Flat-bottomed, 96-well plates, made from polystyrene or polyvinyl chloride, are used in the vast majority of ELISA assays. Alternatively a strip well plate can be used. This is a frame in the size of a 96-well plate that is populated with as many 8-well or 12-well strips as the experiment requires. Further variants are 384-well and 1536-well plates; these have the same footprint as the traditional 96-well plates but obviously are able to process more samples per plate. For optimum use they require automated handling and hence are near exclusively used in high throughput screening. Some enzyme substrates, such as those that produce fluorescent or chemiluminescent signals may require opaque plates for optimal results.
It is important to use plates designed for ELISAs because they are manufactured to maintain consistency, minimizing edge effects and providing optimal optical conditions for data collection. It is a good idea to test plates from several manufacturers for batch-to-batch and plate-to-plate variability, especially if an assay is being developed for commercial, diagnostic, or quality control uses. The usual expectation is a 5% or lower variation in common controls across 2 plates. Standard polystyrene ELISA plates fall into the low to medium binding type, meaning that they will capture around 100–200 ng of IgG/cm2. Modification of the polystyrene yields binding capacities of 400–500 ng of IgG/cm2, these are commercially available as high-binding plates. Finally, antigen or antibody pre-coated plates are commercially available although most often as part of an optimized ELISA kit with all components included.
Several different buffers are used during an ELISA: one for coating, another for blocking, another for washing, and perhaps another for sample and antibody dilution. Buffers can be produced in house or sourced from a variety of commercial antibody and reagent suppliers. Basic ELISA buffer recipes can be found on our ELISA protocols page.
Coating is the first step in any ELISA and is the process where a suitably diluted antigen or antibody is incubated until adsorbed to the surface of the well. Adsorption occurs passively as the result of hydrophobic interactions between the amino acids side chains on the antibody or antigen used for coating, and the plastic surface. It is dependent upon time, temperature, and the pH of the coating buffer, as well as the concentration of the coating agent.
Typical coating conditions involve adding 50-100 μl of coating buffer, containing antigen or antibody at a concentration of 1-10 μg/ml, and incubating overnight at 4°C or for 1-3 hours at 37°C. Alternative temperatures, times, buffers, and coating agent concentrations can be used and should be tested by experimentation. During coating, it is important to maintain a moist environment in the well to minimize evaporation; plate sealers are generally used to achieve this in a repeatable and constant fashion.
It is best to test a range of concentrations of coating agent since higher concentrations of antibody/antigen may actually have a negative effect on coating, leading to oversaturation of the wells, which can inhibit antibody binding due to steric hindrance.
Conversely, when crude antigen or antibody preparations are used for coating, it is possible that the effective antigen/antibody concentration may be low and outcompeted by contaminating proteins, making the specific assay signal too low to be useful. In this case a sandwich assay is more suitable.
Coating buffers stabilize the antigen or antibody which is used to coat the ELISA multiwell plate, maximizing adsorption to the plate and optimizing interactions with the detection antibody. It is imperative that no other proteins are included in the coating buffer as these will compete with the antigen for binding to the plate.
The two most common coating buffers are bicarbonate buffer at pH 9.6 or PBS; basic buffer recipes can be found on our ELISA protocols page.
Blocking is often necessary to prevent the non-specific binding of detection antibodies to the multiwell plate surface itself. There are two main types of blocking agents, proteins and detergents. Proteins are classified as permanent blocking agents and hence added after the capture antibody has adsorbed to the well surface. Detergents only block temporarily, meaning their blocking function disappears during washing steps. As there is no ideal universal blocking buffer, blocking is a compromise between achieving the desired sensitivity and reduced background. Starting with a buffer, that contains an unrelated protein or a protein derivative that does not react with any of the antibodies being used in the detection step, is a recommended starting point for finding an effective blocking buffer.
When a plate is fully blocked, assay sensitivity will be enhanced since additional non-specific signal will be reduced. The most basic blocking buffer contains 1% BSA or milk proteins dissolved in PBS. Usually 150 μl of blocking buffer is added to the well to incubate for one hour at 37°C in order to fully block the plate.
Since the ELISA uses surface binding for separation, wash steps are repeated between each step to remove unbound materials. The wash steps are a critical part of the process and entail filling the wells entirely with buffer, usually PBS, with a small concentration of a non-ionic detergent such as Tween-20.
Washing is typically repeated 3-5 times between each step in the ELISA to thoroughly remove unbound material. Usually the wash solution is only briefly retained on the plate. Excess wash solution must be removed in the final wash step to prevent the dilution of the reagents added in the subsequent stage. This is accomplished most simply by tapping the washed plate upside down on an absorbent paper to remove excess liquid or by careful aspiration. It is crucial not to let the plate dry out.
The antibodies used in ELISA assays can be monoclonal, polyclonal, or a combination of both. Each antibody type offers distinct advantages in the development of ELISAs, so it is important to appreciate the differences between them and how these can be used to obtain an advantage during ELISA development.
The interaction between antibodies and their antigens is described in three ways: specificity, affinity, and avidity. During ELISA development these factors influence the amount of optimization of, e.g. antibody concentration and buffers, required (see ELISA Optimization Section).
Specificity is an indication of whether an antibody binds solely to a unique epitope from a single antigen in a single species, or whether it binds to similar epitopes present on several molecules from a few different species. Cross-reactivity is the opposite of specificity.
Affinity describes the strength of binding of an antibody to a single epitope. Since binding is reversible, affinity determines how much antigen is bound by an antibody, how quickly binding occurs, and for how long the binding lasts. High affinity antibodies are the best choice for all types of immunoassay because they rapidly produce the greatest number of stable immune complexes and therefore provide the most sensitive detection.
Avidity is a more complex term that accounts for the total stability of the antibody-antigen interaction. It is based upon affinity, but is also influenced by the valency of the antibody, or total number of antigen binding sites. Thus, avidity varies with isotype and whether the antibody is intact or fragmented. There is also a contribution made by the spatial arrangement of the whole complex.
Monoclonal antibodies are homogeneous by definition, with specificity for a single epitope or small region of a protein. As a result, they are less likely to interact with closely-related proteins and are not generally expected to trigger non-specific signals in a given immunoassay.
Monoclonal antibodies can be used for all antibody-containing steps in all types of ELISAs. They are commonly used in sets as matched pairs in sandwich ELISAs, but can be used for capture or detection, in conjunction with a polyclonal antibody to enhance signal or to provide a greater chance of capturing antigen from a complex solution.
Polyclonal antibodies are complex antibody pools which represent a collection of specificities to various epitopes found in a single antigen. Some epitopes predominate or there may be wide representation of the epitopes available in any given antigen. Polyclonals can vary significantly from batch-to-batch, and must be tested and validated thoroughly.
As a result of their heterogeneity and the wide representation of epitopes present, polyclonal antibodies can be powerful tools for the thorough detection of an antigen, often yielding higher signal levels. It is also rare that they will fail to bind due to a single blocked antibody binding site, antigen configuration change, or misfolding. However, polyclonals are also more likely to share one or more epitopes with closely-related proteins, resulting in higher non-specific signal. One solution to reduce this problem is to use affinity purified or cross-absorbed polyclonal antibodies.
Sometimes the detection method for an ELISA is switched from direct to indirect detection, and thus from a monoclonal to a polyclonal, in order to increase assay sensitivity due to higher levels of polyclonal antibody binding to the target antigen.
Polyclonal antibodies bring an additional aspect to ELISAs. They can be used as capture and detection antibodies. Antibodies from the same polyclonal batch can both capture the analyte and subsequently also detect it, in a biotin conjugated format.
Matched pairs are the basis of many sandwich ELISAs, either in kits or for in house assay set up. The name refers to sets of antibodies which are known to be capable of detecting different epitopes on the same protein antigen, so they can be used together for the capture and detection of a single antigen in a sandwich ELISA or related immunoassay. Matched pairs can consist of two monoclonals, two polyclonals, or a combination of both.
A wide variety of samples can be tested in an ELISA and the choice of assay conditions will depend upon the complexity of the sample and the expected amount of antigen present.
Samples are usually considered to be homogeneous or heterogeneous, depending on their complexity. This is essentially equivalent to a purified antigen vs. a crude unpurified mixture. In the simplest case, ELISA samples are diluted in PBS, wash buffer, or other specialty buffers and applied in a final volume of 100 μl. Blood presents special challenges due to the proteins present that can disrupt the assay results. Since sera can contain antibodies, there can also be unexpected cross-reactivity. Hence, special treatments and buffers are sometimes needed for the dilution of blood samples in order to obtain optimal results.
It is possible to use the samples to coat the wells themselves, as in a direct ELISA, or to capture and quantitate the antigen samples using a sandwich assay if a matched pair is available. A complex, heterogeneous protein mixture would be less suitable for coating a plate for direct ELISA detection unless the protein of interest is over-expressed and thus the majority of protein present in the sample.
It is important to test all samples in duplicate or triplicate in conjunction with a known standard to ensure the accuracy of results and for quantitation. If possible, it is better to test several dilutions of a sample to make sure the final results fall within the linear portion of the standard curve. This is because highly concentrated samples can underestimate concentration, while highly diluted samples can overestimate it. This will avoid the Hook effect, observed when very high levels of antigen are present in the sample, leading to reduced specific binding of the antigen, that is insufficient to match analyte levels and showing lower signal intensity than expected.
It is generally advantageous to standardize the detection antibodies and source them from a commercial supplier, for consistency and convenience. In certain cases, e.g. direct ELISA it may be impossible to obtain a labeled detection antibody; in these cases the chosen antibody needs to be labeled.
If the antibody is purified and in 10-50 mM amine-free buffer (e.g. HEPES, MES, MOPS and phosphate) at a pH range of 6.5-8.5, it can be quickly and conveniently labeled with HRP using Bio-Rad’s LYNX Rapid HRP Antibody Conjugation Kit® or to alkaline phosphatase with the LYNX Rapid Alkaline Phosphatase Antibody Conjugation Kit.
The final step in an ELISA is the enzyme catalyzed reaction to obtain a colored end product that can be read in a spectrophotometer as absorbance values, representing the analyte concentration. Bio-Rad supplies a range of substrates for HRP and AP enzyme based detection systems.
TMB Core+ (BUF062) is a high-performance TMB (3,3´,5,5´- tetramethylbenzidine) solution, recommended for use in ELISAs as a substrate for HRP. TMB Core+ contains TMB, substrate buffer and hydrogen peroxide and has been optimized for increased sensitivity, minimal background and rapid development. It produces a deep blue color read at 655 nm. The reaction may be stopped with sulfuric acid, resulting in a yellow color read at 450 nm.
TMB CORE (BUF056) is a high performance substrate for HRP that contains TMB, substrate buffer and hydrogen peroxide in a safe, ready-to-use solution.
TMB SIGNAL+ (BUF054) has been optimized to enable increased sensitivity and enhanced stability. It also provides minimal background and rapid development times.
pNPP (BUF044) is a high-performance p-NitroPhenyl Phosphate (pNPP) solution formulated to increase pNPP activity and stability. It is ready-to-use and recommended for ELISAs as a substrate for AP for kinetic and endpoint tests.