Enzyme-Linked Immunosorbent Assay (ELISA)

Direct ELISA

An antigen coated to a multiwell plate is detected by an antibody that has been directly conjugated to an enzyme. This can also be reversed, with an antibody coated to the plate and a labeled antigen used for detection, but the second option is less common. This type of ELISA has two main advantages: It is faster, since fewer steps are required It is less prone to error, since there are fewer steps and reagents

Indirect ELISA

Antigen coated to a polystyrene multiwell plate is detected in two stages or layers. First an unlabeled primary antibody, which is specific for the antigen, is applied. Next, an enzyme-labeled secondary antibody is bound to the first antibody. The secondary antibody is usually an anti-species antibody and is often polyclonal. This method has several advantages: Increased sensitivity, since more than one labeled antibody is bound per primary antibody Flexibility, since different primary detection antibodies can be used with a single labeled secondary antibody Cost savings, since fewer labeled antibodies are required

Competition or Inhibition ELISA

This is the most complex ELISA, and is used to measure the concentration of an antigen (or antibody) in a sample by observing interference in an expected signal output. Hence, it is also referred to as an inhibition ELISA. It can be based upon any of the above ELISA formats, direct, indirect, or sandwich, and as a result it offers maximum flexibility in set up. It is most often used when only one antibody is available to the antigen of interest or when the analyte is small, i.e. a hapten, and cannot be bound by two different antibodies. A simple example of a competitive ELISA is shown in figure 7. In this case samples are added to an ELISA plate containing a known bound antigen. After coating, blocking, and washing steps, unknown samples are added the plate. Detection then follows pretty much as with other ELISA formats. If the antigen in the sample is identical to the plate-adsorbed antigen, then there will be competition for the detection antibody between the bound and free antigen. If there is a high concentration of antigen in the sample, then there will be a significant reduction in signal output of the assay. Conversely, if there is little antigen in the sample, there will be minimal reduction in signal. Therefore, with a competition ELISA, one is actually measuring antigen concentration by noting the extent of the signal reduction. If the detection antibody is labeled, then this would be a direct competition ELISA and if unlabeled, then this would be an indirect competition ELISA. For further examples of competition ELISAs, and a thorough explanation of this technique, please refer to The ELISA Guidebook (Crowther 2001).

ELISA

enzyme-linked immunosorbent assay

Sandwich ELISA Sandwich ELISAs

typically require the use of matched antibody pairs, where each antibody is specific for a different, non-overlapping part (epitope) of the antigen molecule. The first antibody, termed the capture antibody, is coated to the polystyrene plate. Next, the analyte or sample solution is added to the well. A second antibody layer, the detection antibody, follows this step in order to measure the concentration of the analyte. Polyclonals can also be used for capture and/or detection in a sandwich ELISA provided that variability is present in the polyclonal to alow for both capture and detection of the analyte through different epitopes. If the detection antibody is conjugated to an enzyme, then the assay is called a direct sandwich ELISA. If the detection antibody is unlabeled, then a second detection antibody will be needed resulting in an indirect sandwich ELISA. This type of assay has several advantages: High specificity, since two antibodies are used the antigen/analyte is specifically captured and detected Suitable for complex samples, since the antigen does not require purification prior to measurement Flexibility and sensitivity, since both direct and indirect detection methods can be used

ELISA

used to measure the concentration of an analyte (usually antibodies or antigens) in solution. The basic ELISA, or enzyme immunoassay (EIA), is distinguished from other antibody-based assays because separation of specific and non-specific interactions occurs via serial binding to a solid surface, usually a polystyrene multiwell plate, and because quantitative results can be achieved. The steps of the ELISA result in a colored end product which correlates to the amount of analyte present in the original sample. ELISAs are quick and simple to carry out, and since they are designed to rapidly handle a large numbers of samples in parallel, they are a very popular choice for the evaluation of various research and diagnostic targets.