The ELISA, or enzyme linked immunosorbent assay, is the gold standard immunoassay for detection of small quantities of protein in samples as varied as serum, urine, saliva, and more. The ELISA is a labeled assay, which means that some type of label is needed to detect protein binding events. These labels are typically fluorescent, chromatic, or chemiluminiscent, and require the use of a plate reader to quantify the amount of protein in the sample. The major benefit of the ELISA is that low concentrations (often down to pg/mL) of protein are easily quantified. One disadvantage to ELISA is that many steps and reagents are required throughout the protocol. However, this can be mitigated by purchasing an ELISA kit that is pre-bound with capture antibodies and contains a detailed protocol for using all of the included buffers in a clear, easy to follow format. The use of an ELISA kit can improve diagnostic results from assay to assay because the kits are all validated between lots and come with protein standards. This means that a standard curve (detected signal vs. protein concentration) is generated during each assay and this standard curve can be checked against the expected values to ensure that the kit is still functioning as expected. The kit streamlines the process and takes the guesswork out of protocol design.
ELISA kits are commercially available for a wide range of target molecules and for direct and competitive assays. Direct ELISAs measure the amount of protein in the sample by measuring how much target protein binds to the capture antibodies. These assays have a direct relationship between amount of protein and signal produced: more protein, more signal. Competitive assays have an inverse relationship between amount of protein and signal produced: more protein, less signal. ELISA kits are available for use with a variety of samples:
- Cell culture extracts
Most kits are shipped with capture antibody pre-coated on the wells. This saves time for the user, but it also means that kits must be carefully selected for the target protein. It is essential that the capture antibody has specific and high affinity for the target molecule. Luckily, a wide variety of ELISA kits are available for detection of target proteins:
- immune markers/cytokines
- cancer markers
- T cells
- signal transduction pathways
- cardiovascular markers
- blood markers
- stem cells
- cell surface markers
- signaling pathway specific proteins
All standards, blocking, and washing solutions are included in the kit along with a detailed protocol for use. Also, enzyme tagged detection antibody and substrate for the colorimetric reaction. The instructions guide the user through proper dilutions of standard and sample. The standard curve is important to ensure that the kit is working properly because you can compare it to the published standard curve for the kit, but it is also used for quantification of protein concentration of the sample. Once the standard curve is generated and values have been generated for the sample, the concentration of protein in the sample is interpolated from the standard curve.
Although the ELISA kit contains all of the antibodies, buffers, enzymes, and substrates necessary for a successful assay, there are some things that the user must provide. These are things that are found in any biochemical laboratory: pipettes, a microplate reader, tubes for dilutions, and water. A commercially available ELISA kit may be a good choice if high quality, consistency, ease-of-use, and simplicity are desired.
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