Enzyme-Linked Immunosorbent Assay (ELISA) is used mainly to detect and quantify proteins, antibodies, peptides, or hormones in a sample. In ELISA, antigens are immobilized on a solid support, either coated directly or through specific capture antibodies. Primary detection antibodies are then applied, forming a complex with the antigen. If conjugated with an enzyme, the detection antibody can directly be used to quantify the antigen, or can itself be quantified by another enzyme-conjugated secondary antibody. The method chosen depends on which target you are investigating.
Types of ELISA Methods:
Direct ELISA: Antigens are immobilized and enzyme-conjugated primary antibodies are used to detect or quantify antigen concentration. The specificity of the primary antibody is very important.
PROS: minimum procedure; avoids cross-reactivity from secondary antibody.
CONS: requires labeling of all primary antibodies - high cost; not every antibody is suitable for labeling.
Indirect ELISA: Primary antibodies are not labeled, but detected instead with enzyme-conjugated secondary antibodies that recognize the primary antibodies.
PROS: secondary antibodies are capable of signal amplification; many available secondary antibodies can be used for different assays; unlabeled primary antibodies retain maximum immunoreactivity.
CONS: cross-reactivity may occur.
Sandwich ELISA: The antigen to be measured is bound between a layer of capture antibodies and a layer of detection antibodies. The two antibodies must be very critically chosen to prevent cross-reactivity or competition of binding sites.
PROS: sensitive, high specificity, antigen does not need to be purified prior to use.
CONS: antigens must contain at least two antibody binding sites.
Competitive ELISA: The antigen of interest from the sample and purified immobilized antigen compete for binding to the capture antibody. A decrease in signal when compared to assay wells with purified antigen alone indicates the presence of antigens in the sample.
PROS: crude or impure samples may be used, high reproducibility.
The indirect ELISA is used primarily to determine the strength and/or amount of antibody response in a sample. In the assay, the antigen of interested is immobilized by direct adsorption to the assay plate. Detection of the antigen can then be performed by using a matched set of primary antibody and conjugated secondary antibodies.
Sandwich ELISA is performed to determine the amount and serological class of antibodies made by an immunized animal or present in the serum of patients. The anti-immunoglobulin antibodies used have high specificity and sensitivity.
In competitive ELISA, unlabeled antibody is incubated in the presence of its antigen. Then these bound antibody/antigen complexes are then added to an antigen coated well. After washing, unbound antibody is removed. The more analyte in the sample, the less antibody will be able to bind to the antigen in the well. The signal is then detected using a labeled secondary antibody and the decrease in signal is compared to a control. The major advantage of a competitive ELISA is the ability to use crude or impure samples and still selectively bind any antigen that may be present.