Enzyme linked immunosorbent assay, or ELISA, is a powerful qualitative and quantitative tool for measuring proteins in biologic samples. Multiple variants of ELISA immunoassays exist, and are chosen based on the samples being tested.
Since its development during the 1960s and 1970s1, ELISA has become established as a method that uses a specific antibody to detect the presence of a protein using chromogenic, fluorescent, or luminescent readout2. Because ELISA kits and ELISA sets come with standards – solutions of the target protein at known concentrations – ELISAs can also be used to calculate the absolute concentration of a protein in test samples.
Direct ELISA is used to detect antigen that is directly bound to a plate – for example, on whole cells or viruses – while indirect ELISA, which usually takes the format of a sandwich ELISA, uses a multi-step protocol to capture the target protein using a second antibody to detect. This method is used for crude liquid samples including serum and urine.
In a sandwich ELISA, a 96-well plate is coated with an antibody specific to the protein of interest. The plate is then incubated with a blocking buffer containing irrelevant proteins, to block non-specific binding of proteins from the test sample to the plate. Samples, plus appropriate controls and standards, are then added to the plate, where the target protein is captured by the antibody coating the plate. Next, the detection process begins. A primary “detection antibody” – also specific to the protein of interest – is added. This antibody is either directly conjugated to, or then bound by a secondary antibody conjugated to, an enzyme – such as HRP for a chromogenic reaction – followed by the addition of its corresponding substrate.
The combination of enzyme with substrate initiates the reaction whose product is detected by a plate reader to give the readout used to calculate the absolute protein concentration. For chromogenic reactions, the readout is in OD, or optical density – a measure of the absorbance of a specific wavelength of light by the solution in each well. To convert the OD values into protein concentrations, the OD values for the standards are plotted against their known concentrations, giving a standard curve unique to the plate from which it was read. Next, the OD values for the samples are plotted along the linear portion of that standard curve, and the protein concentrations extrapolated from the corresponding point along the second axis.
Because of its qualitative and quantitative applications, ELISA is a robust technique that allows scientists to both detect the presence of a specific protein in samples and determine the amount of protein present. Experimentally, this means that ELISA can be used to compare differences in protein concentration between control and experimental groups in an in vivo or in vitro experiment, or between healthy donor and patient samples in clinical studies.
Bethyl Laboratories sells high quality ELISA kits. These products have recently been used to study: