Enzyme-linked immunosorbent assay sandwich technique

Several heterogeneous electrochemical enzyme immunoassays have been demonstrated. These are based on the enzyme-linked immunosorbent assay (ELISA) technique in which antibody is immobilized on the walls of a small volume plastic vessel. The ELISA technique can follow either a competitive equilibrium or a sandwich format. Both formats have been used with electrochemical detection. The general protocol for these two formats is shown in Fig. 9. 

Figure 8.3. Schematic representation of a non-competitive enzyme-linked immunosorbent assay using the sandwich technique.

The dual-antibody sandwich enzyme-linked immunosorbent assay (DAS ELISA Fig. 1) is a sensitive and specific technique for quantifying molecules in solution. The technique is dependent upon the availability of two antibodies recognizing separate epitopes upon the antigen to be measured such that they are able to bind to the molecule simultaneously (1,2). The capture antibody specific to the substance to be measured is first coated onto a high-capacity... 

Protein arrays with 10-500 antibodies printed onto an array use detection techniques, including fluorescence and multiple sandwich enzyme-linked immunosorbent assays, but the broad application of these assays is restricted by lack of suitable antibodies for laboratory animals and some potential cross-reactivities between antibodies with similar affinities. Calibration, reproducibility, and identification of proteins are common problems for all of these technologies. A number of databases are available to help investigators identify the numerous proteins found using these separation techniques, particularly for mass spectrometer data. 

The sandwich technique can be improved even further if the second antibody is attached to an enzyme, such as alkaline phosphatase. The enzyme rapidly converts an added colorless substrate into a colored product, or a nonfluorescent substrate into a highly fluorescent product. These changes can be quantitated if the degree of change in color or fluorescence is proportional to the amount of hormone present in the patient sample. Less than a nanogram (10 g) of a protein can be measured by such an enzyme-linked immunosorbent assay (ELISA). 

Another technique, the enzyme-linked immunosorbent assay (ELISA), combines the specificity of antibodies with the sensitivity of an enzyme assay. The ELISA can be performed in a variety of combinations that involve either a specific antibody or the total cellular protein immobilized on a solid support, such as the wells of a plastic microplate. In one version of the method, the sandwich ELISA, the primary antibody is bound to the wells. When a mixture of proteins is added, the protein of interest binds to the antibody, and other proteins are washed away. A second labeled antibody, specific to a different epitope on the protein, is added, and the amoruit of signal is proportional to the amoimt of the particular protein in the sample. The method can be modified to detect specific antibodies in a mixture by using their antigen as the immobilized bait. ELlSAs also have the advantage of being able to be performed in 96-weU plates so many samples can be analyzed in one experiment. 

This is directly proportional to the quantity of analyte present. This technique advanced rapidly when monoclonal antibodies became available, since this made it possible to produce unlimited quantities of antibodies with clearly defined characteristics [20]. This second type of assay is also known as a sandwich immunoassay, and is best suited to the assay of proteins rather than haptens. The widespread ELISA (Enzyme Linked Immunosorbent Assay) test belongs to this category [21-24]. 

Immunoassays vary by the different labels they use. The most common labels include chromophores, fluorophores, radioisotopes and enzymes. Of those labels, enzyme immunoassay or enzyme-linked immunosorbent assay (ELISA) is the most popular technique. ELISA has as an advantage the amplification of the analytical signal and/or increase of the sensitivity of the immunoassay. There are four types of ELISA direct ELISA, indirect ELISA, sandwich ELISA and competitive ELISA. 

The immunometric-type assay has also been adapted for use with nonisotopic labels and is typically carried out in a heterogeneous format in which the antibody is immobilized on a solid support, such as a microtiter dish, membrane, or collection of beads. The canonical clinical immunoassay format in toady s laboratories is the enzyme-linked immunosorbent sandwich assay, which employs two antibodies, one to capture the analyte and the other to detect and quantify it. More details of the principles of these and other immunoassay techniques are given elsewhere in this encyclopedia. 

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