The type of antigen immobilization used, as well as the detection method (chemiluminescence, fluorescence, or chemifluorescence) can all be changed in an ELISA protocol. The following section details several ELISA protocol changes and applications.
Researchers can extend the variety of detection methods available to them by modifying the ELISA protocol with new technologies. We have selected a few widely researched techniques below.
- Chromogenic Assay
The result of chromogenic tests is a colourful reaction product that absorbs visible light. Washing removes the antigen-antibody complex formed on the solid carrier from other components. Enzyme is being used to label the antibody. Following the addition of the enzyme’s substrate, the substrate undergoes catalysis by the enzyme, resulting in a coloured product that is proportional to the amount of test substance. The optical density of the reaction product is typically proportional to the amount of analyte being measured. Due to the very high efficiency of the enzyme, the reaction can be greatly enlarged with a high sensitivity.
The two most popular enzymes labeling the antibodies are alkaline phosphatase (AP) and horseradish peroxidase (HRP)
Alkaline Phosphatase
Because the substrate p-nitrophenyl phosphate yields a water-soluble yellow reaction product, most ELISA assays have used alkaline phosphatase coupled antibodies as the detection technique. It can be added to the finished plate to completely dissolve the oxidation product and stop the reaction.
Horseradish Peroxidase
One commonly used enzyme conjugate in ELISA is horseradish peroxidase. Horseradish peroxidase (HRP) is a 40,000 Dalton protein, which catalyzes the reduction of hydrogen peroxide (H2O2) to water (H2O)
- Chemifluorescence
Fluorescent immunoassays are simply a variation of colorimetric ELISA. When activated by light of a specific wavelength, an enzyme converts a substrate into a reaction product that fluoresces. The amount of analyte being measured is usually proportional to the number of relative fluorescence units (emitted photons of light) detected. Fluorescent immunoassays are only significantly more sensitive than colorimetric ELISAs. They do, however, increase the assay’s dynamic range by allowing very high results to be recorded reliably, as compared to the 2.0 to 4.0 OD limit imposed on colorimetric assays.
Chemiluminescence
Chemiluminescence (also chemoluminescence) is the emission of light (luminescence) as the result of a chemical reaction. There may also be limited emission of heat. Given reactants A and B, with an excited intermediate,
[A] + [B] → [◊] → [Products] + light
CLIA is a better alternative method over conventional or traditional assays particularly ELISA for detection of antigen and / or antibodies of blood borne viruses. It can help in detecting early infection compared to ELISA and is suitable in large sample volume laboratories.
The widely used enzymes for luminescent immunoassays are also AP and HRP. HRP can be used with either bio- or chemiluminescent systems and is easily enhanced to allow prolonged detection of intense light (glow luminescence) which makes it compatible with all size microplate assay formats.
- Multiplex Assay
In the biological sciences, a multiplex assay is a type of immunoassay that uses magnetic beads to simultaneously measure multiple analytes in a single experiment. A multiplex assay is a derivative of an ELISA using beads for binding the capture antibody. Multiplex assays are still more common in research than in clinical settings.
In a multiplex assay, microspheres of designated colors are coated with antibodies of defined binding specificities. The results can be read by flow cytometry because the beads are distinguishable by fluorescent signature. The number of analytes measured is determined by the number of different bead colors.
