A method for monitoring formation of latex particle pairs by chemilumnescence is described. Molecular oxygen is excited by a photosensitizer and an antenna dye that are dissolved in one ofthe particles. 1A02dlffse to the second particle and initiates a hi quantum yield chemilu ent reaction of an olefin that is dissolved in it. An alternative homogeneous method utilizes immunochemical aggregation of ligand-or receptor-labeled particles (latex agglutination) (3). Detection of agglutination by conventional light scattering requires formation of large aggregates and has limited sensitivity. Low-angle light scattering measurements provide higher sensitivity but require rigorously exclusion of adventitious particles (4,5).In the present study, nonenzymatic channeling of 1.02 is used to provide exquisitely sensitive real-time monitoring of particle-particle interactions. The prepared by adding concentrated ammonia to 500-kDadextran (Pharmacia) that had been activated by reaction with epichlorohydrin and Zn(BF4)2. The product was purified by precipitation with methanol. Fluorescein-biotin (Fl-biotin) was prepared by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC) coupling of Fl-5'-COOH to 1,12-diamino-4,9-dioxadodecane followed by reaction with biotinyl-6-aminohexanoic acid N-hydroxysuccinimide ester (biotin-LC-NHS, Pierce). Bis-(6-hydroxyhexyl) disulfide and 6-amino-

Luminescent oxygen channeling assay (LOCI) is a homogeneous immunoassay method capable of rapid, quantitative determination of a wide range of analytes--including high and very low concentrations of large and small molecules, free (unbound) drugs, DNA, and specific IgM. Assays have been carried out in serum and in lysed blood. Reliable detection of 1.25 microU/L thyrotropin (TSH) and 5 ng/L hepatitis B surface antigen (HBsAg) corresponds to detection limits approximately 3- and approximately 20-fold lower, respectively, than those of the best commercially available assays. An assay of chorionic gonadotropin is capable of quantification over a 10(6)-fold range of concentrations without a biphasic response. Latex particle pairs are formed in the assay through specific binding interactions by sequentially combining the sample and two reagents. One particle contains a photosensitizer, the other a chemiluminescer. Irradiation causes photosensitized formation of singlet oxygen, which migrates to a bound particle and activates the chemiluminescer, thereby initiating a delayed luminescence emission. Assay times range from 1 to 25 min.

Gene expression analysis has become an invaluable tool for understanding gene function and regulation. However, global expression analysis requires large RNA quantities or RNA preamplification. We describe an isothermal messenger RNA (mRNA) amplification method, Ribo-SPIA, which generates micrograms of labeled cDNA from 5 ng of total RNA in 1 day for analysis on arrays or by PCR quantification. Highly reproducible GeneChip array performance (R2 > 0.95) was achieved with independent reactions starting with 5-100 ng Universal Human Reference total RNA. Targets prepared by the Ribo-SPIA procedure (20 ng total RNA input) or the Affymetrix Standard Protocol (10 microg total RNA) perform similarly, as indicated by gene call concordance (86%) and good correlation of differential gene expression determination (R2 = 0.82). Accuracy of transcript representation in cDNA generated by the Ribo-SPIA procedure was also demonstrated by PCR quantification of 33 transcripts, comparing differential expression in amplified and nonamplified cDNA (R2 = 0.97 over a range of nearly 10(6) infold change). Thus Ribo-SPIA amplification of mRNA is rapid, robust, highly accurate and reproducible, and sensitive enough to allow quantification of very low abundance transcripts.

Background: Simplified and cost-effective methods for the detection and quantification of nucleic acid targets are still a challenge in molecular diagnostics. Methods: Luminescent oxygen channeling assay (LOCITM) latex particles can be conjugated to synthetic oligodeoxynucleotides and hybridized, via linking probes, to different DNA targets. These oligomer-conjugated LOCI particles survive thermocycling in a PCR reaction and allow quantified detection of DNA targets in both real-time and endpoint formats. The endpoint DNA quantification format utilized two sensitizer bead types that are sensitive to separate illumination wavelengths. These two bead types were uniquely annealed to target or control amplicons, and separate illuminations generated time-resolved chemiluminescence, which distinguished the two amplicon types. Results: In the endpoint method, ratios of the two signals allowed determination of the target DNA concentration over a three-log range. The real-time format allowed quantification of the DNA target over a six-log range with a linear relationship between threshold cycle and log of the number of DNA targets. Conclusions: This is the first report of the use of an oligomer-labeled latex particle assay capable of producing DNA quantification and sequence-specific chemiluminescent signals in a homogeneous format. It is also the first report of the generation of two signals from a LOCI assay. The methods described here have been shown to be easily adaptable to new DNA targets because of the generic nature of the oligomer-labeled LOCI particles.

No abstract

No abstract

The system described here can distinguish between single and agglutinated erythrocytes by use of a non-flow fiber optic fluorometer. The method is capable of detecting cell-surface antigens and antibodies to cell-surface antigens present in blood. Significant features include high-efficiency fluorescent dyes that intercalate into cell membranes, a stretching membrane for transport and mixing of samples, charged colloidal magnetite for magnetic separation of erythrocytes, and an immersible fiber optic probe for measuring fluorescence associated with cells in a 1-nL volume of a bulk solution. We describe the application of the system to automation of ABO/Rh grouping and antibody screening.

No abstract