Enzyme-linked immunosorbent assays (ELISAs) are used for detecting analytes of interest in solution, typically in 96-well or 384-well microplate formats. They are one of the most popular types of immunoassays due to their high specificity and sensitivity, ease of use, and the fact that they are able to provide quantitative data. ELISAs can be used with a broad range of samples, including cell lysates, culture supernatants, and tissue homogenates, as well as serum, urine, and saliva, and have applicability for basic research through to clinical testing. ELISAs are classified into four main groups (direct, indirect, sandwich, and competitive) depending on how they are configured. In a direct ELISA, the antigen (along with any other proteins in the sample) is bound to the microplate wells and detected with a labeled primary antibody. An indirect ELISA is similar, but includes an extra protocol step since a labeled secondary antibody is used for detection. Sandwich ELISAs typically use matched pairs of antibodies for analyte capture and detection and are the most popular ELISA format due to their superior specificity. Direct, indirect, and sandwich ELISA can all be converted into a competitive ELISA, which is distinct from the other ELISA types in that it measures signal interference rather than signal accumulation. Specifically, a competitive ELISA is based on competition between a target analyte and a reference molecule for binding to a relevant partner, which could be either a protein antigen or an antibody depending on how the assay is configured. A common way of setting up a competitive ELISA involves coating the microplate wells with a known amount of a reference antigen before washing and blocking. The sample is then added to the wells along with a labeled detection antibody, which may either bind to the immobilized reference antigen or to the antigen that is free in solution. In this scenario, the more antigen there is in the sample, the less antibody is captured on the microplate wells and the lower the assay signal. Because competitive ELISAs are generally more challenging to develop and optimize than the other ELISA formats, they tend to be reserved for situations where only one antibody is available for the target, or for detecting small antigens that cannot be bound by two different antibodies simultaneously.