ELISA AND ELISPOT PRODUCTS (Handbook and Technical Guid)
Enzyme-linked immunosorbent assays (ELISAs) are designed for detecting and quantitating substances such as peptides, proteins, antibodies and hormones. Other names, such as enzyme immunoassay (EIA), are also used to describe the same process. In an ELISA, an antigen must be immobilized to a solid surface. The antigen is then complexed with an antibody that is linked to an enzyme. Detection is accomplished by incubating this enzyme-complex with a substrate that produces a detectable product. The most crucial element of the detection strategy is a highly specific antibody-antigen interaction.
Most commonly, ELISAs are performed in 96-well (or 384-well) polystyrene plates, which will passively bind antibodies and proteins. It is this binding and immobilization of reagents that makes ELISAs so easy to design and perform, as first described by Eva Engvall, et al.1. Having the reactants of the ELISA immobilized to the microplate surface makes it easy to separate bound from unbound material during the assay. This ability to wash away nonspecifically bound materials makes the ELISA a powerful tool for measuring specific analytes within a crude preparation.
A detection enzyme may be linked directly to the primary antibody or introduced through a secondary antibody that recognizes the primary antibody. It may also be linked to a protein such as streptavidin if the primary antibody is biotin-labeled. However the enzyme is incorporated, the most commonly used enzymes are horseradish peroxidase (HRP) and alkaline phosphatase (AP). Other enzymes have been used as well, but they have not gained widespread acceptance because of limited substrate options. These include β-galactosidase, acetylcholinesterase and catalase. A large selection of substrates is available for performing the ELISA with an HRP or AP conjugate. The choice of substrate depends upon the necessary sensitivity level of the detection and the instrumentation available for detection (e.g., spectrophotometer, fluorometer or luminometer).
Enzyme-linked immunospot assays (ELISPOTs) are a subclass of ELISAs that are used to count the number of cytokine-producing cells within a cell suspension. The difference between the two is that in ELISA, the substance containing the “unknown” is stuck at the bottom of the well, whereas in ELISPOT the substance within the “unknown” is placed in the well after the bottom of the well has been coated with cytokine-specific antibody. In both cases, the wells are typically contained within a generic microplate.
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