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.

Luminescent oxygen channeling assay (LOCI): sensitive, broadly applicable homogeneous immunoassay method.

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.

Anti-immune complex antibodies enhance affinity and specificity of primary antibodies.

Antibodies have previously been described that enhance the binding of a second antibody to its antigen. The origin of this effect has been variously ascribed to binding to a neodeterminant on the Fc region, to a combined determinant representing portions of the second antibody and the immunogen, and to a ligand-induced conformation of the Fab fragment. This paper describes an antibody that recognizes an immune complex of an antibody to tetrahydrocannabinol (THC). The antibody binds the anti-THC antibody at an epitope recognized by an anti-idiotype antibody that is capable of blocking THC binding. The ability of various THC derivatives to enhance or inhibit binding taken together with equilibria and kinetic data support a model in which the anti-immune complex antibody interacts through adventitious binding to pendant groups on the THC derivatives. This type of interaction offers the opportunity to increase the sensitivity and specificity of immunoassays beyond the limits imposed by normal antibody binding. The implications of these findings with regard to earlier observations of anti-immune complex antibodies are discussed.

Quantitative homogeneous enzyme immunoassays for amitriptyline, nortriptyline, imipramine, and desipramine.

We describe specific EMIT homogeneous enzyme immunoassays for amitriptyline, nortriptyline, imipramine, and desipramine in patients' serum samples. Before analysis, an easily performed extraction step involving the use of 500 microL of sample and a 1-mL disposable column eliminates cross-reacting polar metabolites. The range of the standard curve for the first three drugs is 25 to 250 micrograms/L, and for desipramine is 50 to 500 micrograms/L. Within-run and between-run CVs are less than 10% throughout the range of the assays. Results for patients' samples obtained by this method and by "high-performance" liquid chromatography compare well, showing a slope range of 0.94-1.04 and correlation coefficients ranging from 0.93 to 0.96, depending on the assay.

The chemistry and conformational and biological analysis of vitamin D3, its metabolites and analogues.

The chemical properties, stereochemical relationships and solution conformation, as assessed in part by proton NMR spectroscopy, for vitamin D3, its major metabolites [including 1alpha,25-(OH)2D3, its hormonally active form] and a number of A-ring and side chain analogues are evaluated and discussed in relation to their biological activity. In particular the relative ability of many of these seco-steroids to compete both with 25-OHD3 for its chick serum binding protein and 1alpha,25-(OH)2-D3 for its chick intestinal cytosol-chromatin receptor system was quantitated, in vitro. Further, the relative effectiveness of all these metabolites and analogues to mediate in vivo intestinal calcium absorption and bone calcium mobilization was determined. Collectively these chemical and biological studies constitute a "systems analysis" of the various steroid structural parameters both required and tolerated by the multi-stepped endocrine system associated with the biological actions of vitamin D.

Vitamin D in solution: conformations of vitamin D3, 1alpha,25-dihydroxyvitamin D3, and dihydrotachysterol3.

Solution conformations of the A and seco B rings of vitamin D(3), 1(alpha), 25-dihydroxyvitamin D(3), 1(alpha)-hydroxyvitamin D(3), and dihydrotachysterol(3) have been established by high resolution, 300-megahertz proton magnetic resonance spectroscopy. The A ring of these steroids is dynamically equilibrated between two chair conformers. For vitamin D(3), 1(alpha)-hydroxyvitamin D(3), and 1(alpha),25-dihydroxyvitamin D(3) the relative proportions of the two conformers are 1 : 1, whereas dihydrotachysterol3 exists principally as only one conformer. Thus, the substituent groups on the A ring may be either equatorially or axially oriented, and suggests a refinement of the existing topological model for vitamin D hormonal activity.