Ouchterlony Method

Double diffusion in two dimensions

A simple procedure invented by the Swedish scientist, Ouchterlony

Ag and Ab solutions are placed in separate wells cut in an agarose plate

The reactants diffuse from the wells toward each other and precipitate where they meet at equivalent proportions and form a single precipitation line.

Radial Imunodiffusion (RID)

Can quantitatively determine the concentration of an antigen

Often used clinically to detect patient levels of blood proteins.

Antibody is incorporated into molten agarose which is poured into a Petri dish and allowed to solidify.

Small wells are cut into the agarose and are filled with known concentrations of Ag

Samples of unknown concentrations are placed in similar wells.

The Ags in solution then diffuse outwards from the well in a circular pattern surrounding the well.

Diffusion of Ag continues until a stable ring of precipitate forms