Types of ELISA

There are many ELISA tests for particular molecules that use the matching antibodies. ELISA tests are broken into several types of tests based on how the analytes and antibodies are bonded and used. The major types are described here.

Direct ELISA

The steps of direct ELISA follows the mechanism below:

A buffered solution of the antigen to be tested for is added to each well (usually 96-well plates) of a microtiter plate, where it is given time to adhere to the plastic through charge interactions.

A solution of nonreacting protein, such as bovine serum albumin or casein, is added to each well in order to cover any plastic surface in the well which remains uncoated by the antigen.

The primary antibody with an attached (conjugated) enzyme is added, which binds specifically to the test antigen coating the well.

A substrate for this enzyme is then added. Often, this substrate changes color upon reaction with the enzyme.

The higher the concentration of the primary antibody present in the serum, the stronger the color change. Often, a spectrometer is used to give quantitative values for color strength.

The use and meaning of the names “indirect ELISA” and “direct ELISA” differs in the literature and on web sites depending on the context of the experiment. When the presence of an antigen is analyzed, the name “direct ELISA” refers to an ELISA in which only a labelled primary antibody is used, and the term “indirect ELISA” refers to an ELISA in which the antigen is bound by the primary antibody which then is detected by a labeled secondary antibody. In the latter case a sandwich ELISA is clearly distinct from an indirect ELISA. When the “primary” antibody is of interest, e.g. in the case of immunization analyses, this antibody is directly detected by the secondary antibody and the term “indirect ELISA” applies to a setting with two antibodies.

Sandwich ELISA

A sandwich ELISA.
(1) Plate is coated with a capture antibody;
(2) sample is added, and any antigen present binds to capture antibody;
(3) detecting antibody is added, and binds to antigen;
(4) enzyme-linked secondary antibody is added, and binds to detecting antibody;
(5) substrate is added, and is converted by enzyme to detectable form.

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

Competitive ELISA

A third use of ELISA is through competitive binding. The steps for this ELISA are somewhat different from the first two examples:
Unlabeled antibody is incubated in the presence of its antigen (sample).
These bound antibody/antigen complexes are then added to an antigen-coated well.
The plate is washed, so unbound antibodies are removed. (The more antigen in the sample, the more Ag-Ab complexes are formed and so there are less unbound antibodies available 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.
The reaction is stopped to prevent eventual saturation of the signal.

Some competitive ELISA kits include enzyme-linked antigen rather than enzyme-linked antibody. The labeled antigen competes for primary antibody binding sites with the sample antigen (unlabeled). The less antigen in the sample, the more labeled antigen is retained in the well and the stronger the signal.