Abstract: Methods of detecting antibodies to one or more glycosphingolipid(s) of interest in a sample are disclosed which comprise using a solid-phase reactant having carbonyl groups attached thereon, and the glycosphingolipid(s) of interest linked to the solid-phase reactant by an amide bond between an amino group of the glycosphingolipid of interest and a carbonyl group attached to the solid-phase reactant. The methods of detecting antibodies to glycosphingolipid(s) of interest can be used in methods of diagnosing autoimmune diseases in an individual.

Abstract: Methods and compositions are provided for determining the potential of a membrane. In one aspect, the method comprises: (a) introducing a first reagent comprising a hydrophobic fluorescent ion capable of redistributing from a first face of the membrane to a second face of the membrane in response to changes in the potential of the membrane, as described by the Nernst equation, (b) introducing a second reagent which labels the first face or the second face of the membrane, which second reagent comprises a chromophore capable of undergoing energy transfer by either (i) donating excited state energy to the fluorescent ion, or (ii) accepting excited state energy from the fluorescent ion, (c) exposing the membrane to radiation; (d) measuring energy transfer between the fluorescent ion and the second reagent, and (e) relating the energy transfer to the membrane potential. Energy transfer is typically measured by fluorescence resonance energy transfer.

Abstract: In a homogeneous immunoassay, fluorophore-conjugated lipopolysaccharide derived bacterial antigens are reacted with antibodies specific for the antigens. Quantitative detection of the formation of an immune complex is obtained by measuring the change in fluorescence polarization after complex formation. The reaction occurs quickly (less than two minutes to equilibrium), and involves the addition of only one reagent to a diluted serum specimen. The absence of a solid phase separation step eliminates false positive results and increases throughput.

Abstract: A novel cyanine dye having the formula is useful for labeling biological and nonbiological molecules.

Abstract: The present invention provides a method for identifying a test compound that binds to a target species. The method includes: incubating at least one test mixture under isothermal denaturing conditions, each test mixture comprising at least one test compound, and at least one target species, wherein the isothermal denaturing conditions are effective to cause at least a portion of the target species to denature to a measurable extent; detecting a denaturation signal of each target species in the presence of the at least one test compound by a change in the diffusion properties of the target molecule using fluorescence correlation spectroscopy; and comparing the denaturation signal of each target species in the presence of at least one test compound with a denaturation signal of the same target species in the absence of the at least one test compound under the same isothermal denaturing conditions.

Abstract: The preparation of phosphorescent metalloporphyrin labelling reagents and their use for preparation of phosphorescent conjugates with biomolecules. The labelling reagents obtainable are water soluble monofunctional derivatives of Pt- and Pd-coproporphyrins, where the term “monofunctional” refers to the number of reactive groups in the porphyrin moiety.

Abstract: The invention provides heterobifunctional cross-linking reagents and methods of using the cross-linking reagents. The cross-linking reagents of the invention combine a nucleophilic hydrazide residue with an electrophilic maleimide residue allowing coupling of aldehydes to free thiols. In the methods of the invention, human immunodeficiency virus (HIV) infected cells can be detected using conjugates that include CD4 molecules conjugated to detectable markers via the disclosed cross-linking reagents.

Abstract: The invention describes reactive dyes having an alkyl spacer attached via a sulfonamide bond to a sulforhodamine 101 fluorophore, and a variety of useful conjugates prepared therefrom. The increased length of the covalent linkage due to the alkyl spacer results in dye-conjugates having a number of surprisingly advantageous properties relative to previous sulforhodamine 101-labeled conjugates, including enhanced solubility and increased fluorescence. The reactive dyes of the present invention are more stable than the known compound sulforhodamine 101 sulfonyl chloride. Novel reactive dyes are described for selective modification of groups other than amines, including thiols and photoreactive derivatives.

Abstract: The present invention describes novel methods for making a substrate for selective cell patterning, and the substrates themselves, wherein the method comprises contacting reactive hydroxyl groups on the surface of a substrate with a hydroxyl-reactive bifunctional molecule to form a monolayer, and using stencils to deposit cell repulsive or cell adhesive moieties in controlled locations on the cell culture substrate. Methods comprising selective differentiation of stem cells to create tissue specific and organ-specific cell substrates, as well as the cell substrates themselves are also provided.

Abstract: To improve the detection of LSD, and LSD metabolites in biological samples, antibodies are raised to 2-oxo-3-hydroxy-LSD conjugated to a protein carrier. Selected antibodies are matched with an immunoassay reagent in which the 2-oxo-3-hydroxy-LSD is conjugated in the same position to a labeling or separation means. The set of reagents can be used in immunoassays for detecting or confirming the presence of LSD or LSD metabolites in a sample potentially containing interfering substances.

Abstract: Disclosed is a fluorescent conjugate comprising a biological reagent, a fluorescent molecule, and a means for impeding phototoxic degradation of the biological reagent. The impeding means can be a cross-linking substance having a long molecular distance, whereby the cross-linking substance links the fluorescent molecule and the biological reagent; a quencher of singlet oxygen; a quencher of a free radical; or a combination thereof. Also disclosed is a solution comprising a fluorescent conjugate of a biological reagent and a fluorescent molecule together with an oxygen depleting system.

Abstract: The invention provides methods, compositions, and apparatus for performing sensitive detection of analytes, such as biological macromolecules and other analytes, by labeling a probe molecule with an up-converting label. The up-converting label absorbs radiation from an illumination source and emits radiation at one or more higher frequencies, providing enhanced signal-to-noise ratio and the essential elimination of background sample autofluorescence. The methods, compositions, and apparatus are suitable for the sensitive detection of multiple analytes and for various clinical and environmental sampling techniques.

Abstract: Sensitive methods using a variety of biomarkers in oral saliva of humans and animals to detect, measure, and quantify the presence of infectious and non-infectious agents and the functional status of living tissues in both (1) biomedical and (2) environmental applications. With the in vivo biomedical applications, saliva samples are taken from human or animal subjects and biomarkers, such as enzyme or antibody levels, are measured. The extent of exposure to an agent is measured by the presence of specific chemical or biological constituents, by the degree of the inhibition of enzymes, or by the changes in the amount of the biochemicals in saliva. With the in vitro environmental applications, enzymes or other biochemicals from animal or human saliva can be used to monitor the presence of toxic or reactive agents in tissue samples, urine, feces, milk, air, water, soil, or plants. The amount of toxicant in samples is estimated from a standard curve for that agent.

Abstract: The present invention provides an immunoassay method for the highly sensitive detection of amphetamines, methamphetamines, and methylenedioxy designer amphetamines in urine samples. Commercially available reagents for the determination of amphetamines and methamphetamines are used with a calibrator comprising a known amount of a substance selected from the group consisting of methylenedioxy designer amphetamines.

Abstract: An immunoassay for selectively measuring human C-peptide as well as a kit therefor is disclosed. In the method, human C-peptide contained in a sample, a first anti-human C-peptide antibody, and a second anti-human C-peptide antibody which is immobilized on a solid support are reacted to form an immune complex among these three components. The formed immune complex is separated from the non-reacted antibodies and sample; and then the separated immune complex is quantified. The first antibody recognizes an epitope existing in the region from 1st to 16th amino acid residue from the N-terminal of the human C-peptide, and the second antibody recognizes an epitope existing in the region from 1st to 16th amino acid residue from the N-terminal of human C-peptide; with the proviso that the first and second antibodies do not recognize the same epitope so that they can simultaneously bind to said human C-peptide.

Abstract: The present invention relates to a method of detecting whether a target animal is Bovine Viral Diarrhea Virus (BVDV) positive or negative by determining whether a gp48 protein-specific reagent binds to a gp48 Bovine Viral Diarrhea Virus protein or protein fragment, which retains antigenic specificity, from a target animal's hair sample.

Abstract: The present invention provides luminescent lanthanide metal chelates comprising a metal ion of the lanthanide series and a complexing agent comprising at least one phthalamidyl moiety. Also provided are probes incorporating the phthalamidyl ligands of the invention and methods utilizing the ligands of the invention and probes comprising the ligands of the invention.

Abstract: Use of pregnancy-associated plasma protein-A as a marker for inflammatory conditions, and in particular, for acute coronary syndromes is described.

Abstract: The invention concerns a method for the determination of an analyte in which rheumatoid factors or rheumatoid-factor-like substances are added as an interference reducing reagent to reduce of avoid a hook effect. The invention in addition concerns suitable reagent kits for carrying out the method.

Abstract: Disclosed are fluorescent energy transfer dyes which are capable of moving between a more stacked configuration to exhibit fluorescent quenching and a more spaced configuration to exhibit fluorescence can be conjugated to a peptide epitope for use in the detection of an unknown antibody in bulk solution. The resulting labeled peptide reagent can be used in an immunoassay procedure by placing it in bulk solution along with the unknown antibody to be detected. When the antibody binds to the peptide epitope, the pair of dyes carried by the peptide epitope will have their configuration altered from a stacked to an unstacked configuration and will exhibit a fluorescent increase in response thereto.

Abstract: This invention provides a method for measuring an antigen concentration in a sample, which comprises: preparing VH-domain polypeptide and VL-domain polypeptide of an antibody specific to the antigen; labeling one of the polypeptides with a reporter molecule to form labeled polypeptides, and immobilizing the other polypeptide onto solid-phase to form immobilized polypeptides; contacting the antigen-containing sample and the labeled polypeptides with the solid-phase; and measuring the reporter molecule of the labeled polypeptides bound to the immobilized polypeptides. The present invention permits simpler and quicker sandwich ELISA for measurements of an antigen concentration in high sensitivity.

Abstract: The present invention provides low molecular weight fluorescent labeling complexes with large wavelength shifts between absorption of one dye in the complex and emission from another dye in the complex. These complexes can be used, for example, for multiparameter fluorescence cell analysis using a single excitation wavelength. The low molecular weight of the complex permits materials labeled with the complex to penetrate cell structures for use as probes. The labeling complexes are synthesized by covalently attaching through linkers at least one cyanine fluorochrome to another low molecular weight fluorochrome to form energy donor-acceptor complexes. Resonance energy transfer from an excited donor to fluorescent acceptor provides wavelength shifts up to 300 nm. The fluorescent labeling complexes preferably contain reactive groups for the labeling of functional groups on target compounds, such as derivatized oxy and deoxy polynucleic acids, antibodies, enzymes, proteins and other materials.

Abstract: The present invention is directed to a fluorescence polarization immunoassay for barbiturates, to the various components needed for preparing and carrying out such an assay, and to methods of making these components. Specifically, tracers, immunogens and antibodies are disclosed, as well as methods for preparing them and a reagent kit containing them. The tracers and the immunogens are made from substituted barbiturate compounds. A fluorescein moiety is included in the tracer, while a poly(amino acid) forms a part of the immunogen. The assay is conducted by measuring the degree of polarization retention of plane—polarized light that has been passed through a sample containing antiserum and tracer.

Abstract: This invention relates to novel tracers and their synthesis and use in an immunoassay for the detection of controlled drugs such as amphetamine (APM), methamphetamine (MAPM) and their derivatives, in a biological or aqueous sample. In particular, this invention provides methods for synthesizing novel tracers in which a non-controlled substance is both the starting material in tracer synthesis and the binding site on the resulting novel tracer for the antibody, thereby eliminating the necessity of using controlled substances as starting materials. In addition, the novel tracers of the present invention can be used as an analyte analog in an immunoassay, such as a continuous flow displacement immunoassay. It was unexpectedly discovered that the novel tracers of the present invention substantially improve the performance of the continuous flow displacement immunoassay as compared with conventionally designed tracers.

Abstract: The present invention provides novel 1,2-dioxetanes derived from spiro-fused ketones with or without &pgr;-electrons in the ring or with carbon-carbon double bond(s) in the spiro-fused ring. Additionally, these new dioxetanes have electron donating or withdrawing groups at the four-membered peroxide ring to render these dioxetanes active at all sites.

Abstract: Various classes of dyes are provided having acid, ester or amide groups for covalent linking to biomolecules. The dyes may be prepared by use of a compound of formula (I) where R1 comprises a linker and a carboxy including acid, salt, ester including N-hydroxysuccinimide, activated ester or amide group; R2, R3, R4 and R5 are H, C1-C10 alkyl or aralkyl or a group to modify solubility or electronic or spectral properties or a functional linking group: or R4-R5 and/or R2-R4 and/or R2-R3 are linked to form an extended ring system; and R6 is H or CHO or NO.

Abstract: The present invention is a method and kit for analyzing a sample, preferably a liquid environmental sample, which may comprise a toxic xenobiotic element or compound, i.e., an environmental pollutant. The method and kit preferably utilize a portion of a molecule comprising a peptide zinc binding domain, known as a zinc finger.

Abstract: The present invention features calcilytic compounds. “calcilytic compounds” refer to compounds able to inhibit calcium receptor activity. Also described are the use of calcilytic compounds to inhibit calcium receptor activity and/or achieve a beneficial effect in a patient; and techniques which can be used to obtain additional calcilytic compounds.

Abstract: The invention concerns the stabilization and amplification of electrochemiluminescence signals in detection methods.

Abstract: A method for determining a base sequence of a nucleotide strand in a sample utilizes a probe including a fluorescent metal-ligand complex coupled to a first oligonucleotide having a sequence complementary to the first fragment of the strand. The first mixture is exposed to an exciting amount of radiation, and the fluorescence of the metal-ligand complex is detected. The first base sequence of the first fragment is identified based on fluorescence of the metal ligand complex. A second probe differing from the first by at least one base is provided. A second base of the second fragment is identified based on the fluorescence of the metal-ligand complex of the second probe. The second base sequence is compared to the first base sequence to identify a difference between the first and second sequences to determine a base sequence of the nucleotide strand.

Abstract: The invention relates to a novel competitive or non-competitive immunoassay comprising the steps of immobilizing an assay specific component on a solid phase in a reaction well, adding a labelled assay specific component, drying said components, adding the sample containing the marker to be analyzed, allowing the marker to react with the assay specific components, and detecting the signal from the label. Furthermore, the invention relates to a device useful in carrying out the novel assay.

Abstract: A process and a kit are provided for detecting differences in two or more samples of protein, including proteins bearing post-translational modifications and peptides. Proteins are prepared, for example, from each of a different group of cell samples or body fluid samples to be compared. Each protein extract is labeled with a different one of a luminescent dye from a matched set of dyes. The matched dyes have generally the same ionic and pH characteristics but emit light at different wavelengths to exhibit a different color upon luminescence detection. The labeled protein extracts are mixed together and separated together by electrophoresis or a chromatographic method. The separation is observed to detect proteins unique to one sample or present in a greater ratio in one sample than in the other. Those unique or excess proteins will fluoresce the color of one of the dyes used. Proteins common to each sample migrate together and fluoresce the same.

Abstract: The invention provides fluorescent analogues of thyroid hormones T3 and T4. Also provided are assays for thyroid hormones utilizing the compounds of the invention. The assays of the invention are in both one-step and two-step formats and allow the determination of both T3 and T4 concentration in a sample.

Abstract: Histamine may be quantitatively measured by performing the following steps. First, an oocyte that expresses histamine receptors is held in a recess formed at the bottom of a vessel. Then, first and second electrodes are inserted into the oocyte. Subsequently, the membrane potential of the oocyte is measured by using the first electrode to stabilize this membrane potential at a predetermined level by driving a current through the second electrode using circuitry for clamping the membrane potential of the oocyte. A sample is then infused into a fine reacting tube having an antigen immobilized on its inner surface together with some buffer solution to promote a histamine releasing reaction. The solution containing histamines that is released in the fine reacting tube is transferred to the vessel to make contact with the oocyte in the vessel.

Abstract: The present invention provides systems, methods, screens, reagents and kits for optical system analysis of cells to rapidly determine the distribution, environment, or activity of fluorescently labeled reporter molecules in cells for the purpose of screening large numbers of compounds for those that specifically affect particular biological functions.

Abstract: We have identified and purified to homogeneity from lymphatic tissues a 8.4 kDa immunophilin that specifically and avidly binds the immunosuppressant drugs FK-506 (Kd=0.8 nM) and rapamycin (Kd=0.08 nM) and their pharmacologically active metabolites and derivatives, but does not bind cyclosporin A. The isolated 8.4 kDa protein appears to be identical to authentic human and bovine ubiquitins in all measured respects (partial amino acid sequence, molecular weight, binding constants, binding specificity, biochemical aspects, and utility as the protein binding reagent in binding assays for immunosuppressant drugs in fluid samples, including patient blood). The availability of commercial quantities of human recombinant ubiguitin removes a supply barrier to the use of immunophilin protein binding assays for the estimation of FK-506, rapamycin and pharmacologically active metabolites and derivatives in the clinical setting.

Abstract: A method of measuring the amount of cyclosporin in a sample suspected of containing cyclosporin is disclosed. A method of inactivating interfering cross-reactive material in an assay for measuring the amount of cyclosporin in a sample suspected of containing cyclosporin is also disclosed. Compositions wherein cyclosporin is conjugated to an immunogenic carrier or a label, optionally through a linking group, at an alanine nitrogen atom of the cyclic backbone of cyclosporin are also disclosed. Compositions wherein atiocyclosporin is conjugated, optionally through a linking group, to an immunogenic carrier or a label are also disclosed. Where cyclosporin is conjugated to an immunogenic carrier, the conjugates may be used as immunogens for the preparation of antibodies which are capable of recognizing cyclosporin.

Abstract: Bridged fluorescent dyes of the cyanine and squaraine families are disclosed. The dyes are useful as markers in assay techniques and offer advantages of undergoing excitation at a common wavelength but emitting at structure dependent different wavelengths.

Abstract: The invention provides methods, compositions, and apparatus for performing sensitive detection of analytes, such as biological macromolecules and other analytes, by labeling a probe molecule with an up-converting label. The up-converting label absorbs radiation from an illumination source and emits radiation at one or more higher frequencies, providing enhanced signal-to-noise ratio and the essential elimination of background sample autofluorescence. The methods, compositions, and apparatus are suitable for the sensitive detection of multiple analytes and for various clinical and environmental sampling techniques.

Abstract: The quenching compounds of the invention are nitrogen-substituted xanthenes that are substituted by one or more aromatic or heteroaromatic quenching moieties. The quenching compounds of the invention exhibit little or no observable fluorescence and efficiently quench a broad spectrum of luminescent compounds. The chemically reactive quenching compounds possess utility for labeling a wide variety of substances, including biomolecules. These labeled substances are highly useful for a variety of energy-transfer assays and applications.

Abstract: The present invention provides a novel chemiluminescent reagent producing chemiluminescence in the presence of hydrogen peroxide, extent of which depends on peroxidase concentration, chemiluminescent analysis method using the same, in particular useful for detection and quantitative analysis of various types of materials by measuring peroxidase enzyme activity or enzyme immunoassay with peroxidase enzyme as the marker. More particularly, the present invention provides a chemiluminescent reagent containing, as the major ingredients, a charge-transferring complex of N,N′-disubstituted-9,9′-bisacridinium salt and N,N-disubstituted carboxylic amide compound; chemiluminescent reagent containing further containing a specific aminoalcohol compound, in addition to the above; and method for measuring peroxidase activity at a high sensitivity in the presence of a peroxide, using the above chemiluminescent reagent.

Abstract: Disclosed herein are methods and reagent kits for the quantitative in vitro determination of the functional determination of multi-drug resistance in cells, as well as for the clinical screening of potential modulators of multi-drug resistant transport activity in cells. The method of the invention is based on the measurement of the accumulation rate of free calcein within the cells of the specimen (advantageously by fluorescence measurement), after exposing the cells in vitro to a cell permeable form of calcein that is a good substrate for MDR proteins present in the sample. The cell permeable form of calcein is converted within the cell by intracellular enzymes to free calcein. Comparison of free calcein accumulation in the presence and absence of a potential inhibitor of transport activity permits the rapid screening of such inhibitors.

Abstract: Disclosed is a modular fluorescence sensor having the following general formula: where Fl is a fluorophore, N is a nitrogen atom, Bd1 and Bd2 are independently selected binding groups, Sp is an aliphatic spacer, and An is an anchor group for attaching the sensor to solid substrates. n=1 or 2, m=1 or 2, x is an integer, and y=1 or 2. The binding groups are capable of binding an analyte molecule to form a stable 1:1 complex. In a preferred embodiment, the Bd1 is R1—B(OH)2 and Bd2 is R2—B(OH)2. R1 and R2 are aliphatic or aromatic functional groups selected independently from each other and B is a boron atom. The present invention also provides methods of synthesizing a modular fluorescence sensor and its use in labeling solid substrates.

Abstract: The present invention relates to compositions comprising novel reduced cortisol conjugates, methods for their preparation and use in immunoassays for cortisol. In another aspect, it relates to conjugates of reduced cortisol as immunogens or haptens for eliciting anti-cortisol or anti-reduced cortisol antibodies.

Abstract: The inventors herein disclose new heterobifunctional chromophores that are capable of coupling with two distinct moieties. One moiety may be either a signal-enhancing agent or a blocking agent. The second moiety may be one member of a specific binding pair. The invention is based in part on the surprising result that when a chromophore is used as a “cross-linker” between a signal-enhancing agent and a member of a binding pair (essentially being buried between the two), the signal of the chromophore is not quenched. This arrangement, wherein the chromophore acts simultaneously as a cross-linker and a detectable compound, provides significant advantages over previously known compounds since the chromophore is sterically hindered from interacting non-specifically with substances present in the test systems. Moreover, the chromophore can be used as a cross-linker with little or no loss of detectable signal.

Abstract: A method and library for determining the sequence of monomers in a polymer which is complementary to a receptor. The method provides for formation of pooled (6) and separate (10, 12) products. Separate products are subjected only to subsequent pooled coupling steps. Each pooled product is subsequently divided for formation of pooled and separate products. The resulting polymer library includes groups of polymer products. A first group of products (42) is used to identify the monomer at a first location in a polymer that is complementary to a receptor. A second group of products (44) is used to identify the monomer at a second location in a polymer that is complementary to a receptor.

Abstract: The present invention provides internally calibrated competitive assays for use on a solid support. Additionally, the invention provides a method of using such assays.

Abstract: The present invention is directed to novel tricyclic antidepressant drug derivatives synthesized for covalent attachment to proteins or polypeptide antigens for use in the preparation of antibodies or receptors to tricyclic antidepressant drugs and tricyclic antidepressant metabolites. The new derivatives are characterized by a saturated double bond on the amitriptyline portion of the molecule and are represented by the structure where R1 is a saturated or unsaturated, substituted or unsubstituted, straight or branched chain of 0-10 carbon or heteroatoms, X is a linker group consisting of 0-2 substituted or unsubstituted aromatic rings, and Y is an activated ester or NH—Z, where Z is a poly(amino acid).

Abstract: 4-(4-hydroxystyryl) pyridine-containing compounds are disclosed as substrates for use in assays. Also, a method for detecting or quantitating an analyte is provided which includes reacting an enzyme with a 4-(4-hydroxystyryl) pyridine-containing compound to form a reactive intermediate wherein the reactive intermediate deposits covalently on a surface by binding to a receptor, wherein deposited reactive intermediates either directly or indirectly through a label generate a signal which can be detected or quantitated.

Abstract: A method and compositions for the detection and/or quantification of an analyte through the use of a plurality of labeled probes, with two or more said probes targeted to different regions of said analyte. In specific embodiments, the labels are separately distinguishable, and/or are present at different specific activities on the labels.