Fluorogenic ELISA assays

Employing variations of the ELISA technique, the fluorogenic ELISA assays are based on the capture of antigens or antibodies from a solution by an insoluble solid phase. These assays are used to determine concentrations of high molecular weight analytes, to indicate the presence of specific antibodies, or for competitive analysis of low-concentration haptens. A linear capillary 

The component parts of the test module used in fluorogenic ELISA assays are shown in Fig. 30. Variations in the use of the different ports and wells allow these test modules to be utilised for different types of ELISA reactions. The use of the ports and wells as described below is specific for a sandwich-type ELISA reaction. 

In the sample port the sample is introduced into the test module and the reaction takes place. The fibrous matrix is a glass fibre which is coated with antibody. The conjugate well is prefilled with enzyme conjugate. The substrate well is prefilled with a substrate solution that is used as both substrate and wash solution. The wash port serves to dispense the substrate. 

OPI Grenner, G., Inbar, S., Meneghini, F.A., Long, E.W., Yamartino, E.J., Bowen, M.S., Blackwood, J.J., Padilla, A.J., Maretsky, D., and Staedter, M. (1989). Multilayer fluorescent immunoassay technique. Clin. Chem. 35, 1865-1868. 

Vaupel, U., Case, A., Farrell, K., Inbar, S., and Metzmann, E. (1990). Rapid self- contained immunoassay for digoxin. Clin. Chem. 36, 1028, Abstr. 359. 

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The reagents used for the Opus system are totally self-contained in the test modules. There are two types of assay methodologies used with the analyzer multilayer film assays and fluorogenic ELISA assays. 

In heterogeneous immunoassays in batch format the problem of background fluorescence is reduced by separating the analyte from other fluorescent species present in the sample matrix. The use of enzymes with fluorogenic substrates in fluorescence enzyme-linked immunosorbent assays (F-ELISA) and Eu(in)-chelates in time-resolved fluoroimmunoassay (TRFIA) permits to achieve very low LODs for pesticides (e.g., between 0.023-0.3 and 0.05-0.4pgl for F-ELISA and TRFIA, respectively). 

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