Heterogeneous enzyme immunoassays competitive assays

Fig. 13. General protocol for heterogeneous enzyme immunoassay preparation of reagent cuvettes by coating Ab, competitive assay format, and sandwich assay format. (Reprinted with permission from W. R. Heineman and H. B. Halsall, Anal. Chem, 1985, 57, 1321A. Copyright 1985, American Chemical Society)...

Capillaries modified as above have been used in several heterogeneous enzyme immunoassays, both competitive and sandwich. The calibration curve for a rabbit IgG assay in human semm controls is shown in Figure 12. The zero dose-response was 7.5 nA using the ion-pairing agent pentane sulfonate. The final... 

Methods based on chemiluminescent and bioluminescent labels are another area of nonisotopic immunoassays that continue to undergo active research. Most common approaches in this category are the competitive binding chemiluminescence immunoassays and the immunochemiluminometric assays. Chemiluminescence and heterogenous chemiluminescence immunoassays have been the subject of excellent reviews (91, 92). Detection in chemiluminescence immunoassays is based on either the direct monitoring of conjugated labels, such as luminol or acridinium ester, or the enzyme-mediated formation of luminescent products. Preparation of various derivatives of acridinium esters has been reported (93, 94), whereas a variety of enzyme labels including firefly or bacterial luciferase (70), horseradish peroxidase (86, 98), and alkaline phosphatase are commercially available. 

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. 

In most cases, competitive immunoassays for the analysis of small molecules are carried out in microtitre plates. The majority of these assays use an enzyme as label, thus leading to the term enzyme immunoassay, and the most commonly used is the ELISA (enzyme-linked immunosorbent assay) that has a heterogeneous format (separation of bound and unbound). This format can be set up either in the enzyme-tracer format (Figure 3.3.1 A) or in the coating antigen format (Figure 3.3. IB). The result shows a... 

Table 1 The plethora of formats of immunoassay. Ab = antibody, Ag = antigen. Noncompetitive and competitive heterogenous binding assays where the probe is labelled with an enzyme are known as enzyme-linked immunosorbent assay (ELISA)...

New immunoassay concepts were described in which immobilized affinity ligands are used in unique flow-through systems (flow-injection immunoassay, FIIA). These systems are exclusively heterogeneous. Both immobilization of a specific antibody or a hapten on the solid support is possible. In most cases the scheme of the assay used is based on a sequential competitive enzyme immu-noas.say procedure [66], [67],... 

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