ELISA

This technique is considered the gold standard for the identification and quantification of a soluble antigen in a complex liquid matrix, such as cell lysate or biological fluids. It is based on the ability of polystyrene plates to bind proteins. The method can also be used to detect peptides, antibodies, and hormones. 

The capture of an antigen on a solid surface is followed by incubation with an antibody that specifically recognizes and binds to the target molecule. The detection is usually colorimetric and is accomplished by measuring the absorbance of the product of the conjugated enzyme activity. Quantitation can be achieved by building a standard curve with antibody or antigen dilutions. The detection antibody is often labeled with HRP horseradish peroxidase or AP Alkaline phosphatases. The most crucial element of the detection strategy is a highly specific antibody-antigen interaction. 

Fluorescent immunoassays (ELFIA) are simply a variation of colorimetric ELISA. An enzyme converts a substrate to a product that fluoresces when excited by light of a particular wavelength. In comparison to the colorimetric ELISA, fluorescent immunoassays have a broader dynamic range, expanding the quantifiable concentration range.

Direct ELISA

Detection is achieved with an antigen-specific antibody conjugated with HRP, AP, or a molecule for fluorogenic or chemiluminescent detection.

Sandwich ELISA

This requires two antibodies specific for different epitopes of the antigen. One of the antibodies is immobilized on the surface of a multi-well plate and used as a capture antibody. This binds to the target antigen contained in a liquid sample. A labeled antibody is then used for the detection.