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ELISA (Enzyme-Linked Immunosorbent Assay)

Enzyme-linked Immunosorbent Assays (ELISAs) combine the specificity of antibodies with the sensitivity of simple enzyme assays, by using antibodies or antigens coupled to an easily assayed enzyme that possesses a high turnover number. ELISAs can provide a useful measurement of antigen or antibody concentration.

Millipore offers a variety of highly validated preconfigured ELISA assays, reagents and accessories for the study of metabolic disease markers and other vital research targets. This guidebook will provide you with an overview of ELISA methodologies, as well as highlighting Millipore’s industry-leading ELISA kits and filter plates. Be sure to check http://www.millipore.com for the latest ELISA product listings.

General Methodology

ELISAs are used to determine the concentration of specific molecules in a sample. This method is typically highly sensitive while usually simple to perform. This solid-phase assay is possible due to the fact that proteins (antibodies) can be positively attached to plastics (96-well microtiter plate).

Sandwich ELISA Assays

One of the most useful of the immunoassays is the two antibody ‘sandwich’ ELISA. This assay is used to determine the antigen concentration in unknown samples. This ELISA is fast and accurate, and if a purified antigen standard is available, the assay can determine the absolute amount of antigen in an unknown sample. The sandwich ELISA requires two antibodies that bind to epitopes that do not overlap on the antigen. This can be accomplished with either two monoclonal antibodies that recognize discrete sites or one batch of affinity-purified polyclonal antibodies. To utilize this assay, one antibody (the ‘capture’ antibody) is purified and bound to a solid phase typically attached to the bottom of a plate well. Antigen is then added and allowed to complex with the bound antibody. Unbound products are then removed with a wash, and a labeled second antibody (the ‘detection’ antibody) is allowed to bind to the antigen, thus completing the “sandwich”. The assay is then quantitated by measuring the amount of labeled second antibody bound to the matrix, through the use of a colorimetric substrate. Major advantages of this technique are that the antigen does not need to be purified prior to use, and that these assays are very specific. However, one disadvantage is that not all antibodies can be used. Monoclonal antibody combinations must be qualified as “matched pairs”, meaning that they can recognize separate epitopes on the antigen so they do not hinder each other’s binding. Unlike Western blots, which use precipitating substrates, ELISA procedures utilize substrates that produce soluble products. Ideally the enzyme substrates should be stable, safe and inexpensive. Popular enzymes are those that convert a colorless substrate to a colored product, e.g., pnitrophenylphosphate (pNPP), which is converted to the yellow p-nitrophenol by alkaline phosphatase. Substrates used with peroxidase include 2,2’-azo-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), o-phenylenediamine (OPD) and 3,3’5,5’-tetramethylbenzidine base (TMB), which yield green, orange and blue colors, respectively.

Sensitivity of Sandwich ELISA is Dependent on

1. The number of molecules of the first antibody that are bound to the solid phase.
2. The avidity of the first antibody for the antigen.
3. The avidity of the second antibody for the antigen.
4. The specific activity of the second antibody. The amount of the capture antibody that is bound to the solid phase can be adjusted easily by dilution or concentration of the antibody solution. The avidity of the antibodies for the antigen can only be altered by substitution with other antibodies. The specific activity of the second antibody is determined by the number and type of labeled moieties it contains.

Competitive ELISA Assays

When two “matched pair” antibodies are not available for your target, another option is the competitive ELISA. Another advantage to the competitive ELISA is that non-purified primary antibodies may be used. Although there are several different configurations for competitive ELISAs, below is an example for one such configuration. In order to utilize a competitive ELISA, one reagent must be conjugated to a detection enzyme, such as horseradish peroxidase. The enzyme may be linked to either the immunogen or the primary antibody. The protocol below uses a labeled immunogen as the competitor.

Briefly, an unlabeled purified primary antibody is coated onto the wells of a 96 well microtiter plate. This primary antibody is then incubated with unlabeled standards and unknowns. After this reaction is allowed to go to equilibrium, conjugated immunogen is added. This conjugate will bind to the primary antibody wherever its binding sites are not already occupied by unlabeled immunogen. Thus, the more immunogen in the sample or standard, the lower the amount of conjugated immunogen bound. The plate is then developed with substrate and color change is measured.

Factors that Influence Assay Performance

1. The greatest impact of the behavior of the assay is typically based on the antibody pair itself. If the pair is a “good” match for the desired analyte, other unwanted factors are greatly minimized. If the pair is a “poor” match with lower affinity for the desired analyte, it may take a lot of time and creativity to achieve acceptable parameters.
2. Non-specific binding can cause high background values and poor recovery and linearity, which can often be controlled with IgG antibodies or serum.
3. False positive values can occur on Non-specific binding of samples to one or both of the antibodies present which is often detected when performing spiked recovery and linearity testing. In the ELISA format, these samples can typically be controlled by the use of IgG antibodies and/or heterophilic blockers such as Ultra HBR.
4. Hook Effect may give incorrect sample values for highly concentrated samples and can usually be detected during linearity studies.

Validation of ELISA Assays

1. Spiked recovery tests are performed using the blank, low standard, medium standard and high standard points spiked into at least 8 different samples in duplicate. This test should be repeated at least two times to determine if results are reproducible and within acceptable criteria for the coefficient of variation (CV). Acceptable criteria are CVs of 80-120%.
2. Linearity tests are performed on at least 8 different samples in duplicate. At least three dilution of the neat samples should be performed and more if possible. This test should be repeated at least two times to assure that the results are reproducible and acceptable (80-120% CVs).
3. Intra-assay variation is tested by running 8 different samples (of varying concentration) in replicates of ten across the microtiter plate and determining the % CVs of the samples. Acceptable criteria are typically CVs of 80-120%.
4. Inter-assay variation is determined by evaluating at least 8 samples (of varying concentration) in duplicate on at least three different microtiter plates on different days using the same reagent lots. The % CVs are then calculated. Acceptable criteria is typically CVs of 80-120%.
5. The microtiter plates coated with the capture antibody must also be validated by running the blank, low, and high standard points in replicates of 32, on a minimum of three plates, and the % CVs calculated. Acceptable criteria is typically CVs of =10%.
6. The sensitivity of the assay is determined by evaluating the blank and the lowest standard point in replicates of at least 20. The following calculation is used to determine the sensitivity of the assay:

   

7. Further specificity of the assay is evaluated by running standards of several similar analytes of available species in the assay to determine if any similar analytes can be detected by the assay.
8. If available, the sample values are compared to sample values in the most popular competitor kit or our own Millipore RIA kit to determine the correlation of sample values.
9. Compare serum vs. plasma values and determine the correlation. Also evaluate if assay can be performed on cell extracts or cell culture media.