Caitlin Smith has a B.A. in biology from Reed College, a Ph.D. in neuroscience from Yale University, and completed postdoctoral work at the Vollum Institute.
Enzyme-linked Immunosorbent Assay (ELISA) kits make it easier to use ELISAs for detecting molecules or performing ligand-binding assays successfully. Most ELISA kits are designed to determine the concentration of an antigen in an unknown sample, and they are faster and more convenient (possibly also more accurate) than developing your own ELISAs from scratch. Amidst the broad swath of available ELISA kits, what’s most important to look for? Obviously, a first criterion must be that the ELISA detects what you want it to detect. Beyond this, here are four important features to look for when shopping for an ELISA kit.
1. Assay format
ELISA kits come in many forms. A common characteristic, however, is capturing your antigen, either by adsorbing it to the assay plate or by capturing it with an antibody that is attached to the assay plate. You'll need to ensure that you get the right format for your experiment, as well as the right container system. Usually ELISAs are done in 96- or 384-well plates, but other variants include in-cell and enzyme-linked immunospot (ELISPOT) formats. In-cell ELISAs detect the antigen of interest using an antibody in fixed and permeabilized cells cultured in microplate wells. ELISPOT assays are reminiscent of Western blots, because spots of secreted antigens are detected on a membrane contained within the microplate wells in which the cells are cultured.
A commonly used ELISA format is the “sandwich” assay, so named because the antigen is bound by (or sandwiched between) two different antibodies—one for capturing and one for detecting. This format is favored by many because it tends to be both sensitive and robust. However, there are times when a sandwich assay is not the best choice, for example if your antigen is especially small (which makes it difficult to attach two bulky antibodies to it) and/or it has only one antibody binding site. In such cases, a better choice may be a competitive ELISA in which purified, labeled antigen competes with unlabeled antigen from samples, for binding to the capture antibodies.
2. Types of antibodies
You can use monoclonal and polyclonal antibodies in ELISAs. In fact, sometimes a combination is useful. But there are a few points to watch for when choosing them. For a sandwich assay, it is sometimes helpful to use a polyclonal antibody for capture and a monoclonal antibody for detection. This enables you to capture all the antigen with the polyclonal antibody and then use the monoclonal antibody to detect only a specific subset of antigens that have a particular epitope, for example.
Another important point to look for in a sandwich ELISA is whether your capture and detection antibodies (whether poly- or monoclonal) make a good team. In a sandwich assay, you don’t want your capture and detection antibodies to compete for the same binding site on the antigen. Therefore, the capture and detection antibodies must recognize different epitopes on the antigen that do not overlap. Interference between the antibodies will skew the assay results. A sure way to avoid this problem is to purchase a “matched pair” of capture/detection antibodies that have been shown to work well together (many antibody suppliers provide this information specifically for this purpose).
3. Cross-reactivity and interference
The specificity of your assay can be confounded by interference or antibody cross-reactivity in your assay. One source of cross-reactivity is compatibility of antibodies from host animals. Though it is increasingly possible to purchase antibodies produced from the animal you need, it is still a good idea to verify whether cross-reactivity might present a problem. For example, when detecting the signal in a sandwich ELISA, the secondary antibody used for detection must be specific for the primary antibody used for detection and not for the capture antibody. If the secondary detection antibody binds the capture antibody, specificity is reduced. Usually this can be ameliorated by obtaining capture and primary detection antibodies (the two slices of bread in the sandwich) from different host animals.
Likewise, it is prudent to know what is in the kit’s buffers (such as the washing and blocking buffers), in case they contain a compound that might alter the interaction between the antigen and antibody and thus skew results.
4. Detection
There are many ways to detect binding in ELISAs today, so the first thing to look for is a kit that uses the type of detection you want to use. Traditionally, ELISA readouts are a soluble product of an enzymatic reaction. The enzyme (such as the commonly used horseradish peroxidase) is attached to one of the antibodies, and the substrate (such as diaminobenzidine) is added to the reaction. Ideally, the enzymatic reaction produces a product of a characteristic color, which can be measured, for example, with a spectrophotometer. Another common enzyme is alkaline phosphatase. The enzyme can be attached to the primary antibody or to a secondary antibody that binds to the primary antibody. Alternatively, a streptavidin-tagged enzyme can bind to a biotin-labeled primary antibody. Or ELISAs may use radioactive, fluorescent, chemiluminescent or chromogenic forms of detection instead.
Each one of these four points could be expounded upon greatly, so if you have any reason to suspect a potential problem or have any questions about the ELISA kits you are perusing, do ask the company representatives, who are knowledgeable about the types of kits to use in particular situations. Or check out the technical literature on their websites. Both can give you the information you need to choose the right kit for you.
The image at the top of the page is from Life Technologies' AMPK [pT172] ELISA Kit.