Different types of ELISA detection strategies
ELISA procedures can be described as a biochemical process that is frequently used to analyse the concentration of a specific antigen or an antibody that is present in each sample. The general principle for this procedure involves an unknown amount of antigen that is attached to the surface of the microwell plate, this antigen can bind to a specific antibody. A particular enzyme (usually HRP or AP) that can be conjugated to the detecting antibody will determine the readout that is obtained from the ELISA protocol.
The three most frequently used detection strategies can be classified as chromogenic, fluorescent, and chemiluminescent.
This is by far the most common method that is used, it is described as a colorimetric assay that results in the production of a coloured reaction end-product which absorbs light in the visible range. The optical density reading for the reaction product is known to be directly proportional to the amount of analyte that is being detected.
For this procedure either horseradish peroxidase (HRP) or alkaline phosphatase (AP) conjugated antibodies are mostly used together with a chromogenic substrate solution (3,3′,5,5′ tetramethylbenzidine, TMB). TMB is regarded as an extremely sensitive substrate, it can produce a blue colour that can be measured at a 650nm wavelength.
For majority of colorimetric assays, the use of either peroxidase or phosphatase is sufficient. Both enzymes can be used with many different substrates to generate either qualitative or quantitative ELISA results. Another important point is that these substrates can yield coloured soluble reaction products. This is idea when preforming an ELISA test.
There are many factors that can affect enzymatic activity measurement. Some of the most common factor responsible for ELISA include temperature, pH conditions, reaction time, substrate depletion, buffer composition, ionic strength, denaturation of the enzyme, exposure to light, build up of protein inhibitors and increase in back reaction as there is an increase in product concentration. For all commercially available kits all these conditions and factors have been optimised to ensure accurate results are generated each time.
Furthermore, this method is well suited for any kinetic analysis since it offers a rapid reaction rate. This procedure relies of using the absorbance to determine the readout, it can then determine the unknown concentration of the analyte using a standard curve.
These immunoassays are a simple variation of the chromogenic assay. The only difference is that the HRP or AP conjugated detection antibodies use fluorescent substates rather than colorimetric. In general, this method offers a slightly higher signal and a broader dynamic range. This can allow much higher readings to be measured more accurately in comparison to the 2.0-4.0 OD limit that is set by the colorimetric methods. One disadvantage of this method is the shorter half-life of fluorescent substrates when compared to the colorimetric substrates.
Some of the properties of this assay are it is offering 100 times greater sensitivity when compared to the colorimetric method, it is highly efficient, there are no radioactive material being used, it is safe and relatively easy to use. However, it displays background fluorescence interference (this can be minimised by using black microwell plates) and requires a fluorometer to read the signal (this is considerably a more expensive equipment).
At present there are many different fluorometers that are available on the market. Some of the features to consider when looking at a particular instrument include presence of an adjustable light detector, a thermal consistency within the reading chamber, a numerical adjustable gain, mix and match emission and excitation wavelengths, an internal background control mechanism, a choice of top plus bottom detection, an option to select duration and number of read times per well.
Luminescent assays are regarded as variation of the standard ELISA procedure, these are quite like that of fluorescent immunoassays. Essentially, an enzyme is used to convert a substrate into a reaction product, this process results in emitting light in the form of protons. Some of the commonly used enzymes for this assay are HRP and AP.
The process of luminescence can be defined as an emission of light from a substance as it returns to the ground state from an electronically excited state. A proton is released when excited intermediates return to the ground state. The released proton can be detected using an equipment that can measure luminescent signals.
There are many different forms luminescence, the most common ones are chemiluminescence, photoluminescence and bioluminescence. The only real difference between them is the way the excited state is reached.
- Chemiluminescence: The light is produced because of a chemical reaction.
- Photoluminescence: This is a simple fluorescence process, and the initiation of excitation is caused by light at a specific wavelength.
- Bioluminescence: This uses compounds that have bioluminescent properties, examples include the firefly luciferase and luciferin.
The most used methods for ELISA assays are the chemiluminescence and the bioluminescence. These have the advantage of providing a high sensitivity and a wide dynamic remeasuring range. The relative light units (RLU) are measured, and they tend to be proportional to the amount of analyte that is present within a sample.
It can be concluded that each of the above detection methods have their own set of requirements, however, with all three it is possible to develop a highly sensitive, precise, and reproducible assay that will general accurate results.
Regardless of which methods is being used, there are some common parameters that will need to be considered for example the use of an enzyme label that is suitable for the method and application being employed, the correct enzyme and substrate are chosen, the optimised condition are created, and finally the correct instrument is used for the measurement.