Streptavidin-coated spot surfaces for sensitive immunoassays using fluorescence surface readout.

Direct measurement of time-resolved fluorescence from a washed surface of an immunoassay well constitutes an advantage compared with label development options involving signal generation in solution. Epi-fluorometric detection collects the signal from only a small part of the microtiter well's bottom surface and it is inadequate for the optimal assay sensitivity when using binding surfaces introduced by large coating volume. This study reports on the use of streptavidin-coated spots intended to condense the binding of the labeled antibodies to coincide with the excitation beam. The spots were generated in special microtiter wells containing 2.5-mm, 3.5-mm, and 4.5-mm diameter indentations by adsorption from liquid droplets containing either native (SAv) or modified high-capacity (GA-SAv) streptavidin. The SAv-coated and GA-SAv-coated spots exhibited maximum Eu-biotin binding densities of 0.080 and 0.47 pmol/mm(2), respectively. A sandwich-type immunoassay of thyroid-stimulating hormone (TSH) provided a fivefold to sixfold increase in the signal-to-background ratios of the spot assay and an equivalent improvement in the detection limit (DL < 0.01 mU/L) compared with a reference assay.

An automated PCR platform with homogeneous time-resolved fluorescence detection and dry chemistry assay kits.

We have developed a novel instrument platform, GenomEra, for small-scale analysis of nucleic acids. The platform combines a rapid thermal cycler, an integrated time-resolved fluorescence measurement unit, and user-friendly software for the analysis of results. Disposable low-cost plastic reaction vessels are designed specifically for the instrument and contain all of the assay-specific reagents in dry form. The appropriate assay protocol is specified on barcodes printed under the vessels and is automatically initiated by the software. Detection is based on the use of sequence-specific probes labeled with intrinsically fluorescent europium or terbium chelates and complementary quencher probes, which enable sensitive, homogeneous closed-tube assays without the risk of carryover contamination. The detection limit of the instrument (background + 3 SD) is approximately 20 pmol/L for both chelates with a dynamic range of nearly four orders of magnitude. The functionality of the platform is demonstrated with a dual-label homogeneous polymerase chain reaction (PCR) assay for the detection of Salmonella using a Magda CA Salmonella assay kit. An internal amplification control is included in each reaction to eliminate false negative results caused by PCR inhibition. Qualitative assay results are automatically interpreted by the software and are available 45 min after sample addition.