Abstract: The present invention discloses novel immunogens, antibodies prepared from such immunogens, and labeled reagents useful in immunoassays for the detection and quantification of testosterone in a test sample. Also disclosed are immunoassays using these reagents and methods for synthesizing these reagents. The immunoassays are preferably microparticle enzyme immunoassays (MEIAs) and fluorescence polarization immunoassays (FPIAs). Further disclosed are novel starting materials for making the above novel immunogens and labeled reagents. Methods for making the novel immunogens and labeled reagents from the novel starting materials are also disclosed.

Abstract: The present invention relates to methods useful for identifying compounds capable of specifically regulating actin polymerization, stress fiber formation or focal adhesion assembly by regulating G.sub..alpha.12 and/or G.sub..alpha.13 activity in cells involved in inflammatory responses, immune responses, allergic responses and neuronal responses, kits to perform such assays and methods to control disease related to such responses.

Abstract: A chemiluminescent assays for the determination of the presence or amount of a biopolymer in bound assays using 1,2-dioxetanes in connection with AttoPhos.TM. as chemiluminescent substrates for enzyme-labeled targets or probes is provided. Further disclosed is a kit for conducting a bioassay for the presence or concentration of a biopolymer comprising a) an enzyme complex; b) a 1,2-dioxetane; and c) AttoPhos.TM..

Abstract: A chemiluminescent analytical method which comprises using a suitable catalyst in a reaction with luminol or a substituted luminol and a perborate in an aqueous solvent and detecting and measuring luminescence produced by the reaction is described. In the method, the aqueous solvent comprises a non-luminescent chelating agent for chelation of an electron-deficient boron atom in the perborate.

Abstract: An in vivo method for depleting mammalian cells of adenosine 5'-monophosphate (AMP) useful in the treatment of certain cancers is provided. According to the method, a population of cells is obtained from a host and assayed for loss of methylthioadenosine phosphorylase (MTAse) activity. MTAse catabolizes methylthioadenosine to adenine for endogenous salvage incorporation into the intracellular AMP pool. The preferred method for assaying loss of MTAse activity is a hybridization technique for detection of a homozygous loss of the gene which encodes MTAse. Hosts having MTAse deficient tumors are treated with a therapeutically effective amount of an agent which inhibits the activity of adenylsuccinate synthetase, which converts inosine 5'-monophosphate to AMP, thus depleting the tumor cells of substrates for de novo AMP production. L-alanosine is the preferred ASS inhibitory agent for use in the method of the invention.

Abstract: This invention relates to InhA enzyme crystals and to methods of growing said crystals. This invention is further directed to the utilization of said crystals to determine the three dimensional structure of InhA enzyme utilizing heavy atom derivatives of said crystals, and to the identification and development of compounds which inhibit the biochemical activity of InhA enzyme in bacteria and plants.

Abstract: Substituted aminostyrylpyridinium salts are fluorescent dyes having advantageous characteristics when employed as viscosity probes for the measurement of viscosity in a liquid, especially in a very small volume, for example, in living cells.

Abstract: A method of detecting nucleic acids, proteins, or protein nucleic acid complexes. The method includes binding an enzyme, such as phosphatase, to a specimen of the nucleic acid, protein, or protein nucleic acid complex. The enzyme is then reacted with a fluorescein derivative phosphate ester to obtain a fluorescein derivative phosphate ester hydrolysate. The hydrolysate is then irradiated with excitation light, and the emitted fluorescein is detected.

Abstract: A chemical sensor includes a patterned layer having discrete sections, a -dimensional detector array, and a two-dimensional lens array that focuses an optical signal from the patterned layer onto the two dimensional detector array. Typically, the two-dimensional detector array is a charge-coupled device array and the lens array is a graded index of refraction lens array. The chemical sensor maintains good resolution throughout its field of view.

Abstract: A homogenous affinity assay method, and sensor using such method, for the detection and measurement of analytes that uses an unmodified polyvalent receptor-bearing molecule and having a receptor with at least two binding sites that has an affinity for an analyte of interest, and two groups of molecules wherein one group of molecules is labeled with a molecule capable of generating a measurable response when in close proximity to a label molecule on the other group of molecules. In one embodiment, the groups of molecules also have grafted on them an analog to the analyte of interest. In the absence of the analyte of interest, members of the molecule groups are brought in close proximity to each other through affinity binding to receptor-bearing molecule. This affinity binding results in the formation of a binding complex consisting of members of the first and second groups of molecules and receptors and which results in a response between the labels on the molecules which is capable of measurement.

Abstract: Methods and compositions are provided for improved kinetics of light production from luciferase activity in beetle luciferase-luciferin reactions utilizing a luciferase inhibitor. Thus, the invention provides methods and compositions for assaying samples for the presence of a beetle luciferase in conjunction with a luciferase inhibitor.

Abstract: Detection or determination of a protease in a sample by incubating the sample with a substrate of the protease and observing proteolytic cleavage of said substrate. The substrate is a modified proenzyme containing a recognition site, e.g., an activation site, cleavable by said protease. Proteolytic cleavage of the modified proenzyme is detected by observing the resulting activity using a suitable substrate of the activated proenzyme. The protease may be e.g. an aspartic protease or a metalloprotease, and the modified proenzyme e.g. pro-urokinase having a mutant activation site which is cleavable by the protease to be determined.

Abstract: Compositions are disclosed comprising (a) a metal chelate wherein the metal is selected from the group consisting of europium, terbium, dysprosium, samarium osmium and ruthenium in at least a hexacoordinated state and (b) a compound having a double bond substituted with two aryl groups, an oxygen atom and an atom selected from the group consisting of oxygen, sulfur and nitrogen wherein one of the aryl groups is electron donating with respect to the other. Such composition is preferably incorporated in a latex particulate material. Methods and kits are also disclosed for determining an analyte in a medium suspected of containing the analyte. The methods and kits employ as one component a composition as described above.

Abstract: Methods for ascertaining the sensory irritation of chemicals in vitro are described. The methods include the cultivation of neuronal cells alone, with target tissue cells, and with target tissue cells and mast cells; the introduction of a chemical to be tested; and the measuring of neuronal response in the form of ion uptake or change in membrane potential. A co-culture system of neuronal and target tissue cells for performing said methods is also described.

Abstract: A rapid single step assay suitable for the detection or quantification of enzymes, in particular, hydrolases, especially, aminopeptidases and esterases. The enzymatic reaction causes the cleavage of a metal ligand labelled hydrolase substrate. The cleaved ligand alters the electrochemiluminescence of bidentate aromatic heterocyclic nitrogen-containing ligand reagent. The change in electrochemiluminescence correlates to the presence of hydrolase activity present in the sample. The assay can be performed on an IGEN Origen.RTM. Analyzer.

Abstract: The invention relates to a method for determining the extent of sepsis and/or an infection in a human or animal patient by detecting the amount of an antigen indicative of such infection. The amount of the antigen is detected in a patient blood derived test sample containing blood cell fractions.

Abstract: Methods for internally and externally photostandardizing chemical assays, e.g. an ATP-bioluminescence assay. A pre-determined amount of a photosensitive derivative of the analyte of interest is used in the assay protocol which releases a known amount of the free analyte when it is exposed to a flash of visible light of pre-determined duration and intensity. By monitoring the response of a test property of the assay to the release of a known quantity of analyte, a standard value can be calculated which allows either direct determination of the amount of analyte originally present in the assayed sample or production of a photocalibration series against which results of an assayed sample can be compared.

Abstract: To sequence DNA automatically, DNA marked with far infrared, near infrared, or infrared fluorescent dyes are electrophoresed in a plurality of channels through a gel electrophoresis slab or capillary tubes wherein the DNA samples are resolved in accordance with the size of DNA fragments in the gel electrophoresis slab or capillary tubes into fluorescently marked DNA bands. The separated samples are scanned photoelectrically with a laser diode and a sensor, wherein the laser scans with scanning light at a wavelength within the absorbance spectrum of said fluorescently marked DNA samples and light is sensed at the emission wavelength of the marked DNA.

Abstract: An apparatus for monitoring a substance in a living organism. The apparatus can be used to provide an assessment of the feeding rate of the organism and can be used to measure water toxicity.

Abstract: A method is disclosed for conditioning samples (of e.g. milk or meat) containing fat globules and somatic cells and/or protein particles before they are subjected to fluorescence measurements in order to determine the bacterial content, as well as methods for performing the determination of bacterial content in such samples. The conditioning method involves the treatment of the samples with an ion-chelating agent, a proteolytic enzyme, detergent, and a bacteriologically specific fluorochrome such as ethidium bromide. Detergent is used in a concentration resulting in substantially no dissolution of the fat globules and the conditioned samples thus loses insignificant amounts of fat globules. The assessment of fluorescence is preferably performed in a conventional flow cytometer. As no separation of fat globules is necessary, the methods are simple and fast. The bacterial determinations have proved reliable when compared to standard methods.

Abstract: An energy transfer fluorescent probe for detecting a reagent is provided which includes a fluorescent reporter molecule and a quencher molecule positioned on the probe relative to the reporter molecule such that the quencher molecule quenches the fluorescence of the reporter molecule when in a first state, the quencher molecule being converted by the reagent to a second state which has a reduced ability to quench the reporter molecule. Examples of conversions of the quencher molecule from a first state to a second state include reductions, oxidations, hydrolyses, phosphate cleavages, and the conversion of amides to amines. In one embodiment, the quencher molecule is a substrate for an enzyme which converts the quencher from a first state to a second state. For example, the enzyme may be an reductase, an oxidase, hydrolytic, a peptidase or a phosphorylase. The probe is used to fluorescently detect a reagent in a sample.

Abstract: A microbiological assay for chemicals, which uses a cell and a reducing dye to quantitatively measure inhibition of electron transport in the cell membrane as a function of chemicals in the substance being tested, is disclosed. This assay and method is reliable, simple, fast, and inexpensive, requires a minimum amount of durable equipment, and avoids the need for the use of live animals as the indicator organisms. The assay is particularly useful for testing for toxicity in food products, environmental, medical and industrial processes, sewage treatment, effluent, agricultural wastes, and chemical dumps.

Abstract: A "reagentless" chemiluminescent biosensor and method for the determination of hydrogen peroxide, ethanol and D-glucose in water. An aqueous stream is basified by passing it through a solid phase base bed. Luminol is then dissolved in the basified effluent at a controlled rate. Oxidation of the luminol is catalyzed by the target chemical to produce emitted light. The intensity of the emitted light is detected as a measure of the target chemical concentration in the aqueous stream. The emitted light can be transmitted by a fiber optic bundle to a remote location from the aqueous stream for a remote reading of the target chemical concentration.

Abstract: Assays and reagents are provided for the measurement of components that are involved in either an enzyme-catalyzed degradation of a protein substrate, or an enzyme-catalyzed polymerization of a substrate. The method involves using a fluorescent-labeled protein substrate in an enzyme-catalyzed reaction, and measurement of the component's effects on the fluorescence emission of the enzyme-catalyzed reaction such as quenching or dequenching, as a measure of the component's activity.

Abstract: The invention comprises a culture medium and method for distinguishing bacteria of Salmonella species from other gram-negative bacteria, especially those belonging to the family Enterobacteriaceae. It is based on the ability of salmonellae to utilize melibiose, mannitol, and sorbitol and convert them into acids, together with a chromogenic substrate used for identifying .beta.-galactosidase. Other bacteria of the family Enterobacteriaceae, most of which are .beta.-galactosidase-positive, appear as brown, blue, or green colonies, depending on the chromogenic substrate used. Apart from Salmonella species, other .beta.-galactosidase-negative bacteria, such as Proteus species, appear as colorless colonies. Salmonellae can be identified directly on the culture medium after incubation, by their characteristic bright red color.

Abstract: A method which utilize chemiluminescence for analyte detection and the detection of bacteria on surfaces. The method uses a device comprising a sampling portion made of a first adsorbent material, a container, and a second adsorbent material located within the container. The sampling portion collects analytes from a test sample which includes a surface or volume of a liquid. The second adsorbent material holds one or more chemiluminescent components including luciferase enzyme and cofactors in a dry state. In a preferred embodiment, the sampling portion is swabbed over a suspected contaminated surface. A bacteriolytic solution is then added to the adsorbent and releases ATP from any bacteria present. The ATP diffuses into the second adsorbent and mixes with the reagents therein to produce detectable light.

Abstract: Method for detecting peroxidase or hydrogen peroxide with high sensitivity. Both peroxidase and hydrogen peroxide are prepared such that one of them is overabundant to the other. Phenoxy radicals are produced from a p-substituted phenol compound by the action of peroxidase in the presence of hydrogen peroxide. The free radicals are trapped by a hydroxy amine compound, and stable radical species are produced. Electron spin resonances of the stable radical species are measured.

Abstract: The invention relates to a target microbe-specific medium for detecting the presence or absence of a target microbe in an environmental or biological sample, the medium including: a) an effective amount of vitamin, amino acid, element and salt ingredients operable to allow viability and log phase reproduction of the target microbe in the presence of a nutrient-indicator and to aid the target microbe through lag phase and into log phase of growth in the sample; and b) an effective amount of a nutrient-indicator which is provided in an amount sufficient to support log phase growth of the target microbe of a sample until a detectable characteristic signal is produced in the medium/sample mixture during the log phase growth; the nutrient-indicator being incapable of supporting continued logarithmic growth of any viable non-target microbes in the medium/sample mixture to produce a detectable characteristic signal; and the nutrient-indicator being operable to alter a detectable characteristic of the medium/sample m

Abstract: Modified apo-peroxidases have been found useful in analytical methods to remove the bias caused by the presence of hemoglobin in the test specimens. While apo-peroxidases are known to remove serum interferents, they fail to overcome the hemoglobin bias, particularly when dry coated analytical elements are used in the assays. Blocking the nitrogen atoms of the imidzolyl groups of the histidine amino acids of the apo-peroxidase prevents catalytic activity of the protein in the presence of hemoglobin.

Abstract: A bacteria impermeable container or ampule (10) contains a liquid growth medium and a substrate-indicator complex. The complex includes a substrate component, e.g., starch, and an indicator molecule, e.g., a dye, a fluorescent molecule, or the like, which are tightly bound and complexed, but which are cleavable by a preselected enzyme. A sterilant passes over a carrier (20) for microorganisms which, upon germination, are capable of rapidly generating large quantities of the preselected enzyme. Following the sterilization process, the carrier is immersed in the liquid growth medium. Any viable surviving microorganisms grow, generating the preselected enzyme. The enzymes cleave the bound indicator molecule from the substrate, resulting in a measurable property change in a couple of hours. Typical property changes include fluorescence, a color change, a change in pH which triggers a pH indicator color change, and the like.

Abstract: A method for simultaneously testing a sample for the presence of multiple rget antigens or antibodies in the sample, which comprises presenting the sample to a plurality of different binding sites, wherein at least two of the sites are binding sites for different known target antigens or antibodies, each known binding site is composed of at least one molecule of a ligand-enzyme complex attached to a support, and the ligand-enzyme complex is a ligand attached to an enzyme in proximity to the enzyme's active site such that the enzymatic activity of the ligand-enzyme complex is changed when the target antibody or antigen is present in the sample. The ligand-enzyme complex embraces nonenzymatic reporter molecules such as electrochemiluminescent compounds. The method also includes assaying each binding site for a change in enzymatic or other reporter activity compared to a control value. A device for performing simultaneous detection of multiple target antigens or antibodies is also disclosed.

Abstract: The invention provides novel methods and compositions for detecting kinase activity in solution, without the use of radioactivity. The methods marry the kinetic advantages of solution-based reaction with the efficiency and high-throughput adaptability of solid-phase wash and detection steps, yet is conveniently practiced in a single tube. The methods may be used to assay for kinase activity per se or, by controlling for the kinase activity, for modulators of kinase activity. The method is exemplified with a preferred chemiluminescent protein kinase assay using biotinylated substrate peptides captured on a streptavidin coated microtiter plate and monoclonal antibodies to detect their phosphorylation.

Abstract: A method of measuring an analyte present in animal (e.g., human) tissue or fluids such as blood or plasma. The analyte is multi-photon excitable (e.g., two-photon excitable) at a first wavelength at which the animal tissue is substantially non-absorbing. The analyte fluoresces at a second wavelength upon being excited at the first wavelength. The animal tissue is irradiated with radiation at the first wavelength so as to excite the analyte through absorption by the analyte of two or more photons of the radiation at the first wavelength. Excitation of the analyte results in a fluorescent emission from the analyte of radiation at the second wavelength. The emission at the second wavelength is detected, and the concentration of analyte determined based on the detected emission.

Abstract: A method is provided for detecting an analyte in a liquid sample, the method using light energy propagational properties of an analyte-responsive polymer. The analyte-responsive polymer is interfaced with a means to detect changes in the light propagation of the analyte-responsive polymer and the changes are correlated to the amount of analyte in the liquid sample.

Abstract: A composition for use in a specific binding assay comprising each of a plurality of substances with one or more components each capable of taking part in a respective distinguishable chemiluminescent reaction.

Abstract: A chemiluminescent assays for the determination of the presence or amount of a biopolymer in bound assays using 1,2-dioxetanes in connection with AttoPhos.TM. as chemiluminescent substrates for enzyme-labeled targets or probes is provided. Further disclosed is a kit for conducting a bioassay for the presence or concentration of a biopolymer comprising a) an enzyme complex; b) a 1,2-dioxetane; and c) AttoPhos.TM..

Abstract: Acridans which are reactable with a peroxidase and peroxide. The acridans are characterized by having an aromatic leaving group ArO which is a di- or polyhalosubstituted phenoxy group. The compounds are useful in assays where one member of a binding pair is linked to the peroxidase and for detecting the peroxidase. The method can also be used to detect hydrogen peroxide.

Abstract: A rapid, sensitive, non-radioactive diagnostic kit for the direct detection of both Shiga-like toxin, type I, and Shiga-like toxin, type II, produced by enterohemorrhagic Escherichia Coli in food and clinical samples. This diagnostic kit is comprised of a monoclonal antibody capable of detecting Shiga-like toxin, type I, and a monoclonal antibody capable of detecting Shiga-like toxin, type II, together with a chemiluminescing detection reagent with a sensitivity enhancer.

Abstract: The present invention relates to screening methods for the identification of compounds and compositions useful as novel antibiotics and antibacterial agents. In particular, the present invention relates to methods utilizing two-component regulatory switches, for example, those comprising a prokaryotic enzyme such as histidine protein kinase that is activated to autophosphorylate by a signal transduction mechanism. The invention also relates to methods of identifying inhibitors of enzyme activity, particularly in bacterial cells. In particular, a high-throughput assay system useful in the large-scale screening of protein kinase inhibitors is disclosed.

Abstract: The present invention relates to a method for determination of numbers of croorganisms present in materials and for predicting their growth capability in the materials such as foodstuffs, growth media and in the presence of chemical agents. The invention also provides immobilizing cassettes and optical apparatus for use in this method.

Abstract: A dry, removable analytical element can be used to detect chemiluminescent signals produced from the reaction of peroxidase and a chemiluminescent detection system. The analytical element contains at least two layers, the outer layer being non-tacky and water-soluble or water-permeable, and used to contact a gel plate or transblotting membrane in which multiple analytes are located. The resulting signal can be recorded using a photosensitive element. Test kits include the various packaged components needed to use the analytical element for analyte detection. Within the element are critical amounts of oxidase and an oxidase substrate for highly sensitive analyte detection.

Abstract: A method and device for determining the presence and concentration of total microbial contamination or the presence and concentration of a specific microbial species on a surface is described. The method consists of a means of a collection device and fluid for removing the microbes from the surface and suspending them in a fluid phase. An aliquot of the fluid phase is introduced into a disposable test ticket which allows filtration of the sample to remove extraneous substances including somatic cells, and concentration of the microbes. The total concentration of microbes is determined by adding a somatic and bacterial releasing reagent to a disposable test device which comprises a membrane containing the luminescent reagents luciferin and luciferase, and introducing the disposable test device into a luminometer that can read the luminescence from the underside.

Abstract: A system for evaluating one or more biochemical markers for evaluating individual cancer risk, cancer diagnosis and for monitoring therapeutic effectiveness and cancer recurrence, particularly of bladder cancer. The system uses automated quantitative fluorescence image analysis of a cell sample collected from a body organ. Cells are treated with a fixative solution which inhibits crystal formation. Cell images are selected and stored as grey level images for further analysis. Cell images may be corrected for autofluorescence using a novel autofluorescence correction method. A neural net computer may be used to distinguish true-positive images from false-positive images to improve accuracy of cancer risk assessment. Cells having images positive for a marker amy be compared to threshold quantities related to predetermined cancer risk.

Abstract: A machine-based method for collecting and interpreting quantitative data on cells and tissues so that a diagnosis will obtain as to the existence or non-existence of disease in an human. Vibrational spectroscopy is used and the spectra generated by such spectroscopy are compared with stored spectra to provide whether cells or tissues are diseased, and if diseased to what degree. It, therefore, is possible to provide a basis for immediate diagnostic decisions for patients and physicians, leading in turn to immediate implementation of next-step procedures and treatment all in one visit to the doctor's office. This means that patients and the examining clinician can know almost instantly whether or not the cells or tissue examined are normal or diseased, and the level of disease present if found.

Abstract: An assay compound or a salt thereof for assaying the activity of an enzyme inside a metabolically active whole cell is disclosed. The assay compound includes a leaving group and an indicator group. The leaving group is selected from the group comprising amino acids, peptides, saccharides, sulfates, phosphates, esters, phosphate esters, nucleotides, polynucleotides, nucleic acids, pyrimidines, purines, nucleosides, lipids and mixtures thereof. The indicator group is selected from compounds which have a first state when joined to the leaving group, and a second state when the leaving group is cleaved from the indicator group by the enzyme. Preferably, the indicator compounds are rhodamine 110, rhodol, and fluorescein and analogs of these compounds. A method of synthesizing the compound as well as methods of using these compounds to measure enzyme activity are also disclosed.

Abstract: A method of assay for a ligand in a sample is described in which calibration occurs within the assay. This is achieved utilizing a measurement region and one ore more calibration regions. In at least one of the calibration regions a non-zero signal results, either because of the presence of a calibration reagent or as a result of a binding reaction analogous to that which takes place in the measurement region.

Abstract: Disclosed are new fluorogenic substrates for assay of angiotensin converting enzyme, a process for preparing them and methods for using them to assay angiotensin converting enzyme and to screen antihypertensive agents which inhibit angiotensin converting enzyme.

Abstract: A chemiluminescent assay method, compositions, kits and chemiluminescent acridan compounds are described which use a two-step chemiluminescent reaction process. The reaction involves an acridan compound, preferably a derivative of an N-alkylacridan-9-carboxylic acid, which undergoes a reaction with a peroxide compound, a peroxidase enzyme and an enhancer under conditions of time, temperature and pH which permit the accumulation of an intermediate compound, which is subsequently induced to produce a burst of light by raising the pH. The result is generation of very high intensity light from the reaction. The peroxidase enzyme is present alone or linked to a member of a specific binding pair in an immunoassay, DNA probe assay or other assay where the hydrolytic enzyme is bound to a reporter molecule. The method is particularly amenable to automated assays because of the separation of the incubation and light generating steps.

Abstract: The present invention provides for novel reagents whose fluorescence increases in the presence of particular proteases. The reagents comprise a characteristically folded peptide backbone each end of which is conjugated to a fluorophore. When the folded peptide is cleaved, as by digestion with a protease, the fluorophores provide a high intensity fluorescent signal at a visible wavelength. Because of their high fluorescence signal in the visible wavelengths, these protease indicators are particularly well suited for detection of protease activity in biological samples, in particular in frozen tissue sections. Thus this invention also provides for methods of detecting protease activity in situ in frozen sections.

Abstract: The present invention is drawn to an immunoassay capable of the rapid detection of a variety of test substances that may be present in a test sample. One feature of the invention is that extraction or isolation of the test substance occurs simultaneously with the formation of the primary antigen-test substance complex. The primary antigen-test substance complex is then captured in a solid phase format having a plurality of interstitial spaces which facilitate rapid and efficient detection. The immunoassay of the present invention works over a wide range of environmental conditions and is simple enough to be used in the absence of laboratory facilities.