Enzyme channeling immunoassays

In this method an appropriate enzyme (e.g., GPDase) and the antigen are co-immobilized on a solid phase. Hexokinase-labeled antibody produces an accelerated conversion of glucose, ATP and NAD+ since the enzymes are in each other s vicinity. 

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The original methods of fluorescence exciting transfer immunoassay (Ul) and enzyme channeling immunoassay (L9) are similar to the proximal linkage immunoassay. In the former, two reactants are coupled to solid antigen and antibody and, in the latter, they are coupled to two kinds of monoclonal antibodies. The enzyme enhancement immunoassay (G4) also falls in this category. 

L9. Litman, D. J., Hanlon, T. M., and Ullman, E. F., Enzyme channeling immunoassay A new homogeneous enzyme immunoassay technique. Anal. Biochem. 106, 223-229... 

The general principle of enzyme-channelling immunoassays, in which Ab-Ag binding brings two enzyme labels into close proximity to speed sequential reactions, is described in Fig. 8.4. One enzyme conjugate is... 

Fig. 8.4. Schematic illustration of the principle of the separation-free, amperometric enzyme channelling immunoassay with immobilized glucose oxidase and antibody on the PEI-modified electrode surface.

Fig. 7 The principle of enzyme-channeling immunoassay using an electrode-bound enzyme to make the substrate for the enzyme label on the Ag. ...

Miniaturized proteomic analysis devices include enzyme assays and immunoassays. The enzyme assay chip is mainly a sophisticated incubator and flowthrough system. It can perform multiple functions typically required by the biochemist, namely, diluting substrate and buffer, mixing enzyme and substrate, incubating during conversion, and allowing for detection in a flow channel. Immunoassay chips are similar... 

The plate washing can be conducted automatically if a microplate washing system is available. We used the Perkin Elmer-Getus Pro/Pette instrument with a Pro/Wash head and the Beckman Biomek 1000 Automated Laboratory Workstation in our enzyme immunoassays. If automated instrumentation is not available, liquid transfers and plate washing can be carried out by a 12-channel micropipet. 

Microfiuidic bioreactors are a variety of devices that can be made by immobilizing a variety of biologically active substrates within a microfiuidic device [1]. The ability to create a variety of biologically important devices is critical to enabling the true total analytical system. The variety of devices that can be made in this way ranges from immobilized enzyme reactors to enzymatic biosensors, immunoassays, and affinity chromatographic stationary phases. In order to form a microfiuidic bioreactor, it is necessary to immobilize the active molecule within the device, either directly onto the channel or onto a solid support within the channel such as a bead or a monolith. 

In another study, a microchip-based electrochemical enzyme immunoassay was developed by Chatrathi and colleagues [65] and its performance is demonstrated for the determination of monoclonal mouse IgG as a model analyte. The direct homogeneous immunoassay requires the integration of electrokinetic mixing of ALP-labeled anti-mouse IgG antibody (Ab-E) with the mouse IgG antigen (Ag) analyte in a precolumn reaction chamber, injection of immunochemical products into the separation channel, followed by rapid electrophoretic separation of enzyme-labeled free antibody and enzyme-labeled antibody-antigen complex. The separation is followed by a postcolumn reaction of enzyme tracer with p-aminophenyl... 

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. 

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