ELISA

ELISA

Enzyme-Linked ImmunoSorbent Assay (ELISA) is a biochemical technique used mainly in immunology to detect the presence of an antibody or an antigen in a sample. In simple terms, in ELISA an unknown amount of antigen is affixed to a surface, and then a specific antibody is washed over the surface so that it can bind to the antigen. This antibody is linked to an enzyme, and in the final step a substance is added that the enzyme can convert to some detectable signal. Thus in the case of fluorescence ELISA, when light of the appropriate wavelength is shone upon the sample, any antigen/antibody complexes will fluoresce so that the amount of antigen in the sample can be inferred through the magnitude of the fluorescence.

Types of ELISA

There are 3 types of ELISA. "Indirect", Sandwhich and Competitive ELISA.

"Indirect" ELISA:

The steps of the general, "indirect," ELISA for determining serum antibody concentrations are:

- Apply a sample of known antigen of known concentration to a surface, often the well of a microtiter plate The antigen is fixed to the surface to render it immobile. Simple adsorption of the protein to the plastic surface is usually sufficient.

- A concentrated solution of non-interacting protein, such as bolvine serum albumin(BSA) is added to all plate wells. This step is known as blocking, because the serum proteins block non-specific adsorption of other proteins to the plate.

- The plate wells or other surface are then coated with serum samples of unknown antigen concentration, diluted into the same buffer used for the antigen standards.

- The plate is washed, and a detection antibody specific to the antigen of interest is applied to all plate wells. This antibody will only bind to immobilized antigen on the well surface, not to other serum proteins or the blocking proteins.

- Secondary antibodies, which will bind to any remaining detection antibodies, are added to the wells. These secondary antibodies are conjugated to the substrate-specific enzyme.

- Wash the plate, so that excess unbound enzyme-antibody conjugates are removed.

-Apply a substrate which is converted by the enzyme to elicit a chromogenic or fluorogenic or electrochemical signal.

-View/quantify the result.

The enzyme acts as an amplifier; even if only few enzyme-linked antibodies remain bound, the enzyme molecules will produce many signal molecules. A major disadvantage of the indirect ELISA is that the method of antigen immobilization is non-specific; any proteins in the sample will stick to the microtiter plate well, so small concentrations of analyte in serum must compete with other serum proteins when binding to the well surface.

Sandwhich ELISA:

A less-common variant of this technique, called "sandwich" ELISA, is used to detect sample antigen. The steps are as follows:

- Prepare a surface to which a known quantity of capture antibody is bound.

-Block any non specific binding sites on the surface.

-Apply the antigen-containing sample to the plate.

-Wash the plate, so that unbound antigen is removed.

-Apply primary antibodies that bind specifically to the antigen.

-Apply enzyme-linked secondary antibodies which are specific to the primary antibodies.

-Wash the plate, so that the unbound antibody-enzyme conjugates are removed.

-Apply a chemical which is converted by the enzyme into a color or fluorescent or electrochemical signal.

-Measure the absorbance or fluorescence or electrochemical signal (e.g., current) of the plate wells to determine the presence and quantity of antigen.

The major advantage of a sandwich ELISA is the ability to use impure samples and still selectively bind any antigen that may be present. Without the first layer of "capture" antibody, any proteins in the sample may competitively adsorb to the plate surface, lowering the quantity of antigen immobilized.

Lastly, Competitive ELISA:

A third use of ELISA is through competitive binding. The steps for this ELISA are somewhat different than the first two examples:

-Unlabeled antibody is incubated in the presence of its antigen.

-These bound antibody/antigen complexes are then added to an antigen coated well.

-The plate is washed, so that unbound antibody is removed. (The more antigen in the sample, the less antibody will be able to bind to the antigen in the well, hence "competition.")

-The secondary antibody, specific to the primary antibody is added. This second antibody is coupled to the enzyme.
-A substrate is added, and remaining enzymes elicit a chromogenic or fluorescent signal.

For competitive ELISA, the higher the original antigen concentration, the weaker the eventual signal.

A video on the usage of ELISA (:

References:

google

wikipedia

http://www.youtube.com/watch?v=spQ70PZ3mDg&feature=related