Antibodies for ELISA

Enzyme-linked immunosorbent assay (ELISA) is a microplate-based technique for detecting an analyte of interest within a heterogeneous solution. It is compatible with a broad range of sample types - including cell lysates, culture supernatants, and tissue homogenates, as well as serum, urine, and cerebrospinal fluid - and represents one of the most sensitive and easily automatable immunoassays available. Unlike many other immunoassay techniques, ELISA can yield quantitative data provided a relevant standard curve is included on each plate. ELISA is divided into four main groups based on how it is configured. In a direct ELISA, an antigen (along with any other proteins in the sample) is bound to the surface of the microplate wells and detected with a labeled primary antibody. This type of ELISA benefits from a short workflow but has only limited sensitivity due to using just a single antibody. Indirect ELISA is similar to direct ELISA but incorporates a labeled secondary antibody for detection. It has greater sensitivity, but risks unwanted background signal if the secondary antibody cross-reacts with any antigen present. During a sandwich ELISA, an analyte-specific antibody is bound to the microplate wells and used for target capture. Another analyte-specific antibody (that recognizes a different epitope) is then added. This antibody may be directly labeled, or may instead be detected with a labeled secondary antibody - if this has been cross-adsorbed to prevent it from binding to the capture antibody. Sandwich ELISA is the most popular ELISA format due to its high specificity and sensitivity. Direct, indirect, and sandwich ELISA can all be converted to a competitive ELISA, during which the target analyte competes with a reference for attachment to a known amount of its labeled binding partner. Competitive ELISA is generally reserved for detecting small antigens that cannot be bound by two different antibodies, or for situations where only one antibody is available for the target, since it is more challenging to develop. When selecting antibodies for ELISA, researchers must make several key decisions. As well as whether to use labeled primary antibodies or secondary antibodies for detection, these include which type of readout to choose. Colorimetric detection uses antibodies labeled with enzymes such as horseradish peroxidase (HRP) or alkaline phosphatase (AP) to convert an appropriate substrate into a soluble colored product that can be measured with a standard spectrophotometer. Chemiluminescent detection, which most often relies on HRP-labeled antibodies, offers greater sensitivity but requires specialized reagents and access to a suitable reader. Another important consideration is whether to choose monoclonal or polyclonal antibodies. Because monoclonal antibodies recognize just a single epitope, they may be better able to identify small differences in analyte concentration. Polyclonal antibodies, which bind multiple epitopes, are often used as the capture antibodies in a sandwich ELISA to pull down as much of the target as possible, before a monoclonal antibody is added for specific detection. Whichever antibody reagents are used, it is essential that they are validated for the ELISA application and, in the case of sandwich ELISA, proven to function effectively as a matched pair. Rather than developing an ELISA in-house, researchers may instead decide to purchase a ready-to-use ELISA kit. This can both save time and provide confidence in the accuracy of results since off-the-shelf products will have undergone extensive testing before reaching the market.