1. Setting up your ELISA

Since ELISAs are flexible in their design, it is important to choose the right ELISA format for your experiment. ELISA assays can be of four general types- direct, indirect, sandwich or competitive. It is best to use the simplest ELISA that works with your reagents and provides the output you require. For example, if you are evaluating monoclonal antibody candidates, then a simple direct or indirect ELISA would be best. Both can yield the results you need, but depending on whether you need a more sensitive result, or whether you have labeled primary antibodies, then one type may be more suitable than the other. To learn more about ELISA types, visit our Introduction to ELISA.

2. ELISA Plates

Flat-bottomed, 96-well plates, made from polystyrene or polyvinyl chloride, are used in the vast majority of ELISA assays. 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. Some enzyme substrates, such as those that produce fluorescent or chemiluminescent signals may require opaque plates for optimal results.

3. Consistency between wells

One of the most important aspects of any assay is consistency and standardization of conditions as this will affect the reproducibility and accuracy of your results. In the initial stages of assay development it is important to test a range of parameters, usually by completing a checkerboard dilution series to test various conditions in systematic manner. In addition, buffers, temperature, and humidity must be kept constant between and within experiments in order to produce standardized results.

In a typical ELISA, multiwell plates, multichannel pipets, and plate washers provide for more consistent and faster results, as well as higher throughput. It is very important to make sure that all pipettors used in ELISAs are properly calibrated on a regular basis, or there can be significant variation in the results. Furthermore, it is good technique to observe the level of the liquid in the pipet and the wells while following the procedure, in order to make sure no sample is far out of line with the others. This is particularly important when multi-channel pipets are use, as sometimes the tips in the end rows do not always attach fully to the pipettor.

4. Standard ELISA Errors

ELISA assays are prone to two common types of standard errors, which must be watched out for and controlled against. These are edge effect and hook effect. An edge effect is the result of inconsistencies in the production of ELISA multiwell plates or when assay conditions, such as stacking plates, cause the outer wells to behave differently. As a result, unexpected values can appear in the outer wells which may be out of line with neighboring well. This can best be controlled for by using duplicates or triplicates for all samples, and noting any large variations in the results for a given sample.

A Hook effect, on the other hand, is something that is seen when there are very high levels of antigen in the sample. As a result specific binding of the antigen is insufficient to match analyte levels and signal is lower than expected. The best way to avoid this issue is to test several dilutions of each sample.

5. Specialty buffers

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 they can be sourced from a variety of commercial antibody and reagent suppliers. Basic ELISA buffer recipes can be found on our ELISA protocols page.

Some specialist buffers are multifunctional, allowing for simultaneous coating, stabilization, and blocking. One such example is AbGuard® Plate Stabilizer (BUF063), which preserves the biological activity of adsorbed molecules and prevents degradation, denaturation, and leaching of the coating material. As a result, this buffer can shorten the length of time required for the assay and will increase efficiency by extending the life of coated plates.

Another option is Block Ace (BUF029), a buffer that can be used for blocking, sample dilution, and washing for ELISAs and Western blots. It also improves ELISA results by reducing background and producing sharper standard curves.

6. Antibody considerations

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 advantage during ELISA development.

The interaction between antibodies and their antigens is described in three ways: specificity, affinity, and avidity.

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 very 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.

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 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. Search our collection of validated matched antibody pairs.

7. Coating

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 the plates in a moist environment to minimize evaporation.

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 over saturation 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 likely possible that the effective antigen/antibody concentration may be low and may be 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. In addition, specialist coating buffers are available which have been optimized for ELISA, such as BUF030A which has been developed to stabilize the adsorbed protein, preserving the antigenic regions and allowing greater binding reactivity in order to enhance the specific signal. For more specialty ELISA buffers and substrates, visit our ELISA Buffers Page.

8. Samples

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 ul.

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 in order to make sure the final results fall within the linear portion of the standard curve because highly concentrated samples can underestimate concentration and highly diluted samples can overestimate concentrations.

One type of specialty sample buffer used for sample dilutions is HISPEC assay diluent, BUF049A. This buffer is used to dilute a range of sample types or for the dilution of detection antibodies. It reduces cross-reactivity, non-specific binding, and matrix effects.

ELISAs are often used to detect antigens or antibodies in heterogeneous samples, such as blood, cell culture media, or urine where small quantities of a substance, such as a drug of abuse or a virus particle, might need to be measured. 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. As a result, special treatments and buffers are sometimes needed for the dilution of blood samples in order to obtain optimal results.

BUF037A is a specialized buffer that is recommended for use in sandwich ELISA assays with samples containing plasma, serum, or cell culture supernatant. This buffer contains goat serum proteins which reduce the difference between the sample matrix and the diluent used to generate the standard curve. It also contains a proprietary chelating agent that blocks interference from complement and thrombin in plasma and serum. It is easy to use and is applied by pipetting 50-100 ul directly onto the plate before adding the sample. Since this buffer contains proteins derived from goat, it is not recommended for ELISAs that use anti-goat secondary antibodies for detection, as this will result in non-specific signal.

9. Blocking

Blocking is often necessary to prevent the non-specific binding of detection antibodies to the multiwell plate surface itself. To accomplish this task, blocking buffer usually contains an unrelated protein or a protein derivative that does not react with any of the antibodies being used in the detection step.

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 ul of blocking buffer is added to the well to incubate for a one hour at 37 C in order to fully block the plate. At Bio-Rad we find that the blocking step is not always required with our antibodies.

A wide range of sophisticated commercial blocking buffers are available, some of which are BSA-based (BUF032), but which also contain preservatives to create a stable long-term environment. Another option is to use a high performance commercial blocking buffer, such as ELISA Ultrablock (BUF033), which is an optimized formulation that includes small protein fragments which thoroughly block less accessible surfaces of the multiwell dish. Ultrablock is recommended for use on mammalian samples, particularly, human, pig, and cow, as there is less chance of cross-reactivity. When maximal blocking strength is required, or for most sandwich ELISAs, preparations such as ELISA Synblock (BUF034), an inert, synthetic blocking formula that reduces non-specific interference.

10. Washing

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 absorbant paper to remove excess liquid or by careful aspiration.

For greatest consistency, specialized plate washers are used to add and remove the wash liquid. Specialized wash buffers are also available, such as BUF031, which contains an optimized formulation of pH stabilizers, salts, and detergents that reduce background noise and enhance specific signal.

For a wealth of useful ELISA resources together with antibodies, reagents and kits, go to our ELISA section.