Overview of ELISA ELISA (enzymelinked immunosorbent assay) is a platebased assay technique designed for detecting and quantifying substances such as peptides, proteins, antibodies, and hormones. Other names, such as enzyme immunoassay (EIA), are also used to describe the same technology. In an ELISA, an antigen must be immobilized to a solid surface and then complexed with an antibody that is linked to an enzyme. Detection is accomplished by assessing the conjugated enzyme activity via incubation with a substrate to produce a product that can be measured. The most crucial element of the detection strategy is a highly specific antibodyantigen interaction Page contents Introduction ELISA formats (direct, sandwich, etc.) Direct vs. indirect detection ELISA strategies Other ELISA formats (competitive, ELISPOT, etc.) Complete, readytouse ELISA kits Selecting and coating ELISA plates Precoated ELISA plates Antibodies and probes for ELISA Blocking buffers and wash buffers Detection strategies for ELISA View products Browse ELISA Products Find ELISA Kits by Target Introduction ELISAs are typically performed in 96well (or 384well) 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. Having the reactants of the ELISA immobilized to the microplate surface makes it easy to separate bound from nonbound 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 or other tag can be linked directly to the primary antibody or introduced through a secondary antibody that recognizes the primary antibody. It also can be linked to a protein such as streptavidin if the primary antibody is biotin labeled. The most commonly used enzyme labels 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 βgalactosidase, 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 required assay sensitivity and the instrumentation available for signal detection (spectrophotometer, fluorometer, or luminometer) ELISA formats ELISAs can be performed with a number of modifications to the basic procedure. The key step, immobilization of the antigen of interest, can be accomplished by direct adsorption to the assay plate or indirectly via a capture antibody that has been attached to the plate. The antigen is then detected either directly (labeled primary antibody) or indirectly (labeled secondary antibody). The most powerful ELISA assay format is the sandwich assay. This type of capture assay is called a “sandwich” assay because the analyte to be measured is bound between two primary antibodies—the capture antibody and the detection antibody. The sandwich format is used because it is sensitive and robust Diagram of common ELISA formats (direct vs. sandwich assays) Common ELISA formats. In the assay, the antigen of interest is immobilized by direct adsorption to the assay plate or by first attaching a capture antibody to the plate surface. Detection of the antigen can then be performed using an enzymeconjugated primary antibody (direct detection) or a matched set of unlabeled primary and conjugated secondary antibodies (indirect detection) Direct vs. indirect detection ELISA strategies Among the standard assay formats discussed and illustrated above, where differences in both capture and detection were the concern, it is important to differentiate between the particular strategies that exist specifically for the detection step. However an antigen is captured to the plate (by direct adsorption to the surface or through a precoated "capture" antibody, as in a sandwich ELISA), it is the detection step (as either direct or indirect detection) that largely determines the sensitivity of an ELISA Watch this video about ELISA detection and signal amplification strategies 0:00 / 2:31 The direct detection method uses a labeled primary antibody that reacts directly with the antigen. Direct detection can be performed with antigen that is directly immobilized on the assay plate or with the capture assay format. Direct detection is not widely used in ELISA but is quite common for immunohistochemical staining of tissues and cells The indirect detection method uses a labeled secondary antibody for detection and is the most popular format for ELISA. The secondary antibody has specificity for the primary antibody. In a sandwich ELISA, it is critical that the secondary antibody be specific for the detection primary antibody only (and not the capture antibody) or the assay will not be specific for the antigen. Generally, this is achieved by using capture and primary antibodies from different host species (e.g., mouse IgG and rabbit IgG, respectively). For sandwich assays, it is beneficial to use secondary antibodies that have been crossadsorbed to remove any antibodies that have affinity for the capture antibody Comparison of direct and indirect ELISA detection methods Direct ELISA detection Advantages Quick because only one antibody and fewer steps are used Crossreactivity of secondary antibody is eliminated Disadvantages Immunoreactivity of the primary antibody might be adversely affected by labeling with enzymes or tags Labeling primary antibodies for each specific ELISA system is time consuming and expensive No flexibility in choice of primary antibody label from one experiment to another Minimal signal amplification Indirect ELISA detection Advantages A wide variety of labeled secondary antibodies are available commercially Versatile because many primary antibodies can be made in one species and the same labeled secondary antibody can be used for detection Maximum immunoreactivity of the primary antibody is retained because it is not labeled Sensitivity is increased because each primary antibody contains several epitopes that can be bound by the labeled secondary antibody, allowing for signal amplification Different visualization markers can be used with the same primary antibody Disadvantages Crossreactivity might occur with the secondary antibody, resulting in nonspecific signal An extra incubation step is required in the procedure Fluorescent tags and other alternatives to enzymebased detection can be used for platebased assays. Despite not involving reporterenzymes, these methods are also generally referred to as a type of ELISA. Likewise, wherever detectable probes and specific protein binding interactions can be used in a platebased method, these assays are often called ELISAs despite not involving antibodies Other ELISA formats Besides the standard direct and sandwich formats described above, several other styles of ELISA exist: Competitive ELISA is a strategy that is commonly used when the antigen is small and has only one epitope, or antibody binding site. One variation of this method consists of labeling purified antigen instead of the antibody. Unlabeled antigen from samples and the labeled antigen compete for binding to the capture antibody. A decrease in signal from purified antigen indicates the presence of the antigen in samples when compared to assay wells with labeled antigen alone Watch this video about competitive ELISA methods 0:00 / 1:13 ELISPOT (enzymelinked immunospot assay) refers to ELISAlike capture and measurement of proteins secreted by cells that are plated in PVDFmembranebacked microplate wells. It is a "sandwich" assay in which the proteins are captured locally as they are secreted by the plated cells, and detection is with a precipitating substrate. ELISPOT is like a western blot in that the result is spots on a membrane surface Incell ELISA is performed with cells that are plated and cultured overnight in standard microplates. After the cultured cells are fixed, permeabilized, and blocked, target proteins are detected with antibodies. This is an indirect assay, not a sandwich assay. The secondary antibodies are either fluorescent (for direct measurement by a fluorescent plate reader or microscope) or enzyme conjugated (for detection with a soluble substrate using a plate reader) ELISA is nearly always performed using 96well or 384well polystyrene plates and samples in solution (i.e., biological fluids, culture media, or cell lysates). This is the platform discussed in the remainder of this article Complete, readytouse ELISA kits In addition to the individual components and general principles of ELISA discussed in the remainder of this article, readytouse sandwich ELISA kits are commercially available for detection of hundreds of specific cytokines, neurobiology analytes, and phosphorylated proteins that are common targets of research interest For many targets, two kit types are available: ELISA Kits contain precoated antibodyplates, detection antibodies, buffers, diluents, standards, and substrates Antibody Pair Kits contain only matched antibodies and standard (no plates or detection reagents) Learn more View products ELISA Development and Optimization Search All ELISA Kits by Target Performing and Evaluating Spike and Recovery and Linearity of Dilution for ELISA ReadytoUse ELISA Kits Factors Affecting Signal Generation in ELISA Antibody Pair Kits ELISA Protocols Guide to ELISA Kit Package Formats ELISA Reagents and Accessories Assay Development Technical Handbook The revised Assay Development Technical Handbook is an essential resource for any laboratory using enzymelinked immunosorbent assay (ELISA) and related platebased assay methods. The handbook describes the essential techniques and tools for designing and optimizing ELISA Assays. Featured products include coated microplates, standards, blockers, buffers, probelabeling reagents, secondary antibodies and detection substrates Contents include: Introduction to ELISA, Selecting an ELISA Plate, Thermo Scientific Pierce Microplates, Thermo Scientific Pierce Coated Microplates, Blocking and Washing, Blocking and Washing Reagents, Detection Probes, Antibody Labeling, Choosing a Substrate, Bulk and Custom Offerings, and Recommended Reading Download the Assay Development Technical Handbook Selecting and coating ELISA plates When developing a new ELISA for a specific antigen, the first step is to optimize the platecoating conditions for the antigen or capture antibody. Begin by choosing an assay microplate (not tissue culture treated plates) with a minimum proteinbinding capacity of 400 ng/cm². It is also important that the CV value (coefficient of variation) of the protein binding be low (