Novel biotinylated acridinium derivatives: New reagents for fluorescence immunoassays and proteomics

Background: The favorable properties of acridinium derivatives over conventional fluorescent probes have attracted considerable interest. These conjugates have found wide utility in tests developed for medical diagnosis providing at least comparable sensitivity to radioimmunoassay (RIA) and enzyme immunoassay (EIA) procedures. Methods: In this paper we describe the synthesis, luminescent properties and immunoassay applications of two novel biotinylated acridinium derivatives, 9-(2-biotinyl-oxyethyl)-carboxylate-10-methyl-acridinium triflate, BOCMAT and 9-(2-biotinyl-amidoethyl)-carboxylate-10-methyl-acridinium triflate, BACMAT. Results: The chemiluminescence efficiency of the novel reagents is high in polar aprotic solvents and attains detection limits down to 7.28 × 10- 8 M, while the fluorescence efficiency has its maximum in aqueous solutions with detection limits down to 1.94 × 10- 10 M. The above novel compounds were applied in the fluorimetric determination of solid phase-immobilized biotinylated mouse IgG and the detection limit was found to be ∼ 1 μg/assay (∼ 7 pmol/assay). Conclusions: The new streptavidin-based detection reagents were synthesized can be used for the development of highly sensitive solid-phase fluorescence-based immunoassays and may find important applications in proteomics. © 2005 Elsevier B.V. All rights reserved

10-(2-Biotinyloxyethyl)-9-acridone. A novel fluorescent label for (strept)avidin-biotin based assays

A novel biotinylated fluorophore, 10-(2-biotinyloxyethyl)-9-acridone 3 has been synthesized and its fluorescent properties were examined in the presence and absence of avidin or streptavidin. In aqueous solutions the novel fluorophore 3, as well as its precursor 10-(2-hydroxyethyl)-9-acridone 2 exhibit intense fluorescence and can be detected down to 8.62 × 10-10 and 1.90 × 10-10 M, respectively. A short spacer was chosen in order to minimize steric repulsion between the adjacently bound to avidin or streptavidin residues of acridone. The novel biotinylated fluorophore 3 exhibits rapid, specific and stoichiometric binding to the four biotin binding sites in avidin or streptavidin tetramers, even at low concentrations (20 nM). Preliminary measurements showed that the new conjugate can be applied for the fluorimetric determination of solid-phase immobilized mouse IgG with detection limits down to 1.3 ng per assay. © 2005 Elsevier B.V. All rights reserved