Sandwich immunoassays enzyme linked immunoassay format

Labeling with enzymes has been the most widely employed approach to increase sensitivity in chromatographic immunodetection, probably because of broad experience with the very well known enzyme-linked immunoassays. Enzyme tags have been used to form electroactive, fluorescent, and colored products. Assays developed by de Alwis and Wilson are landmarks in the field of immunodetection. Electrochemical detection of hydrogen peroxide formed by the reaction of a glucose oxidase tag in both sandwich [58] and competitive [59] formats allowed detection of subpicomoles of bovine and human IgG in serum. 

In a direct immunoassay the immobilized antibody binds to the corresponding antigen. The competitive immunoassay relies upon the competition of the analyte with a labelled analyte for antibody binding. These formats are widely used for high throughput affinity arrays. A sandwich immunoassay is based on the trapping or capture of the analyte by another antibody. In ELISA (enzyme linked immunosorbent assays) the second antibody is conjugated with an enzyme. The bound enzyme labelled antibody is detected by its ability to break down its substrate to a colored product. 

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. 

Depending on the experimental design, the number of Abs involved in the immunocomplex formation reaction is varying. A t-Ag, like a bacteria toxin, can be directly immobilized on a reaction substrate and can be quantified with a labeled detection antibody (d-Ab) in a direct immunoassay, involving a single t5q)e of Ab. This technique is however limited, since t-Ags for direct surface immobilization have to be available. A more flexible technique, called sandwich immunoassay, consists to flank the t-Ag to be detected between a capture antibody (c-Ab) linked to a reaction substrate and a labeled d-Ab (see Fig. 3b). In this technique, the t-Ag is specific for both c-Ab and d-Ab. Also an enzyme can be used to label the d-Ab, and, in... 

A companion approach is to form copolymers using pyrrole and pyrrole butyric acid. The copolymer presents available carboxylic acid groups at the surface that may serve to covalently immobilize the IgG. Figure 6 illustrates the covalent tethering of amine-functionalized biotin following EDC/sulfo-NHS activation of the pendant carboxylic acid groups of poly(pyrrole-co-pyrrolylbutyric acid). Subsequent incubation in streptavidin or neutravidin followed by incubation in biotinylated IgG immobilizes the primary antibody to the device surface. This format has been used to build sandwich immunoassays for antigens that are detected by the indirect action of the oxidoreductase enzyme-linked antibodies on the conductivity of the polymer film. 

Immunoassay is well acknowledged since the introduction of ELISA (enzyme-linked immunosorbent assay) which was developed over 20 years ago. ELISA can be classified into three formats direct, sandwich, and competitive [2]. Direct ELISA is a simple process that the excessive antibody is reacted with antigen. After incubating, a washing step is conducted to eliminate the unbound antibody. The sensitivity is proportional to the amount of the present antibody in the solution. In a sandwich immunoassay, the antigen (e.g., sample) is between the primary antibody and the second antibody (detection antibody). Generally, the primary antibody is immobilized on the solid surface and the second antibody is labeled with certain enzyme or fluorophore. The amount of sample is proportional to the amount of the labeled second antibody which is measured by the different detection strategies. In a competitive immunoassay, the... 

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. 

Most immunoassay kits and many commercial immunoassay analyzers are based on heterogenous EIA or FIA. These include an immunoassay system that uses FIA linked to radial partition chromatography of the antibody—antigen complex (39) a system that uses antibody-coated tubes for enzyme immunoassay of a variety of hormones and dmgs (40) and a system that uses either a sandwich or competitive FIA format to measure a variety of analytes (41). 

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