Enzymatic Immunoassay

In enzymatic immunoassays [22,23], the enzyme used as the label does not generate any detection signal, but produces the signal-generating species upon the enzyme-catalyzed reaction. High turnover of the enzyme-catalyzed reaction gives rise to efficient amplification of the detection signal so that... 

M. Maeda and A. Tsuji, Enzymatic Immunoassay of a-Fetoprotein, Insulin and 17-a-Hydroxyprogesterone Based on Chemiluminescence in a Flow-Injection System. Anal. Chim. Acta, 167 (1985) 231. 

GC = gas chromatography (interferences primarily with older methods) HPLC = high-pressure liquid chromatography lA = immunoassay SC = spectrochemical TLC = thin-layer chromatography EC = electrochemical EZ = enzymatic EE = flame emission MEIA = microparticle enzymatic immunoassay. 

The use of solid-phase reactors coupled online to FI manifolds significantly increases the potential of FI by enhancing such basic analytical parameters as sensitivity and selectivity and allowing implementation of specific reactions. These reactors involve (bio)chemical reactions - whether enzymatic, immunoassay, ion exchange, or redox - or act as sorbent extractants or reagent releasers. 

The microplate model has often been used as a starting platform for electrochemical array development. In a report from Tang et al. [14], a multichannel array consisting of eight channels of Pt electrodes, fitted to the dimensions of standard microplate wells, has been developed, with the required multichannel potentiostat for amperometric detection of the product of an enzymatic immunoassay label. Alkaline phosphatase was used as the model immunoassay label in a model rabbit/anti-rabbit IgG study, where the second IgG is labeled with enzyme. The enzyme product provided the amperometric signal with a detection limit in the low pM range. 

Monitoring enzyme catalyzed reactions by voltammetry and amperometry is an extremely active area of bioelectrochemical interest. Whereas liquid chromatography provides selectivity, the use of enzymes to generate electroactive products provides specificity to electroanalytical techniques. In essence, enzymes are used as a derivatiz-ing agent to convert a nonelectroactive species into an electroactive species. Alternatively, electrochemistry has been used as a sensitive method to follow enzymatic reactions and to determine enzyme activity. Enzyme-linked immunoassays with electrochemical detection have been reported to provide even greater specificity and sensitivity than other enzyme linked electrochemical techniques. 

A homogeneous electrochemical enzyme immunoassay for 2,4-dinitrophenol-aminocaproic acid (DNP-ACA), has been developed based on antibody inhibition of enzyme conversion from the apo- to the holo- form Apoglucose oxidase was used as the enzyme label. This enzyme is inactive until binding of flavin adenine dinucleotide (FAD) to form the holoenzyme which is active. Hydrogen peroxide is the enzymatic product which is detected electrochemically. Because antibody bound apoenzyme cannot bind FAD, the production of HjOj is a measure of the concentration of free DNP-ACA in the sample. 

Arenkov, P., Kukhtin, A., Gemmell, A., Voloshchuk, S., Chupeeva, V., and Mirzabekov, A. (2000). Protein microchips Use for immunoassay and enzymatic reactions. Anal. Biochem. 278, 123-131. 

Chemiluminescence reactions are currently exploited mainly either for analyte concentration measurements or for immunoanalysis and nucleic acid detection. In the latter case, a compound involved in the light emitting reaction is used as a label for immunoassays or for nucleic acid probes. In the former case, the analyte of interest directly participates in a chemiluminescence reaction or undergoes a chemical or an enzymatic transformation in such a way that one of the reaction products is a coreactant of a chemiluminescence reaction. In this respect, chemiluminescent systems that require H2O2 for the light emission are of particular interest in biochemical analysis. Hydrogen peroxide is in fact a product of several enzymatic reactions, which can be then coupled to a chemiluminescent detection. 

Competitive immunoassays may also be used to determine small chemical substances [10, 11]. An electrochemical immunosensor based on a competitive immunoassay for the small molecule estradiol has recently been reported [11]. A schematic diagram of this immunoassay is depicted in Fig. 5.3. In this system, anti-mouse IgG was physisorbed onto the surface of an SPCE. This was used to bind monoclonal mouse anti-estradiol antibody. The antibody coated SPCE was then exposed to a standard solution of estradiol (E2), followed by a solution of AP-labeled estradiol (AP-E2). The E2 and AP-E2 competed for a limited number of antigen binding sites of the immobilized anti-estradiol antibody. Quantitative analysis was based on differential pulse voltammetry of 1-naphthol, which is produced from the enzymatic hydrolysis of the enzyme substrate 1-naphthyl phosphate by AP-E2. The analytical range of this sensor was between 25 and 500pg ml. 1 of E2. 

Apart from immunoassays, enzyme assays can also be used to detect certain substrates in a clinical diagnostic setting. The benefits of performing enzymatic assays on microchips are the analytical power and minimal reagent use in microfluidic systems combined with the selectivity and amplification factors that come with biocatalysis. 

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