Competitive inhibition ELISA, also known as blocking ELISA, is a widely used technique in immunoassays. The core principle involves the use of the target antigen or antibody to interfere with a pre-established system, ultimately leading to a colorimetric result that reflects the level of interference. The relationship between the degree of interference and the amount of the target analyte is inverse—higher concentrations lead to lower signal intensity.
This method is highly flexible, allowing for the development of more complex experimental designs, such as direct competitive inhibition ELISA, indirect competitive inhibition ELISA, and sandwich competition ELISA. Below, we explain the principles of two common types: direct and indirect competitive inhibition ELISA.
In **direct competitive inhibition ELISA**, an antigen is first coated onto a solid-phase carrier. An enzyme-labeled specific antibody is then added. During the experiment, the sample containing either the antigen or antibody to be tested is introduced. If the target is an antigen, it competes with the pre-coated antigen for binding to the enzyme-labeled antibody. If the target is an antibody, it competes with the enzyme-labeled antibody for binding to the coated antigen. After washing, unbound enzyme-labeled antibodies are removed, and a substrate is added to develop color. The final color intensity is inversely proportional to the concentration of the target analyte. Notably, this method can detect both antigens and antibodies using the same pre-prepared system.
**Indirect competitive inhibition ELISA** works by introducing the test antigen or antibody into a previously established indirect ELISA system. Here, the antigen is coated on the solid phase, followed by the addition of a specific primary antibody and an enzyme-labeled secondary antibody. In the experiment, the sample is diluted and introduced, where the test antigen or antibody competes with the bound antigen for the primary antibody. This method includes a three-step signal amplification process, making it more sensitive than the direct version.
One of the major advantages of competitive inhibition ELISA is its minimal reagent requirement. Both monoclonal and polyclonal antibodies can be used, and it is suitable for a wide range of targets, including macromolecules, peptides, small molecules, drugs, and even hormones with only one epitope—substances that may not work well in sandwich ELISA formats.
For instance, in the detection of hepatitis B markers, the e antigen is unstable and prone to degradation into the core antigen. As a result, sandwich ELISA cannot be used to detect e antibodies, but competitive inhibition ELISA provides an effective alternative.
Beyond these standard methods, researchers can tailor other ELISA formats based on their needs. For example, the biotin-avidin system can enhance signal amplification, while non-enzyme secondary antibodies like Protein A or G can improve binding efficiency or specificity.
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