What is Competitive ELISA
Enzyme-linked immunosorbent assay (ELISA) is a widely used immunoassay technique that involves the binding of a target antigen or antibody to a solid support, followed by the detection of the bound antigen or antibody using an enzyme-linked detection system. Competitive ELISA is a variation of this technique that is used to measure the concentration of a specific antigen in a sample.
In this assay, a known amount of antigen is added to the sample along with a fixed amount of enzyme-labeled antigen and a limited amount of specific antibody. The antibody in the sample competes with the enzyme-labeled antigen for binding sites on the specific antibody. The more antigen present in the sample, the less enzyme-labeled antigen will bind to the specific antibody. After incubation and washing steps, a substrate is added to the wells, which reacts with the enzyme to produce a signal, usually a color change. The signal is inversely proportional to the concentration of antigen in the sample, as more antigen in the sample will result in less enzyme-labeled antigen binding to the specific antibody and thus less signal.
One of the key advantages of competitive ELISA is that it is highly specific, as the antibody used in the assay is specific to the target antigen. This specificity means that the assay can accurately measure the concentration of the target antigen in the presence of other molecules in the sample. Additionally, the assay is relatively easy to perform and can be adapted to a range of different sample types, including blood, urine, and food samples.
Figure 1. The competitive ELISA technique.
The competitive ELISA technique can be divided into three main steps: coating, incubation, and detection. In the coating step, the specific antibody is immobilized onto a solid support, usually a microtiter plate. The plate is then washed to remove any unbound antibody. In the incubation step, a known amount of antigen is added to the sample along with a fixed amount of enzyme-labeled antigen and the sample is incubated for a defined period of time. During this time, the antigen in the sample competes with the enzyme-labeled antigen for binding sites on the specific antibody. The more antigen present in the sample, the less enzyme-labeled antigen will bind to the specific antibody. In the detection step, a substrate is added to the wells, which reacts with the enzyme to produce a signal, usually a color change. The signal is inversely proportional to the concentration of antigen in the sample, as more antigen in the sample will result in less enzyme-labeled antigen binding to the specific antibody and thus less signal. The amount of antigen in the sample can be determined by comparing the signal of the sample to a standard curve generated by known concentrations of the antigen. This standard curve can be generated by performing the assay with known concentrations of the antigen and plotting the resulting signal against the concentration of the antigen.
The sensitivity of competitive ELISA can be improved by optimizing detection conditions, such as incubation time, antibody concentration, and enzyme-labeled antigen concentration. In addition, the use of more sensitive enzymes or substrates can also increase the sensitivity of the assay.
In summary, competitive ELISA is a highly specific and widely used immunoassay technique that can be used to measure the concentration of a specific antigen in a sample. The assay is relatively easy to perform and can be adapted to a range of different sample types. Assay sensitivity can be improved by optimizing assay conditions and using more sensitive enzymes or substrates. Competitive ELISAs can be used in biomedical research, clinical diagnostics, and food safety testing.
