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ELISA (Enzyme-linked Immunosorbent Assay) Detection Strategies

The type of enzyme (most commonly HRP or AP) that is conjugated to the detection antibody dictates the type of readout of an ELISA experiment. Three of the most common types of detection include chromogenic, fluorescent, and chemiluminescent—the choice of which depends on the required sensitivity and signal-to-noise ratio.

Chromogenic Assay

The most common type of ELISA detection uses a colorimetric assay. Generally, horseradish peroxidase (HRP-) or alkaline phosphatase (AP-) conjugated antibodies are used in combination with a chromogenic substrate (eg, TMB) solution. This detection method uses absorbance to determine the readout and compares samples to each other or determines concentrations based on a standard curve.

CST provides researchers with a line of ELISA kits across a variety of biological targets, including targets in critical signaling pathways, that are developed, produced, and validated in-house to ensure consistent results. Our Pathscan® and FastScan™ ELISA kits are available for a wide selection of antibody assays measuring total proteins, cleavage products, and post-translational modifications.

Chemiluminescent Assay

A second methodology uses the peroxidase-conjugated detection antibodies (ie, AP and HRP) in a chemiluminescent reaction. In this type of experiment, a luminol-based enhancer solution is added along with a substrate, and the signal is read using a luminometer. Chemiluminescent detection is often used if a large dynamic range is needed.

CST produces a number of PathScan® Chemiluminescent Sandwich ELISA Kits that have a wider dynamic range and greater sensitivity than conventional chromogenic detection systems. These kits include low volume microplates, which provide increased signal and sensitivity while using half the sample volume of traditional colorimetric ELISA microplates.

Fluorescent Assay

A third methodology uses the peroxidase-conjugated detection antibodies (ie, AP and HRP) with fluorescent substrates. This method may provide a higher signal and broader dynamic range; however, fluorescent substrates may also have a shorter half-life than colorimetric substrates.

Fluorescent assays require the use of black multiwell plates in order to minimize background. In addition, the use of a fluorometer is required to read the signal.