Enzyme-linked immunosorbent assay
Enzyme-linked immunosorbent assay (ELISA) hinges on specific antibodies binding to the target antigen in order to both indicate the presence of, and quantify peptides, antigens, proteins, antibodies and glycoproteins. The procedure was developed from the radioimmunoassay (RIA) described by Berson and Yalow in 1960, as a safer method to detect and measure biological molecules. ELISA’s are all conducted on a microtiter plate (usually 96 well), coated with an antigen or antibody.
The plate coating is attained through passive absorption (hydrophobic interactions between the plastic of the microtiter plate and the protein molecule). Plates are incubated for several hours before the coating solution is removed and a blocking buffer added to cover any unbound surfaces of the plastic well. Plates are often brought pre-coated with an optimised concentration in order to reduce both turnaround times and any errors which may arise from passive absorption, denaturation, poor immobilisation and binding of contaminants along with the target antigen or capture antibody.
There are a few different methods of ELISA the simplest being direct ELISA. In which a specific antigen is immobilised before antibody conjugate enzyme (normally alkaline phosphate or horseradish peroxidase) is added. The plate is washed in order to remove any unbound antibodies, addition of a subsequent chromogenic (colourless) substrate yields a visible colour change in the presence of the enzyme thus indicating the presence of the specific antigen in the sample. The colour intensity can then be measured using spectrometry to measure the concentration of antigen in the sample. A direct ELISA is the fastest method as it only uses one antibody, however this can be a disadvantage in that it has lower sensitivity than other ELISA methods.
Other ELISA technologies include: Indirect, Sandwich and Competitive ELISA’s. Where a direct ELISA is generally used to determine the concentration of high-molecular weight antigens by directly coating the microtiter plate with the antigen. An indirect ELISA is a two-step process, in which a primary antibody (in the test sample) binds to the immobilised antigen before incubating and washing. A secondary antibody with the enzyme conjugate and substrate enables the concentration to be determined; the intensity of the colour directly correlates to the concentration of the primary antibody. Indirect ELISA’s have a high level of sensitivity as the dual antibody bonding allows more than one enzyme conjugated antibody to bind and thus a greater number of bonding sites for the substrate, furthermore less enzyme conjugated antibodies are required for the reaction and allows for flexibility in the primary detection antibody. However, this method is more time consuming than a direct ELISA and allows for cross – reaction and nonspecific signals due to multiple antibodies. Therefor optimisation of this type of assay is important to reduce background signals.
A sandwich ELISA measures the concentration of the antigen between two antibodies, consequently it has a high specificity and is more sensitive (2-5 times) than other ELISA methods. The microtiter wells are coated with the first capture antibody and incubated, after washing a blocking buffer is used to block any unbound protein sites before adding the sample, incubating, washing and adding the enzyme conjugated antibody (the detection antibody) repeating the incubation and washing steps and adding the substrate. The sandwich ELISA is highly flexible as both direct and indirect ELISA methods can be used, however optimisation can be difficult; as cross reactivity may occur between the capture and detection antibodies.
Unlike the other methods a competitive ELISA, involves adding the sample and an enzyme conjugate concurrently. The wells coated with specific antigen for which the molecules compete in order to bind. Because there is an Inverse proportion between analyte concentration and absorbance the antigen concentration can be calculated. The competitive ELISA is more robust due to its high level of sensitivity allowing complex mixtures to be analysed with a single antibody thus enabling quantification of low amounts of antibody which aren’t necessarily suitable for a sandwich method due to cross reactivity.
The ElIspot (enzyme – linked immunospot) assay is a modification of the ELISA principle, which combines the immunoassay with bioassay in order to quantify the number of cells which produce a specific molecule (secreted proteins). Developed by Cecil Czerkinskdy in 1983 its highly sensitive and specific, measuring the target cytokine using the ELISA principle to bind the cytokine after having stimulated its release from the T-cell, before its dispersed thus enabling a more accurate depiction and quantification of the immune response. Its sensitivity lends itself to the analysis and detection of small cell numbers within specific immune responses such as transplantation risks, analysis of cancer antigens and vaccine development. This method is fairly expensive and difficult to perform.