Improved detection by time-resolved fluorometry of specific DNA immobilized in microtiter wells with europium/metal-chelator labelled DNA probes.

Recent ly , a cloned DNA probe l a b e l l e d wi th m e t a l c h e l a t o r s ( d i ethylentriamine pentaacetate, DTPA) was used in a dot-blot hybridization assay for the non-radioactive detection of down to 1 attomol target DNA by europium time-resolved fluorometry (1). We now report modifications of the procedure which led to a more than tenfold increase in sensitivity. A DTPA-labelled, HBV-specific plasmid DNA probe (pKKHBs34) was prepared as described (1). The hybridization and detection procedure was changed in three points: i) Polystyrene microtiter wells (Titertek 1x12 Well Microstrips, Flow Laboratories) instead of ni t rocel lulose f i l ters were used as the solid support. Target DNA was immobilized by UV-light (2). i i ) A lower Eu-concentration (lOuM instead of lOOuM Eu-EDTA) was used for the chelation of the hybrid-bound DTPA ligands after the hybridization reaction, i i i ) Washing conditions after the chelation step were changed to 10nM EDTA, 0.1 M Tris-HCl (pH 7.8) to remove unspecifically bound Eu more efficiently. The samples in the microt i ter s t r ips were di rect ly measured in an "Arcus 1230" timeresolved fluorometer (LKB-Wallac) after releasing the europium ions into an enhancement solution (0.1 M acetate-phthalate buffer, pH 3.2, 0.1% Triton X-100, 15 uM 2-naphtoyltrifluoroacetone, 50 uM t r i n octylphosphinoxid). 0.5 pg (0.1 attomol) of t a rge t | plasmid DNA were clearly detectable Jo (8131 cps against 2135 cps background without DNA). Heterologous DNAs gave ;" only blank signals (lpg calf thymus DNA | | 2354 cps, l^g pBR322 DNA 2844 cps). The «I dose-response curve was linear and proportional (see figure) while in the f i l t e r a s s a y s a tenfold increase in \~ fluorescence required a lOOOfold increase in DNA (1). " " •»•! » ^ P<<«M