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Antibodies for ELISA

Enzyme-linked immunosorbent assay (ELISA) is a microplate-based technique for detecting an analyte of interest (typically a protein) in solution. Briefly, it involves capturing the analyte on the surface of the microplate wells and detecting it with an enzyme-labeled antibody. However, depending on the assay configuration, ELISA can be used for simply confirming the presence of the analyte in a sample or can allow for determining its concentration.

Advantages of ELISA include its high sensitivity, ease of use, and compatibility with automation. In addition, ELISA allows for testing a broad range of sample types, including cell lysates, culture supernatants, and tissue extracts, as well as bodily fluids such as serum, urine, and saliva. While it is common for ELISA to be run in 96-well plates, the method is easily adapted to a 384-well format to save on limited sample material and increase throughput.

When developing an ELISA, it is important to establish which assay configuration best meets your needs, as well as decide whether to use a colorimetric or chemiluminescent readout. Another key consideration is the types of antibodies to use, which includes choosing between monoclonal and polyclonal antibodies and deciding whether to perform direct detection (with enzyme-labeled primary antibodies) or indirect detection (with enzyme-labeled secondary antibodies).

Whichever experimental approach is selected, high quality reagents are essential to generate accurate, reproducible results. Our ELISA-validated products include over 400 primary antibodies, from hosts such as mouse, rat, goat, and hamster, and more than 800 secondary antibodies, many of which have been cross-adsorbed against purified immunoglobulins and serum proteins from multiple species to minimize potential cross-reactivity.

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Frequently Asked Questions

ELISA is grouped into four main categories, based on assay configuration:

  • Direct ELISA

During a direct ELISA, the analyte (along with other sample components) is bound to the microplate wells, before being detected with an enzyme-labeled primary antibody. While direct ELISA benefits from a relatively short protocol, it has limited sensitivity and may exhibit high background due to non-specific antigen immobilization. Direct ELISA is commonly used for assessing the immune response to an antigen, such as during allergy testing.

  • Indirect ELISA

Indirect ELISA is similar to direct ELISA, but combines an unlabeled primary antibody with an enzyme-labeled secondary antibody for detection. It has higher sensitivity than direct ELISA and removes the need to source labeled primary antibodies, but it involves more protocol steps and has an associated risk of cross-reactivity between the secondary antibody and the target analyte. Indirect ELISA is well suited for measuring the antibody concentration in a sample.

  • Sandwich ELISA

In a sandwich ELISA, the microplate wells are coated with an analyte-specific antibody to enable target capture from solution. Another analyte-specific antibody is then added, which may be either enzyme-labeled or detected with a labeled secondary antibody – provided the secondary antibody has been cross-adsorbed to prevent non-specific binding. When using two monoclonal antibodies for sandwich ELISA, it is essential for each to recognize a different epitope to avoid competition for target binding.

With polyclonals, the same antibody can be used for both capture and detection, if required. If a monoclonal and a polyclonal will be combined in the same assay, it is recommended that the monoclonal be used for analyte capture. This is because monoclonals recognize just a single epitope, which the polyclonal antibody might also bind, thus resulting in low or no signal. A main advantage of sandwich ELISA is its high specificity, which makes it the most widely used ELISA format.

  • Competitive ELISA

Direct, indirect, and sandwich ELISA can all be converted to a competitive ELISA, during which the analyte of interest (either a protein or an antibody) competes with a reference for a known amount of its labeled binding partner. While competitive ELISA is more challenging to develop and optimize than the other ELISA formats, it is useful when only one analyte-specific antibody is available or when there is a need to detect small analytes, such as a banned drug substance in blood or an environmental contaminant in river water.

Most ELISA tests use immunoglobulin G (IgG) antibodies, since they are the most widely available. However, these could be either monoclonal or polyclonal, and be either enzyme-labeled or require a secondary antibody for their detection. If a monoclonal antibody will be paired with a polyclonal antibody for sandwich ELISA, it is recommended that the monoclonal be used for analyte capture. This is because monoclonals have an inherent monospecificity toward a single epitope, which the polyclonal antibody might also recognize, thus resulting in low or no signal. Polyclonals can be especially useful for detecting a newly-discovered target, as their shorter production times compared to monoclonals mean they are developed sooner.

A major advantage of secondary antibodies is their capacity to provide signal amplification, which they achieve by binding to multiple epitopes on the primary antibody. This is particularly important in ELISA assays for detecting low-abundance analytes, such as certain cytokines or growth factors. When using secondary antibodies for ELISA, it is essential to select products that have been cross-adsorbed to minimize the risk of off-target binding. For example, when performing a sandwich ELISA with indirect detection, the secondary antibody should be cross-adsorbed against the sample species and the host species of the capture antibody.

A direct ELISA involves just one antibody – an enzyme-labeled primary antibody with specificity for the target of interest. Following sample addition to the microplate, and incubation to allow for protein binding, any unbound material is washed away, then the labeled primary antibody is used for analyte detection.

Both direct and indirect ELISA are used for detecting antibodies in a sample. The main difference between the two methods is that a direct ELISA uses an enzyme-labeled primary antibody for analyte detection, while an indirect ELISA uses an unlabeled primary antibody and an enzyme-labeled secondary antibody. Indirect ELISA is more sensitive than direct ELISA, due to signal amplification from the secondary antibody, and can also be a more flexible option as it eliminates the need to source conjugated primary antibodies.

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