Abstract: New methods are provided for the post-genomic era that will permit the analysis of the dynamic expression and localization of gene products in living cells. Herein we propose the development of such a method from a bioorganic approach involving organic synthesis and protein engineering. Specifically, novel compounds bearing two maleimide groups attached directly to fluorescent cores will be prepared, whose latent fluorescence is quenched until their maleimide groups undergo a specific thiol addition reaction. Complementary a-helical proteins are designed bearing two cysteine residues appropriately positioned to react with our novel fluorogens. Genetically fusing our helical probe peptides to test proteins of interest, we can selectively label the target sequence in living cells with our small synthetic fluorogenic molecules. The scope of this technique is described in the context of studying protein localization and protein-protein interactions in living cells.

Abstract: The invention provides methods and compositions for simultaneously detecting the activation state of a plurality of proteins in single cells using flow cytometry. The invention further provides methods and compositions of screening for bioactive agents capable of coordinately modulating the activity of a plurality of proteins in single cells. The methods and compositions can be used to determine the protein activation profile of a cell for predicting or diagnosing a disease state, and for monitoring treatment of a disease state.

Abstract: The present invention relates to novel chemiluminescent compounds, to a method for synthesizing these compounds, to derivatives and conjugates comprising these compounds, to the use of these compounds or conjugates thereof in chemiluminescence based assays, especially in immunoassays.

Abstract: The present invention relates to a temperature-stable labeling reagent of formula (0): in which: R1 represents H or an alkyl, aryl or substituted aryl group, R2 represents a detectable marker or at least two detectable markers interlinked by at least one multimeric structure, L is a linker arm comprising a linear chain of at least two covalent bonds and n is an integer equal to 0 or 1, R3 and R4 represent, independently of one another: H, NO2, Cl, Br, F, I, R2-(L)n-Y—X—, OR, SR, NR2, R, NHCOR, CONHR, COOR, —CO—NH—(CH2)3—(O—CH2—CH2)3—CH2—NH—R2, —CO—NH—(CH2)3—(O—CH2—CH2)4—CH2—NH—R2 with R=alkyl or aryl, A is a linker arm comprising at least one covalent double bond enabling the conjugation of the diazo function with the aromatic ring and u is an integer between 0 and 2, preferably 0 or 1, —Y—X— represents —CONH—, —NHCO—, —CH2O—, —CH2S—, —Z— represents —NH—, —NHCO—, —CONH— or —O—, m is an integer between 1 and 10, preferably between 1 and 3, and p is an integer between 1 and 10, preferably between 1 and 3

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: Methods using chemiluminescent label compounds and chemiluminescent labeled conjugates are provided. The compounds comprise an acridan ring bearing an exocyclic ketene dithioacetal group and further contain a labeling substituent which permits attachment to compounds of interest. The novel chemiluminescent compounds and labeled conjugates are convenient to prepare, are highly stable, and generate chemiluminescence rapidly on demand. The compounds and conjugates are useful in assays of an analyte in a sample and in assays employing labeled specific binding pairs.

Abstract: Generally, the present invention relates to lamotrigine analogs that have substituents at the triazine 3-position and on the benzene 4-position and 5-position. The lamotrigine analogs can include immunogenic moieties that can be used to prepare anti-lamotrigine antibodies, or antigenic moieties that can be used in immunodiagnostic assays for lamotrigine. Also, the lamotrigine analog can include tracer moieties for detecting the presence or amount of the analog during an immunodiagnostic assay. Additionally, the lamotrigine analogs can be used in immunodiagnostic assays to compete with lamotrigine for binding with anti-lamotrigine antibodies.

Abstract: Disclosed are high throughput assay systems and methods for identifying agents that alter the level of expression of proteins in mammalian cells, particularly integral membrane proteins.

Abstract: Methods for differentially identifying cells in an instrument employ compositions containing a combination of selected antibodies and fluorescent dyes having different cellular distribution patterns and specificities, as well as antibodies and fluorescent dyes characterized by overlapping emission spectra which form non-compensatable spectral patterns. When utilizing the compositions described herein consisting of fluorescent dyes and fluorochrome labeled antibodies with overlapping spectra that cannot be separated or distinguished based upon optical or electronic compensation means, a new fluorescent footprint is established. This new fluorescent footprint is a result of the overlapping spectra and the combined cellular staining patterns of the dyes and fluorochrome labeled antibodies chosen for the composition. The new fluorescent footprint results in histogram patterns that are useful for the identification of additional cell populations or subtypes in hematological disease.

Abstract: The invention provides peptide synthons having protected functional groups for attachment of desired moieties (e.g. functional molecules or probes). Also provided are peptide conjugates prepared from such synthons, and synthon and conjugate preparation methods including procedures for identifying optimum probe attachment sites. Biosensors are provided having functional molecules that can locate and bind to specific biomolecules within living cells. Biosensors can detect chemical and physiological changes in those biomolecules as living cells are moving, metabolizing and reacting to its environment. Methods are included for detecting GTP activation of a Rho GTPase protein using polypeptide biosensors. When the biosensor binds GTP-activated Rho GTPase protein, an environmentally sensitive dye emits a signal of a different lifetime, intensity or wavelength than when not bound.

Abstract: Topiramate analogs have substituents at the sulfamate group, 9-position, or 10-position. Topiramate analogs may include immunogenic moieties to prepare anti-topiramate antibodies, or antigenic moieties for immunodiagnostic assays. Also, the topiramate analog can include tracer moieties for detecting the presence or amount of the analog during an immunodiagnostic assay. Additionally, the topiramate analogs can be used in immunodiagnostic assays to compete with topiramate for binding with anti-topiramate antibodies. Such an immunodiagnostic assay can be used for detecting the presence of topiramate in a sample obtained from a subject previously administered topiramate by the following: combining an anti-topiramate antibody and a topiramate analog with a sample to form a first composition; allowing any free topiramate from the sample and the topiramate analog to compete for binding with the antibody; detecting binding between the topiramate analog and the antibody.

Abstract: Disclosed are screening processes for identification of compounds that may inhibit complex formation between C-reactive protein and fibronectin. Compounds identified by the disclosed methods may be utilized to inhibit the target analytes that are known to occur during skin aging as well as during the course of several diseases. Accordingly, inhibitory compounds identified by the disclosed methods may be utilized to prevent and treat damaged tissue, inflammatory conditions, cardiovascular conditions, renal conditions, periodontal conditions and obesity, among other conditions. For example, the compounds identified by the disclosed methods may be utilized to treat tissue that has suffered trauma, e.g., burns or wounds, as well as tissue that is inflamed due to any of several causes.

Abstract: A method for detection and/or quantification of cardiac troponin I (cTnI) in a sample derived from an individual's blood, the method being based on a sandwich immunoassay employing at least two capture antibodies and at least one tracer antibody, in which a first capture antibody is directed to the N-terminal part of cTnI, to the C-terminal part of cTnI or to the part of the cTnI midfragment, which is slightly affected by the interfering factor, and a second capture antibody is directed to another of these parts, and a tracer antibody is directed to the N-terminal part of cTnI, to the C-terminal part of cTnI or to TnC, which is complexed with cTnI.

Abstract: Generally, the present invention relates to topiramate analogs that have substituents at the sulfamate group or at the 9-position or 10-position. The topiramate analogs can include immunogenic moieties that can be used to prepare anti-topiramate antibodies, or antigenic moieties that can be used in immunodiagnostic assays for topiramate. Also, the topiramate analog can include tracer moieties for detecting the presence or amount of the analog during an immunodiagnostic assay. Additionally, the topiramate analogs can be used in immunodiagnostic assays to compete with topiramate for binding with anti-topiramate antibodies.

Abstract: The invention relates to a reagent and a process for the identification and counting of biological cells in a sample. This reagent comprises a cell lysing agent selected from at least one detergent in a concentration capable of specifically lysing a given type of cells in the sample, and a stain capable of marking the intracellular nucleic acids of the remaining unlysed cells. Application in particular for the identification and counting of cells using an automated analysis system based on flow cytometry.

Abstract: Systems for detecting molecular modifications and the presence and/or activity of enzymes and/or other agents involved in facilitating or otherwise regulating such modifications.

Abstract: Methods for detecting and quantifying phosphoinositide lipids in a sample are provided, together with novel SidC- and SdcA-derived polypeptide fragments, or fusion proteins comprising such fragments that may be effectively employed as PI(4)P probes in biochemical and cell biological assays. Applications of the disclosed probes include: (i) detection and quantification of PI(4)P in vitro; and (ii) staining of intracellular compartments in live or fixed eukaryotic cells.

Abstract: A method useful for the enumeration of cell populations in a biological sample includes the steps of reacting in a single reaction mixture a sample, a first antibody labeled with a fluorochrome having a first emission spectrum and an additional antibody. The first antibody binds to an antigenic determinant differentially expressed on leukocytes and non-leukocytes. The additional antibody binds to an antigenic determinant differentially expressed on mature and immature granulocytes or myeloid cells, and is labeled either with the first fluorochrome or an additional fluorochrome having an emission spectrum distinguishable from the first emission spectrum. The reaction mixture can be mixed with a nucleic acid dye having an emission spectrum that overlaps with one of the first or additional emission spectra. The reaction mixture may be treated with a lytic system that differentially lyses non-nucleated red blood cells and conserves leukocytes.

Abstract: Disclosed are methods for detecting antibody in a sample, where the antibody targets an antigen expressed by red blood cells or red blood cell ghosts. Rather than detecting the binding events between a particular antigen antibody pair (as in traditional agglutination based assays) the methods herein allow for multiplexed detection of clinically important allo-immune antibodies to blood group antigens. Specifically the method involves generating fluorescently encoded red blood cells or red blood cell ghosts with known antigen presentation and using them to detect the presence of antibody in serum/plasma with a fluorescent sandwich type immunoassay. The assay results can be read using flow cytometric or fluorescent microscope based imaging techniques.

Abstract: A method for the detection or quantitation of unlabeled target analyte in a biological sample, the method comprising labeling a target analyte for a biological sample suspected of containing unlabeled target analyte with an acridinium compound to form a labeled target analyte and providing the labeled target analyte to the biological sample, or providing the labeled target analyte to the biological sample, wherein the acridinium compound comprises an acridinium nucleus having an electron-donating substituent directly attached to the acridinium nucleus, with the electron-donating substituent attached at the C2 position. Chemiluminescent acridinium compounds useful in the method have emission maxima close to or in the near infrared (NIR) region (>590 nm).

Abstract: A method having clinically sufficient degree of diagnostic accuracy for detecting the presence of coronary artery disease in a human patient from the general population and for distinguishing between the stages of the disease in that patient is disclosed. The stages are, first, the non-acute stage, which is either asymptomatic coronary artery disease or stable angina, second, the acute stage known as unstable angina, and, third, the acute stage known as acute myocardial infarction. The diseased state (as opposed to the non-diseased state) is indicated by the clinically significant presence of a first marker in a sample from the patient. The presence of one of the two acute stages, unstable angina or acute myocardial infarction, is indicated by the clinically significant presence of a second marker in a sample from the patient. The presence of the more severe acute stage known as acute myocardial infarction is indicated by the clinically significant presence of a third marker in a sample from the patient.

Abstract: Assay systems and specialized antibodies for the detection and quantitation of troponin I and troponin T in body fluids as an indicator of myocardial infarction. Since troponin I and T exist in various conformations in the blood, the ratios of the monomeric troponin I an T and the binary and ternary complexes, as well as which form of troponin present in the blood, may be related to the metabolic state of the heart. Disclosed is a system to determine the presence of a troponin form or a group of troponin forms in a sample of whole blood, serum or plasma. Disclosed is a stabilized composition of troponin; the stabilized composition can comprise a stabilized composition of troponin I, wherein the troponin I is oxidized, the troponin I can be unbound or the troponin I can be in a complex.

Abstract: Embodiments of the present invention relate to devices and methods for detecting cellular products using detection particles having product-specific detection reagents and having a characteristic spectral feature. In particular, devices and methods are provided for measuring secreted cellular products including cytokines. Detection substrates, include microwells having product-specific capture reagents thereon or comprising hydrophobic membranes are described having greater capability to detect products from individual cells in a mixture of heterogeneous cells. With the use of multiple detection particles, multiple cellular products can be detected in a single well. Additionally, using the inherent spectral properties of detection particles, no enzymatic reactions are needed to visualize a secreted product, thereby increasing the sensitivity, reproducibility and ease of use.

Abstract: An infrared fluorescent particle comprising a functional group or a substance that is capable of binding to an analyte, wherein fluorescence at infrared wavelength is emitted from the particle upon exposure of the particle to excitation light at infrared wavelength. The infrared fluorescent particle of the present invention is capable of binding to the analyte. Due to a high penetration of the fluorescence and the excitation light into biological substances, the infrared fluorescent particle of the present invention can reduce an influence of luminescence, light absorption or light scattering which is occurred due to the analyte and the surrounding substances.

Abstract: The present invention provides a method for controlling the spatial arrangement of an energy donor and an energy acceptor in a reaction complex in order to carry out the measurement with good precision and high sensitivity, and a measurement system using the method. Two types of material having affinity for the subject material to be measured are respectively labeled with a combination of reporters that give rise to energy transfer; those that have been thus obtained by labeling these materials are each further labeled with materials that have weak affinity for each other to give reagents, which are then mixed with a sample to give the reaction complex. Each of the materials is brought into spatial proximity by binding based on the affinity among the materials having weak affinity for each other in the reaction complex, and since that condition is stably maintained, a more efficient energy transfer occurs.

Abstract: A method for identifying or monitoring SLE in an individual is provided. The method includes quantitating complement component C4d on the surfaces of platelets and comparing the amounts of C4d to reference levels of C4d on platelets of individuals without SLE and/or on platelets of the individual obtained at a different time. Kits for use in the above-described methods are provided along with computer readable media tangibly embodying executable instructions to perform the methods.

Abstract: The invention is related to methods of diagnosing inflammatory diseases or conditions by determining levels of components of the complement pathway on the surface of white blood cells.

Abstract: The present invention provides hapten derivatives useful for the preparation of antigens, antibodies and reagents for use in immunoassays for the detection of LSD and 2-oxo-3-hydroxy LSD. In the present invention, the 2-oxy LSD nucleus is derivatized out of the indole nitrogen to form an aminoalkyl derivative. The resulting haptens can then be further modified at this functionalized position for linking to appropriate immunogenic or labeling groups to provide reagents for immunoassays having substantially equal specificity for both LSD and 2-oxo-3-hydroxy-LSD.

Abstract: The invention provides methods and compositions for simultaneously detecting the activation state of a plurality of proteins in single cells using flow cytometry. The invention further provides methods and compositions of screening for bioactive agents capable of coordinately modulating the activity of a plurality of proteins in single cells. The methods and compositions can be used to determine the protein activation profile of a cell for predicting or diagnosing a disease state, and for monitoring treatment of a disease state.

Abstract: The present invention relates to the qualitative and quantitative validation of marker indices, especially for medical diagnosis and particularly for the determination of the growth fraction in a sample with antibodies against the Ki-67 protein. Diagnostic kit for the quantification of a cell fraction labeled by a marker for in vitro diagnosis, characterized in that a pseudo-tissue is used for the intra- and inter-assay standardization of the marker index.

Abstract: Engineered fluorescent proteins, nucleic acids encoding them and methods of use are provided.

Abstract: Disclosed herein are novel probes, which can be used to detect and identify target molecules of interest in a sample. The disclosed probes can be used to monitor conformational changes induced by molecular recognition events in addition to providing signaling the presence and/or identity of a target molecule. Methods, including solid phase synthesis techniques, for making probe molecules that exhibit changes in their optical properties upon target molecule binding are described in the disclosure. Also disclosed herein are novel chromophore moieties, which have tailored fluorescent emission spectra.

Abstract: The present invention provides an assay for detection of M. bovis-infected animals. A tracer, comprising a peptide of M. bovis protein MPB70 conjugated to a fluorophore, is added to a serum sample from an animal to form a mixture. The fluorescence polarization of the mixture in then measured and compared to the fluorescence polarization of a control. The present invention further provides a tracer for use in fluorescence polarization assay to detect antibodies specific for M. bovis. The tracer comprises a peptide of M. bovis protein MPB70 conjugated to a fluorophore, such that the tracer is able to bind to antibodies specific for M. bovis to produce a detectable change in fluorescence polarization.

Abstract: Systems, including compositions, kits, and methods, particularly for photoluminescence applications. The systems may include, among others, (1) organic chelators, (2) complexes between the chelators and lanthanide ions, and (3) precursors, derivatives, and uses thereof. The chelators may include a 1,4,7,10-tetraazacyclododecane ring system, for example, having the formula: Here, R1, R2, and R3 are substituents of the tetraazacyclododecane ring system, that is further substituted at the 10-position by a sensitizer Z that is typically a polyheterocyclic ring system. The organic chelator may be capable of forming a luminescent complex with a lanthanide ion, and is optionally further substituted by a reactive functional group or a conjugated substance. The resulting lanthanide complex may be useful in luminescence-based assays, such as energy transfer assays, among others.

Abstract: Arrays of microparticle populations, each population labeled with a single fluorescent dye, are provided for use in multiplex assays. The populations form a virtual multidimensional array wherein each microparticle is identified by fluorescence intensity in two different fluorescence detection channels. The arrays are useful in a variety of assays, including multiplex, multi-analyte assays for the simultaneous detection of two or more analytes by, for example, flow cytometry, and a labeling reagents in, for example, microscopy. The use of singly-dyed microparticles to form multidimensional arrays greatly simplifies the creation of multiplex assays.

Abstract: Luminescent chemical reagents that include complexes of rare earth metals with ligands such as aromatic heterocyclic nitrogen-containing compounds and semi-aromatic oxygen-containing compounds are used to detect small quantities of complex substances such as pharmaceuticals, metabolites, and microorganisms in complex sample mixtures.

Abstract: The present invention provides methods and systems for performing in vivo flow cytometry. In one embodiments, selected circulating cells of interest of a subject are labeled with fluorescent probe molecules. The labeled cells are irradiated in vivo so as to excite the fluorescent probes, and the radiation emitted by the excited probes is detected, preferably confocally. The detected radiation is then analyzed to derive desired information, such as relative cell count, of the cells of interest.

Abstract: The ionic conjugates include an inorganic particle electrostatically associated with a macromolecule which can interact specifically with predetermined chemical species or biological targets.

Abstract: The present invention provides methods and compositions for detecting bacteria of the Chlamydiaceae family in a biological sample. Methods include, for example, contacting a biological sample with an antibody that specifically binds to a chlamydial antigen displayed on the surface of a chlamydia-infected blood cell; and analyzing the sample using fluorescence microscopy or flow cytometry to detect bound antibody.

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: The proteins in a biological sample that is sought to be analyzed for its protein composition by an electrophoretic or chromatographic procedure are coupled to a dye in an unusually efficient manner by combining the sample with a solid dry composition containing the dye, a buffering agent, and in preferred embodiments, a denaturing agent as well. The solid and dry form of the composition prevents the dye from deteriorating or decomposing, and the combination of components in the composition allows the dye to couple to the proteins in a relatively uniform manner with no overstaining of the protein when the composition and the sample are heated together and held at an elevated temperature for a short period of time.

Abstract: A self-calibrated, magnetic binding assay (e.g., sandwich, competitive, etc.) for detecting the presence or quantity of an analyte residing in a test sample is provided. The magnetic binding assay includes detection probes capable of generating a detection signal (e.g., fluorescent non-magnetic particles) and calibration probes capable of generating calibration signal (e.g., fluorescent magnetic particles). The amount of the analyte within the test sample is proportional to the intensity of the detection signal calibrated by the intensity of the calibration signal.

Abstract: The inventive subject matter relates to a method of measuring non-protein hydrophobic and protein target antigens by competitive fluorescence polarization using peptide mimics as competitors. The peptide mimics used in the inventive method contain conformational epitopes not represented in the linear sequence. The method can be used in the detection of a wide range of agents including steroids, hormones, nucleic acids, proteins and infectious organisms. A method for the detection of cortisol and gamma-interferon is also contemplated in the method. Samples suitable for analysis by the inventive method include environmental samples and bodily fluids including serum and oral fluids including saliva.

Abstract: A method of generating light through chemiluminescence involves providing a stable 1,2-dioxetane of the formula: Wherein (a) R1 and R2 are each, individually, a chemical reactive site or when fused together form a chemical reactive site, and R3 and R4 are each, individually, a chemical reactive site or when fused together form a chemical reactive or (b) R1 has at least two hetero atoms with chemical reactive site and R3 and R4 are inactive site and R2 is a chemical reactive site.

Abstract: A method of generating light through chemiluminescence involves providing a stable 1,2-dioxetane of the formula: Wherein (a) R1 and R2 are each, individually, a chemical reactive site or when fused together form a chemical reactive site, and R3 and R4 are each, individually, a chemical reactive site or when fused together form a chemical reactive or (b) R1 has at least two hetero atoms with chemical reactive site and R3 and R4 are inactive site and R2 is a chemical reactive site.

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 salicylamidyl moiety. Also provided are probes incorporating the salicylamidyl ligands of the invention and methods utilizing the ligands of the invention and probes comprising the ligands of the invention.

Abstract: The invention provides methods and kits for detecting and/or measuring receptor homodimers on a cell surface membrane. In one aspect, the methods employ pairs of probes comprising binding compounds and a cleaving probe, such that at least one binding compound binds specifically to the same epitope of a membrane-bound analyte as the cleaving probe. The binding compound includes one or more molecular tags attached through a cleavable linkage, and the cleaving probe includes a cleavage-inducing moiety that can cleave the linkage when within a defined proximity thereto. Binding of the two probes to a homodimer of a cell surface molecules results in release of molecular tags from the binding compounds, providing a measure of formation of the homodimeric complex.

Abstract: A TBET cassette including at least one xanthine- or pyronin-based compound as a donor or acceptor is disclosed. Also, a method of TBET cassette design in which four criteria may be used is provided. TBET cassettes may be used to label biological molecules, in clothing dyes, and in cosmetics.

Abstract: Methods are provided for detecting the formation of complexes of molecules, especially proteins, in a sample, such as a cell or tissue lysate. In one aspect, a cleaving probe specific for a first protein in a complex and one or more binding compounds specific for one or more second proteins in a complex are provided. Upon binding, the cleaving probe is induced to generate an active species, such as singlet oxygen, that cleaves molecular tags attached to the binding compounds only in the local region of the cleaving probe. The released molecular tags are separated from the assay mixture and from one another to provide a readout that is related to the number and types of proteins present in the complex.

Abstract: The present invention provides an approach for the simultaneous determination of the activation states of a plurality of proteins in single cells. This approach permits the rapid detection of heterogeneity in a complex cell population based on activation states, and the identification of cellular subsets that exhibit correlated changes in activation within the cell population. Moreover, this approach allows the correlation of cellular activities or properties. In addition, the use of potentiators of cellular activation allows for characterization of such pathways and cell populations.