Enzyme linked immunosorbent assay (ELISA) is one of the most widely used immunoassay techniques due to its sensitivity, flexibility, and ease of operation. For accurate results, it is important to select an ELISA that aligns with your experimental needs and to configure it using high-quality reagents. Here, we detail the various types of ELISAs, highlighting their respective advantages and disadvantages, along with factors to consider when choosing an ELISA technique.
ELISA is a microplate-based technique for detecting an analyte in solution. It begins with coating the microplate wells with either an antigen or antibody, followed by blocking to prevent non-specific binding events that could cause unwanted background signal. Next, the sample is introduced to the microplate and incubated to allow for target capture. After washing to remove any unbound material from the wells, a detection reagent is added and the resultant signal is measured with an appropriate microplate reader. The ELISA readout is typically colorimetric, based on the enzymatic conversion of a substrate into a soluble colored product, but it may also be luminescent or fluorescent. ELISA has utility for basic research, as well as for a broad range of diagnostic and clinical applications.
There are four main ELISA formats, as shown in Figure 1. In a direct ELISA, the antigen is bound to the microplate wells and detected with an enzyme-conjugated primary antibody. An indirect ELISA is similar but uses an unlabeled primary antibody and an enzyme-conjugated secondary antibody for detection.
In a sandwich ELISA, the microplate wells are coated with an antigen-specific antibody, which captures the target analyte from solution. Another antigen-specific antibody is then added; this may either be directly labeled or detected with an enzyme-conjugated secondary antibody. Critically, each of the antigen-specific antibodies must recognize a different epitope to avoid competition for target binding. The high specificity of sandwich ELISA makes it the most widely used ELISA format.
All three of these formats can be adapted to form a competitive ELISA, during which the sample analyte (either an antigen or an antibody) competes with a reference for a known amount of its labeled binding partner.
Figure 1. The four main ELISA formats.
When deciding which ELISA format to use, it is important to consider the pros and cons of the available options. These are summarized in Table 1, which also suggests potential applications for each approach.
Table 1. Key features of the main ELISA formats.
Besides the ELISA format, other factors to consider for assay selection include whether a qualitative or quantitative measurement is required and whether to develop an ELISA in-house or use an off-the-shelf kit.
If there is a need for quantitative data, the ELISA should include a standard curve (a serial dilution of the purified analyte) on every plate, along with relevant positive and negative controls. The standard should come from a trusted source and be supplied with detailed batch records.
When developing a sandwich ELISA, researchers can save significant time on assay optimization by purchasing matched antibody pairs. Alternatively, using off-the-shelf kits allows for testing samples straight away for even faster time to results.
Antibodies are pivotal in determining ELISA specificity and sensitivity. Sino Biological offers over 7,000 validated ELISA antibodies and more than 400 matched antibody pairs, as well as an extensive selection of ready-to-use ELISA kits. Discover more by visiting sinobiological.com/elisa-antibody