Enzyme-linked Immunosorbent Assay (ELISA) combines 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. ELISA can provide a useful measurement of antigen or antibody concentration. 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. Generally there are 5 types of ELISA (Figure 1):
Multiplex ELISA (Top)
Figure 1. Types of ELISA.
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Shown in diagram generalizing models of different types of ELISA. Further variation of the assay are there depending upon the labeling and signal detection methodology. The basic approaches stay the same: fixing either antigen or antibody and detecting antibody-antigen complex. (Top)
3.1 Direct ELISA
The direct ELISA uses the method of directly labeling the antibody itself. Microwell plates are coated with a sample containing the target antigen, and the binding of labeled antibody is quantitated by a colorimetric, chemiluminescent, or fluorescent end-point. Since the secondary antibody step is omitted, the direct ELISA is relatively quick, and avoids potential problems of cross-reactivity of the secondary antibody with components in the antigen sample. However, the direct ELISA requires the labeling of every antibody to be used, which can be a time-consuming and expensive proposition. In addition, certain antibodies may be unsuitable for direct labeling. Direct methods also lack the additional signal amplification that can be achieved with the use of a secondary antibody. (Top)
3.2 Indirect ELISA
The indirect, two-step method uses a labeled secondary antibody for detection. First, a primary antibody is incubated with the antigen. This is followed by incubation with a labeled secondary antibody that recognizes the primary antibody. For ELISA it is important that the antibody enzyme conjugate is of high specific activity. This is achieved when the antibody is affinity purified and the enzyme conjugation chemistry preserves antibody specificity as well as enzyme activity. (Top)
Table 2. Comparison of Direct and Indirect ELISA Detection Methods
Advantages of Direct Detection
Quick methodology since only one antibody is used.
Cross-reactivity of secondary antibody is eliminated.
Disadvantages of Direct Detection
Immunoreactivity of the primary antibody may be reduced as a result of labeling.
Labeling of every primary antibody is time-consuming and expensive.
No flexibility in choice of primary antibody label from one experiment to another.
Little signal amplification.
Advantages of Indirect Detection
A wide variety of labeled secondary antibodies are available commercially.
Versatile, since many primary antibodies can be made in one species and the same labeled secondary antibody can be used for detection.
Immunoreactivity of the primary antibody is not affected by labeling.
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 of Indirect Detection
Cross-reactivity may occur with the secondary antibody, resulting in nonspecific signal.
An extra incubation step is required in the procedure.
3.3 Sandwich ELISA
The sandwich ELISA measures the amount of antigen between two layers of antibodies. The antigens to be measured must contain at least two antigenic sites, capable of binding to the antibody, since at least two antibodies act in the sandwich. For this reason, sandwich assays are restricted to the quantitation of multivalent antigens such as proteins or polysaccharides. Sandwich ELISAs for quantitation of antigens are especially valuable when the concentration of antigens is low and/or they are contained in high concentrations of contaminating protein.
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.
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GenWay has successfully applied polyclonal IgY antibodies for development of sandwich ELISAs. The technology can expedite development of ELISA with certain throughput and low cost. The ELISA kits are good enough to reach detection senility at sub-nanogram per ml level and are useful for screening protein targets and quantifying their expression in different conditions. For higher detection sensitivity needed, monoclonal antibodies can be further introduced into the ELISA kit to pair with polyclonal IgY as either capture or detection antibodies.
The sensitivity of the sandwich ELISA is dependent on four factors:
The number of molecules of the first antibody that are bound to the solid phase.
The avidity of the first antibody for the antigen.
The avidity of the second antibody for the antigen.
The specific activity of the second antibody.
3.4 Competitive ELISA
When two "matched pair" antibodies are not available for a target, another option is the competitive ELISA. The advantage to the competitive ELISA is that non-purified primary antibodies may be used. Although there are several different configurations for competitive ELISA, one reagent must be conjugated to a detection enzyme, such as horseradish peroxidase. The enzyme may be linked to either the antigen or the primary antibody. The example shown in Figure 1 is a labeled antigen as the competitor. In this type of ELISA, there is an inverse relationship between the signal obtained and the concentration of the analyte in the sample, due to the competition between the free analyte and the ligand-enzyme conjugate for the antibody coating the microplate, i.e. the more analyte the lower the signal.
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 antigen is added. This conjugate will bind to the primary antibody wherever its binding sites are not already occupied by unlabeled antigen. Thus, the more unlabeled antigens in the sample or standard, the lower the amount of conjugated antigen bound. The plate is then developed with substrate and color change is measured.
3.5 Multiplex ELISA
A logical progression of the widely used microtiter plate ELISA is toward a protein array format that allows simultaneous detection of multiple analytes at multiple array addresses within a single well. There are different types of multiplex ELISA have been developed and in practice. One of the examples is to measure antigens by coating or printing capture antibodies in an array format within a single well to allow for the construction of "sandwich"ELISA quantification assays. Generally, multiplex ELISA can also be achieved through antibody array, where different primary antibodies can printed on glass plate to capture corresponding antigens in a biological sample such as plasma, cell lysate, or tissue extract. Detection method can be direct or indirect, sandwich or competitive, labeling or non-labeling, depending upon antibody array technologies