Immunoassay heterogeneous electrochemical enzyme

Wehmeyer, K.R., H.B. Halsall, and W.R. Heineman. 1985. Heterogeneous enzyme immunoassay with electrochemical detection Competitive and sandwich -type immunoassays. Clin. Chem. 31 1546-1549. 

Several heterogeneous electrochemical enzyme immunoassays have been demonstrated. These are based on the enzyme-linked immunosorbent assay (ELISA) technique... 

K.R. Wenmeyer, H.B. Halsall, W.R. Heineman, C.P. Voile, and I.W. Chen, Competitive heterogeneous enzyme immunoassay for digoxin with electrochemical detection. Anal. Chem. 58, 135-139 (1986). 

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 summary, it can be stated that methods of electrochemical detection are very applicable to enzyme immunoassays. Broadly speaking, the above examples demonstrate that homogeneous EIA are faster and simpler but often less sensitive and more subject to interference than heterogeneous EIA. The latter are less sensitive to interference and electrode fouling because the measuring chamber in front of the electrode is rinsed before determination of the marker activity. However, none of the methods described is suitable for continuous measurement. 

Samples from patients receiving digoxin therapy were analyzed by the heterogeneous immunoassay LCEC method. Samples were diluted 5/1 with pooled human plasma in order to eliminate an observed antibody matrix effect. Digoxin standards in pooled human plasma were treated similarly and a standard curve constructed. Digoxin levels in patient samples were determined by reference to the standard curve. The values obtained for the samples by the electrochemical enzyme immunoassay method were compared to those obtained by radioimmunoassay. The results for the 54 samples analyzed are presented in Fig. 8. A good correlation was obtained between the two methods (r = 0.93). 

This chapter presents an approach to perform enzyme linked immunosorbent assays (ELISA) in a microfluidic format with electrochemical detection. This field of analytical chemistry has shown a strong activity in recent years, and many reports have presented the use of capillary-sized reactors for running immunoassays either in homogeneous format (where the antigen-antibody complex and the labelled revelation reagents are separated prior to detection, as for instance by capillary electrophoresis [1-3]) or in heterogeneous format (where the antibody is immobilised on the inner surface of the microsensor device [4] or on microbeads [5,6]). 

One form of heterogeneous immunoassay is called enzyme-linked immunosorbent immunoassay (ELISA). In one instance, electrochemical immunoassay was performed for anti-ferritin (antibody) in a PDMS/PMMA chip. First, DTSSP was self-assembled on the gold electrode deposited on the PMMA plate. Then horse spleen ferritin (antigen) was attached to the DTSSP layer. A 100-p.g/mL solution of anti-horse ferritin (rabbit serum) was added. Then a secondary anti-rabbit antibody (HRP-linked) was introduced. A substrate (4-CN) was finally added which was converted to a precipitate product. The precipitate caused a reduction... 

Heterogeneous immunoassay has also been conducted without the use of an enzyme label. For instance, electrochemical immunoassay of mouse IgG (antigen) was carried out in glass chip. The chip contained magnetic beads coated with the sheep anti-mouse antibody. After flowing in the secondary antibody (rat antimouse conjugated with PAPP), electrochemical oxidative detection of PAP was achieved (i.e., PAP was oxidized to p-quinoneimine) [1016]. 

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