Overview of ELISA
ELISAs are typically 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. 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 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 required assay sensitivity and the instrumentation available for signal-detection (spectrophotometer, fluorometer or luminometer).
The revised Assay Development Technical Handbook is an essential resource for any laboratory using enzyme-linked immunosorbent assay (ELISA) and related plate-based assay methods. The handbook describes the essential techniques and tools for designing and optimizing ELISA Assays. Featured products include coated microplates, standards, blockers, buffers, probe-labeling 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.
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.
An ELISA can also be performed as a competitive assay. This is common 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 the competitive ELISA
Fluorescent tags and other alternatives to enzyme-based detection can be used for plate-based assays. Despite not involving reporter-enzymes, 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 plate-based method, these assays are often called ELISAs despite not involving antibodies.
ELISA is nearly always performed using 96-well or 384-well 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.
Variants of the standard ELISA also exist:
- ELISPOT (enzyme-linked immunospot assay) refers to ELISA-like capture and measurement of proteins secreted by cells that are plated in PVDF-membrane-backed 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.
- In-cell 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).
Direct vs. Indirect Detection ELISA Formats
Watch this video about ELISA methods
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 cross-adsorbed to remove any antibodies that have affinity for the capture antibody.
|Direct ELISA Detection|
|Indirect ELISA Detection|
Selecting and Coating ELISA Plates
When developing a new ELISA for a specific antigen, the first step is to optimize the plate-coating conditions for the antigen or capture antibody. Begin by choosing an assay microplate (not tissue culture treated plates) with a minimum protein-binding capacity of 400 ng/cm². It is also important that the CV value (coefficient of variation) of the protein binding be low (<5% is preferred) so that there is limited deviation in values that should be identical in the assay results between wells and plates. The choice of plate color depends upon the signal being detected. Clear polystyrene flat bottom plates are used for colorimetric signals while black or white opaque plates are used for fluorescent and chemiluminescent signals. Visually inspect plates before use as imperfections or scratches in the plastic will cause aberrations when acquiring data from the developed assay.
Plate coating is achieved through passive adsorption of the protein to the plastic of the assay microplate. This process occurs though hydrophobic interactions between the plastic and non-polar protein residues. Although individual proteins may require specific conditions or pretreatment for optimal binding, the most common method for coating plates involves adding a 2-10 μg/ml solution of protein dissolved in an alkaline buffer such as phosphate-buffered saline (pH 7.4) or carbonate-bicarbonate buffer (pH 9.4). The plate is left to incubate for several hours to overnight at 4-37°C. Typically, after removing the coating solution, blocking buffer is added to ensure that all remaining available binding surfaces of the plastic well are covered (see subsequent discussion). Coated plates can be used immediately or dried and stored at 4°C for later use, depending on the stability of the coated protein.
It is important to note that optimal coating conditions can vary with each protein. With the exception of competition ELISAs, the plates are coated with more capture protein than can actually be bound during the assay in order to facilitate the largest working range of detection possible. Some proteins, especially antibodies, are best coated on plates at a concentration lower than the maximum binding capacity in order to prevent nonspecific binding in later steps by a phenomenon called "hooking". Hooking results from proteins getting trapped between the coating proteins which prevents effective washing and removal of non bound proteins. When hooking nonspecifically traps detection primary and secondary antibodies, high background signal results lowering the signal to noise ratio and thus sensitivity of an assay.
Pre-coated ELISA Plates
For antibodies and proteins, coating plates by passive adsorption usually works well. However, problems can arise from passive adsorption, including improper orientation, denaturation, poor immobilization efficiency and binding of contaminants along with the target molecule. Antibodies can be attached to a microplate through the Fc region using Protein A, G, or A/G coated plates, which orients them properly and preserves their antigen binding capability. Fusion proteins can be attached to a microplate in the proper orientation using glutathione, metal-chelate, or capture-antibody coated plates. Peptides and other small molecules, which typically do not bind effectively by passive adsorption, can be biotinylated and attached with high efficiency to a streptavidin or NeutrAvidin Protein coated plate. Biotinylated antibodies also can be immobilized on plates precoated with biotin-binding proteins. Using pre-coated plates in this manner physically separates the antigen or capture antibody from the surface of the plate as protection from its denaturing effects.
Antibodies and Probes for ELISA
Either monoclonal or polyclonal antibodies can be used as the capture and detection antibodies in sandwich ELISA systems. Monoclonals have an inherent monospecificity toward a single epitope that allows fine detection and quantitation of small differences in antigen. A polyclonal is often used as the capture antibody to pull down as much of the antigen as possible. Then a monoclonal is used as the detecting antibody in the sandwich assay to provide improved specificity.
An important consideration in designing a sandwich ELISA is that the capture and detection antibodies must recognize two different non-overlapping epitopes. When the antigen binds to the capture antibody, the epitope recognized by the detection antibody must not be obscured or altered. Capture and detection antibodies that do not interfere with one another and can bind simultaneously are called "matched pairs" and are suitable for developing a sandwich ELISA. Many primary antibody suppliers provide information about epitopes and indicate pairs of antibodies that have been validated in ELISA as matched pairs.
Another design consideration in choosing antibodies is cost. A polyclonal antibody is generally less expensive (~5 fold) to produce than a monoclonal. The specificity gained by using monoclonals for both the capture and detecting antibody must be weighed against the cost and time required for producing two monoclonal antibodies. Preparing a “self-sandwich” ELISA assay, where the same antibody is used for the capture and detection, can limit the dynamic range and sensitivity of the final ELISA.
Blocking Buffers and Wash Buffers
The binding capacity of microplate wells is typically higher than the amount of protein coated in each well. The remaining surface area must be blocked to prevent antibodies or other proteins from adsorbing to the plate during subsequent steps. A blocking buffer is a solution of irrelevant protein, mixture of proteins, or other compound that passively adsorbs to all remaining binding surfaces of the plate. The blocking buffer is effective if it improves the sensitivity of an assay by reducing background signal and improving the signal-to-noise ratio. The ideal blocking buffer will bind to all potential sites of nonspecific interaction, eliminating background altogether, without altering or obscuring the epitope for antibody binding.
When developing any new ELISA, it is important to test several different blockers for the highest signal:noise ratio in the assay. Many factors can influence nonspecific binding, including various protein:protein interactions unique to the samples and antibodies involved. The most important parameter when selecting a blocker is the signal:noise ratio, which is measured as the signal obtained with a sample containing the target analyte as compared to that obtained with a sample without the target analyte. Using inadequate amounts of blocker will result in excessive background and a reduced signal:noise ratio. Using excessive concentrations of blocker may mask antibody-antigen interactions or inhibit the enzyme, again causing a reduction of the signal:noise ratio. No single blocking agent is ideal for every occasion and empirical testing is essential for true optimization of the blocking step.
In addition to blocking, it is essential to perform thorough washes between each step of the ELISA. Washing steps are necessary to remove nonbound reagents and decrease background, thereby increasing the signal:noise ratio. Insufficient washing will allow high background, while excessive washing might result in decreased sensitivity caused by elution of the antibody and/or antigen from the well. Washing is performed in a physiologic buffer such as Tris-buffered saline (TBS) or phosphate-buffered saline (PBS) without any additives. Usually, a detergent such as 0.05% Tween-20 is added to the buffer to help remove nonspecifically bound material. Another common technique is to use a dilute solution of the blocking buffer along with some added detergent. Including the blocking agent and adding a detergent in wash buffers helps to minimize background in the assay. For best results, use high-purity detergents to prevent introduction of impurities that will interfere with the assay such enzyme inhibitors or peroxides.
Detection Strategies for ELISA
The final stage in all ELISA systems is a detection step. Unless a radioactive or fluorescent tag was used, this involves the introduction of an enzyme substrate.The enzyme converts the substrate to a detectable product. If an ELISA has been constructed and developed properly, then the intensity of signal produced when the substrate is added will by directly proportional to the amount of antigen captured in the plate and bound by the detection reagents. Enzyme-conjugated antibodies (especially those involving horseradish peroxidase, HRP) offer the most flexibility in detection and documentation methods for ELISA because of the variety of substrates available for chromogenic, chemifluorescent and chemiluminescent imaging.
Though not as sensitive as fluorescent or chemiluminescent substrates, chromogenic ELISA substrates allow direct visualization and enable kinetic studies to be performed. Furthermore, chromogenic ELISA substrates are detected with standard absorbance plate readers common to many laboratories. Fluorescent ELISA substrates are not as common and require a fluorometer that produces the correct excitation beam to cause signal emission to be generated from the fluorescent tag. Though best used with a luminometer plate reader, chemiluminescent substrates can be detected by various means including digital camera systems. Once draw back of using chemiluminescent substrates for ELISA is the signal intensity can vary more with than other substrates. For assays requiring many plates to be read, this can present a problem if the signal begins to decay before plates are read. For this reason, it is important to make sure the assay has been optimized with the substrate in order to ovoid misinterpreting signal-fade in a sample as low antigen abundance.
Specific ELISA Kits and In-Cell ELISA Kits
In addition to the individual components and general principles of ELISA discussed in this article, sandwich ELISA kits are available for detection of specific cytokines and other targets, such as interferon gamma (IFN gamma) and interleukin 6 (IL-6).
For many targets, three kit sizes (package types) are available:
- Complete ELISA Kits contain pre-coated antibody-plates, detection antibodies, buffers, diluents, standards, and substrates
- ELISA Reagent Kits (formerly called "Screening Sets") contain matched antibodies and all other reagents but no coated plates.
- ELISA Antibody Pair Kits (formerly "MiniKits") contain only matched antibodies and standard (no plates or detection reagents).
Specific In-Cell ELISA Kits are available for detection by colorimetric or fluorescent (Near-IR) instrumentation and include all reagents necessary except the primary antibody. The colorimetric version of the In-Cell ELISA is developed using a TMB substrate for HRP, while the Near-IR detection kit uses secondary detection reagents conjugated to DyLight Fluors. Both types of In-Cell ELISA Kits allow multiple target proteins to be compared to each other in the same assay either through normalization or multiplex analysis.
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