Quantification of DNA using the luminescent oxygen channeling assay.

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 (LOCI(TM)) 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.

5-Hydroxytryptamine (5-HT)4 receptors in post mortem human brain tissue: distribution, pharmacology and effects of neurodegenerative diseases.

1. The distribution, pharmacology and effects of neurodegenerative diseases on 5-HT4 receptors in human brain have been characterized in vitro. 2. The 5-HT4 receptor in post mortem human brain tissue was specifically labelled with [3H]-GR 113808. In human putamen, this ligand labelled a homogeneous population of sites, with an apparent affinity (-log Kd) of 10.1 and a density (Bmax) of 5.73 fmol mg-1 tissue. The pharmacology of this site was characterized by use of a series of displacing ligands, and the following rank order of apparent affinities (with mean +/- s.d. -log Ki values in parentheses) was generated: GR113808 (10.05 +/- 0.04) > SDZ 205,557 (8.65 +/- 0.08) > DAU 6285 (7.95 +/- 0.04) > BIMU-1 (7.81 +/- 0.06) > DAU 6215 (7.42 +/- 0.23) > tropisetron (7.39 +/- 0.23) > 5-HT (7.32 +/- 1.00) > BIMU-8 (7.25 +/- 0.04) > (R)-zacopride (5.82 +/- 0.04). The Hill coefficients were not significantly different from unity, consistent with an interaction at a single site. A comparison of the affinities of these compounds with those obtained from guinea-pig striatum indicated no evidence of species differences. 3. The regional distribution of 5-HT4 receptors was assessed by determining the density of binding sites for [3H]-GR 113808. The distribution were as follows (with mean +/- s.d. Bmax values, fmol mg-1 tissue, in parentheses): caudate nucleus (8.7 +/- 1.5), lateral pallidum (8.6 +/- 5.5), putamen (5.7 +/- 3.0), medial pallidum (3.8 +/- 0.9), temporal cortex (2.6 +/- 0.6), hippocampus (2.4 +/- 0.8), amygdala (2.3 +/-1.1), frontal cortex (1.7 +/- 0.5), cerebellar cortex (<1.0). In these studies, the affinities of GR 113808 were not significantly different.4. The density of 5-HT4 receptors selected from regions of post mortem brains of patients with Parkinson's disease, Huntington's disease and Alzheimer's disease were compared to age-matched controls. In Parkinson's disease, there was no significant difference between control or patient values(mean +/- s.d. Bmax values, fmol mg-1 tissue; putamen, control 4.74 +/- 0.07, patient 5.86 +/- 1.48; substantia nigra, control 4.21 +/- 2.56, patient 5.57 +/- 0.10). In Huntington's disease, there was a significant decrease in putamen (control 5.33 +/- 1.08, patient 2.68 +/- 1.08), while in Alzheimer's disease, there was a marked loss of receptors in hippocampus (control 2.34 +/- 0.62, patient 0.78 +/- 0.61), in frontal cortex (control,1.76 +/- 0.19, patient 1.30 +/- 0.22). Receptor density in temporal cortex showed a decrease, but did not achieve statistical significance (control 2.06 +/- 0.21, patient 1.44 +/- 0.64).5. These data suggest a heterogeneous distribution of 5-HT4 receptors in human brain, with high to moderate densities in basal ganglia and limbic structures. These receptors may not be principally co-localized on dopaminergic cell bodies or terminals, given the lack of change observed in Parkinson's disease. The loss of 5-HT4 receptors in the putamen in Huntington's disease raises the possibility of their presence on intrinsic striatal GABAergic or cholinergic neurones. The marked loss of receptors in hippocampal and cortical regions in the brains from patients with Alzheimer's disease is consistent with a role for the 5-HT4 receptor in cognitive processing.

Adsorption losses from urine-based cannabinoid calibrators during routine use.

The major metabolite of cannabis found in urine, 11-nor-delta 9-tetrahydrocannabinol-9-carboxylic acid (delta 9-THC), is the compound most often used to calibrate cannabinoid immunoassays. The hydrophobic delta 9-THC molecule is known to adsorb to solid surfaces. This loss of analyte from calibrator solutions can lead to inaccuracy in the analytical system. Because the calibrators remain stable when not used, analyte loss is most probably caused by handling techniques. In an effort to develop an effective means of overcoming adsorption losses, we quantified cannabinoid loss from calibrators during the testing process. In studying handling of these solutions, we found noticeable, significant losses attributable to both the kind of pipette used for transfer and the contact surface-to-volume ratio of calibrator solution in the analyzer cup. Losses were quantified by immunoassay and by radioactive tracer. We suggest handling techniques that can minimize adsorption of delta 9-THC to surfaces. Using the appropriate pipette and maintaining a minimum surface-to-volume ratio in the analyzer cup effectively reduces analyte loss.