Heterogeneous enzyme immunoassays

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)...

Catalase has also been used as an enzyme label in competitive heterogeneous enzyme immunoassays. Catalase generates oxygen from hydrogen peroxide with the oxygen determined amperometrically with an oxygen electrode. This approach has been demonstrated for a-fetoprotein theophylline and human serum albumin... 

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). 

Gonzalez-Martinez, M., S. Morias, R. Puchades, A. Maquieria, M.-P. Marco, and D. Barcelo (1998). Automation of a heterogeneous enzyme immunoassay for atrazine Comparison of immobilization supports. Fres. J. Anal. Chem., 361 179-184. 

The heterogeneous enzyme immunoassays, which include the enzyme-linked immunosorbent assay (ELISA), are based on the same principles as are used in radioimmunoassays (RIA). In short, after incubation of antigen and antibodies, the antigen-antibody complexes formed are separated from free antigen and antibody by one of a number of different techniques, and the activity in one or both of the fractions is determined. 

E639 Kissel, T. (1990). Simple combination substrate solution for dual-label detection in heterogeneous enzyme immunoassays. Clin. Chem. 36, 1094, Abstr. 666. 

II. Enzyme Label Catalyzing Production of Eiectroactive Product 1. Heterogeneous Enzyme Immunoassays... 

The LCEC method in conjunction with the optimal assay parameters resulted in a very sensitive immunoassay for digoxin in plasma throughout its therapeutic range, with a detection limit of 50 pg/ml. A standard curve is shown in Fig. 7. The relative standard deviation of ip obtained for any given digoxin standard ranged from 4% to 12% on various days and is comparable to results ordinarily obtained by other heterogeneous enzyme immunoassays. Approximately 20 samples can be injected per hour. 

Fig. 6. Heterogeneous enzyme immunoassay with LCEC detection for a series of digoxin standards in plasma solutions. Concentration of digoxin in plasma samples (A), 5.0, (B) 2.0, (C) 1.0, (D) 0.5, and (E) 0 ng/ml. Assay conditions 10 pg/ml antibody coating concentration, 1/125 digoxin-alkaline phosphatase conjugate dilution, 4 h antigen/antibody incubation interval, and 40 min substrate reaction time. (Reprinted with permission from Wehmeyeretal., 1986. Copyright 1986, American Chemical Society.)...

Heterogeneous enzyme immunoassay is by far the most common assay format used in ECI. Most of the heterogeneous assays are based on the enzyme-linked immunosorbent assay (ELISA) technique where Ah is immobilized on the walls of small (<500 gL) microtiter wells or cuvettes [105]. The general procedure of heterogeneous immunoassay is outlined... 

The sandwich immunoassay is the other widely used form of heterogeneous enzyme immunoassay. As shown in Fig.3,... 

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... 

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. 

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