Enzymically amplified time-resolved fluorescence immunoassay with terbium chelates

Abstract

We report an ultrasensitive, enzymatically amplified, time-resolved fluorescence immunoassay with a terbium chelate as the detectable moiety. In this immunoassay, the primary label is the enzyme alkaline phosphatase (ALP). ALP cleaves phosphate out of a fluorogenic substrate, 5-fluorosalicyl phosphate, to produce 5-fluorosalicylic acid (FSA). 5-Fluorosalicylic acid can then form a highly fluorescent ternary complex of the form FSA-Tb(3+)-EDTA, which can be quantified by measuring the Tb3+ fluorescence in a time-resolved mode. In this assay, exceptional sensitivity is achieved because of the enzymatic amplification introduced by ALP and the quantification by laser-induced microsecond time-resolved fluorometry. Time-resolved fluorometry is applicable because of the long fluorescence lifetime of the Tb3+ complexes. It is shown that in a model AFP assay 10(6) or 1.5 x 10(5) molecules can be detected (final assay volume, 100 microL) by using monoclonal or polyclonal detection antibodies, respectively. The assay demonstrates excellent precision (approximately 4%), and it seems to be highly suited for automated, sensitive, and rapid immunoassays.

Transcriptional upregulation of human tissue kallikrein 6 in ovarian cancer: clinical and mechanistic aspects

“…Immediately after incubation, 100 ml of developing solution (1 M Tris-HCl buffer, 0.4 M NaOH, 2 mM TbCl 3 , 3 mM EDTA) was added to each well and incubated for 1 min with shaking at room temperature. The fluorescence was measured with a time-resolved fluorometer, the Cyberfluor 615 Immunoanalyzer (MDS Nordion), as described elsewhere (Christopoulos and Diamandis, 1992). The KLK6 concentrations in ng ml À1 were converted to ng of KLK6 mg À1 of total protein to adjust for the amount of tumour tissue extracted.…”

Identification of heat shock protein 90 and other proteins as tumour antigens by serological screening of an ovarian carcinoma expression library

“…The fluorescence was measured with a time-resolved fluorometer, the Cyberfluor 615 Immunoanalyzer (MDS Nordion, Kanata, ON, Canada). The details of this procedure have been published elsewhere (Christopoulos and Diamandis, 1992). All serum samples were analysed in duplicate.…”

Polyvinylamine-streptavidin complexes labeled with a europium chelator: a universal detection reagent for solid-phase time resolved fluorometric applications

“…Precision was Ͻ 10% at PSA concentrations between 5-10,000 ng/L (ten replicates per concentration) and was 27% at a PSA concentration of 1 ng/L. When we compared this assay with assays using either enzymatically amplified time-resolved fluorometry (8,11) or enzymatically triggered chemiluminescence with dioxetane substrates (12,13) (same reagents and conditions), we obtained comparable results (similar signal to noise ratios and detection limits) but linearity was extended by one decade with the present method.…”

“…We have previously described highly sensitive detection technologies based on enzymatically amplified time-resolved fluorescence using terbium chelates as labels (8). Furthermore, enzymaticallytriggered chemiluminescence is among the most sensitive detection techniques (16 -18).…”

“…Time-resolved fluoroimmunoassays (TR-FIA) using europium and other lanthanide complexes as fluorescent labels have attracted wide interest (2)(3)(4)(5)(6)(7)(8). A Eu 3ϩ chelator, 4,7-bis(chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid (BCPDA) has been synthesized and extensively used (4,9).…”