Abstract: A method of conducting a rapid microbiological assay of gases is disclosed that utilizes an improved gelatin membrane filter that is pre-filtered before casting to remove microscopic-sized particles.

Abstract: Compounds of formula (I) in which R1, R2, R3, R4 and R5 are hydrogen atoms or chromogenic substituents and X is hydroxyl, OR6 wherein R6 is selected from the group consisting of C1-C4 alkyl, or O−Me+ wherein Me+ is a cation derived from an organic or inorganic base; these compounds do not exhibit significant fluorescence but are capable of being cleaved by phosphatidyl-inositol-specific phospholipase C, an enzyme which is indicative of bacterial activity; the umbelliferyl moity resulting from such cleavage is a strong fluorogen thus providing effective test methods for various pathogenic bacteria, such as Listeria, Staphylococcus and Clostridium species. Also disclosed are plating media for detection of microorganisms that are capable of metabolic generation of a phosphatidyl inositol-specific phospholipase C (PI-PLC).

Abstract: A fluorescence polarization process used to identify activity of conjugative enzymes involved in xenobiotic transformations, such as glucuronosyltransferases is provided.

Abstract: The present invention is related to microbiology and forms part of a system for rapid microbiological diagnosis. The invention allows detection of turbidimetric changes due to microbial growth, using equipment comprised of two main devices: a static turbidimetric minireader and a microflow sensor which is fed by a peristaltic pump; this equipment is coupled to a microcomputer with a program package for acquisition, processing and formation of databases used in generating necessary reports. The diagnostic kit has a glass vial with culture medium and a polymer with derepressive activity and two additional substrates for E.coli identification, as well as a set of antibiotic discs arranged in a strip for antibiogram determination from previously isolated colonies or samples obtained directly from their sources, allowing detection of urinary tract infections from direct samples of urine, and additionally simultaneous identification of E.coli.

Abstract: Transmembrane potential measurement methods using cationic dyes, and anionic dyes are provided. Compositions of the cationic and anionic dyes and microfluidic systems which include the dyes and membranes are provided in conjunction with processing elements for transmembrane potential measurements.

Abstract: A method for analyzing a sample containing a plurality of analytes, comprises labelling the analytes with a detectable label, the label being chosen such that its detectability can be influenced by modifying the conditions of detection; separating the labelled analytes, before or after labeling,; and determining the presence of the separated analytes under the modified conditions and also, if desired, the unmodified conditions. Suitable detection apparatus comprises a support for a gel, means for detecting one or more analytes in the gel, and means for reducing the temperature of the gel to below ambient temperature, e.g., to no more than 0°C.

Abstract: The invention provides a compound, useful as an optical probe or sensor of the activity of at least one cytochrome P450 enzyme, and methods of using the compound to screen candidate drugs, and candidate drugs identified by these methods.

Abstract: A rapid method for detecting the presence or absence of coliform bacteria in a liquid or liquified dairy sample, for example, skimmed milk, lowfat milk, or whole milk. A growth medium containing a fluorogenic substrate is combined with the dairy sample and is incubated for a brief period of time, for example for about 4 hours, after which a first fluorescence value of 4-methylumbelliferone is measured. The dairy sample is incubated again for about 2-8 hours after which a second fluorescence value of 4-methylumbelliferone is measured. Total, fecal, or thermotolerant coliform bacteria are determined to be present in the sample if the second fluorescent value exceeds the first fluorescent value by a predetermined 4-methylumbelliferone concentration threshold.

Abstract: The invention provides a method for detecting ATP in a sample, wherein the sample is contacted with a reaction mixture to effect a light signal, the reaction mixture comprising luciferin, luciferase and one or more water-soluble salts, the total salt concentration being at least 0.05 mole/liter, and wherein the light signal is measured. A kit is described for detecting ATP in a sample comprising luciferin, luciferase and a buffer solution comprising salts. A composition for detecting ATP in a sample comprising a total salt concentration of at least 0.05 mole/liter for prolonging a light signal occurring in a luciferin-luciferase reaction is also provided.

Abstract: Disclosed is a method for differentiating human peripheral blood granulocytes which comprises cultivating the granulocytes in the presence of an interferon and acting an immune complex on said granulocytes in the presence of a chemiluminescent substance, and measuring the chemiluminescent amounts induced. It contributes to the screening of patients suffering from serious infectious diseases or a range of inflammations, and is advantageous for monitoring the curative effects for those patients.

Abstract: The present invention provides scintillation proximity assays (SPAs) for measuring the activity of enzymes involved in type II fatty acid biosynthesis. Enzymes which can be assayed in this manner include KASI, KASII, KAS III and MAT. The present invention also provides methods of assaying for ketoacyl synthase activity, malonyl transferase activity, and acyl transferase activity in enzymes using the SPA format. Because the SPA format allows the direct detection of a radiolabeled product, these assay methods are appropriate for use in high throughput screening techniques. The present invention also provides methods for assessing a compound's ability to modulate the activity of a type II fatty acid biosynthetic enzyme.

Abstract: Improvements in calorimetric assays, surfaces for arraying microcolonies and instrument hardware for screening mutagenized enzymes and proteins in a solid phase format are presented. These improvements permit new enzyme activities to be screened. New filter membrane materials and formats for arraying the microcolonies provide higher throughput, better solvent resistance and ease of handling. Modifications to the instrument heating and illumination systems provide improved temperature control and a more compact, folded light path.

Abstract: The present invention provides high throughput screening systems for identifying antifungal compounds. The method can be performed in plurality simultaneously with fluorescence or absorbance readouts.

Abstract: Compositions for monitoring transmembrane potential across cellular membranes. The compositions typically comprise a cell having a plasma membrane that comprises a first leaflet and a second leaflet, the membrane comprising first and second membrane associated components which, when placed adjacent each other either produce or quench a fluorescent signal, wherein. The first membrane associated component translocates from a first leaflet of the membrane to a second leaflet of the membrane in response to an electrical potential gradient across the membrane, the first membrane associated component being selected from a non-fluorescent cationic fluorescence quencher a non-fluorescent anionic fluorescence quencher and a cationic fluorophore.

Abstract: A method for quantitatively and/or qualitatively assaying an analyte in a sample, wherein the analyte is a receptor binding compound, has low detection limits equivalent to those of radioreceptor assays. The method comprises the steps of a) contacting the sample with material comprising a receptor for the analyte in order for receptor-analyte binding to occur and b) further contacting the sample with a detectable ligand for the receptor in order for receptor-ligand binding to occur, followed by c) separating the resulting receptor bound and free fractions, d) subjecting the receptor bound fraction to dissociating conditions releasing the ligand from the receptor and e) assaying for the dissociated ligand in a manner known per se for the detection of the detectable ligand.

Abstract: The present invention clarifies and contrasts stain intact biological tissue samples for microscopic analysis. A biological specimen is depigmented and then contacted with a fluorescent dye, wherein the dye stains the specimen nonspecifically. The depigmented, non-specifically stained specimen is equilibrated in a clearing solution, wherein the refractive index of the clearing solution approximates that of the specimen, thereby preparing the biological specimen for visual analysis.

Abstract: Methods of enhancing sensitivity and specificity of an assay measuring enzymatic activity in a sample by measuring enzymatic activity in the sample in the presence and absence of a specific inhibitor of the enzymatic activity are provided. Methods of measuring carboxypeptidase A levels and total carboxypeptidase A levels, wherein procarboxypeptidase A is converted to carboxypeptidase A by addition of clostripain, in a biological fluid with a carboxypeptidase A substrate, specificity of which is enhanced by addition of a carboxypeptidase A specific inhibitor are also provided. In addition, methods of diagnosing acute pancreatitis by measurement of carboxypeptidase A levels and pancreatic cancer by measurement of total carboxypeptidase A levels are also provided.

Abstract: Novel screening methods for identifying antimicrobial agents involving use of membrane potential indicator dyes are provided.

Abstract: A method for measuring both the anti-migratory and cytotoxic effects of anticancer drugs on cancer tissues measuring the chemosensitivity of human biopsies to therapeutic drugs. Single cell suspension preparations of biopsy tissue is exposed to anticancer drugs and introduced into the first chamber of a migration plate separated from a second chamber by a porous membrane opaque to radiation. A migration stimulus is added to the second chamber. Migrated cells on the side of the membrane facing the second chamber are labeled with a live-cell fluorescent indicator. Non-migrated cells on the side of the membrane facing the first chamber are labeled with a fluorescent indicator of cell death. The emitted fluorescence of both the migrated cells and the non-viable cells is quantified in a fluorescence plate reader. The comparative intensity of fluorescence is an indicator of the susceptibility of the cells to the cytotoxic properties of the drug.

Abstract: A method for monitoring the microbiological contamination of opaque media is described and claimed. In this method, a Fluorogenic Dye is added to an Aliquot of opaque medium. After a certain time period, a fluorometer capable of measuring fluorescent signals in an opaque medium is used to measure the fluorogenic signals of the Fluorogenic Dye and the Reacted Fluorogenic Dye. A Useful RATIO of the fluorescent signal of the Reacted Fluorogenic Dye to the fluorescent signal of the Fluorogenic Dye is calculated and the information gleaned from the RATIO is used to ascertain the state of microbiological contamination in the opaque medium.

Abstract: Novel screening methods for identifying antimicrobial agents involving use of metabolic oxidation-reduction indicator dyes are provided.

Abstract: The present invention relates to a transparent sample container containing, preferably, a liquid bacterial growth media for detecting microbacteria and a process for detecting microbacteria using this sample container. The container is optically transparent, heat resistant, and stable during storage. The container and process provide a bacterial growth medium substantially free of contamination upon prolonged storage of preferably about one year at 40° C.

Abstract: Enzymatic methods to determine the concentration of pyridoxal 5′-phosphate (PLP) in biological fluids are described. The methods of the invention are useful to assess risk for cardiovascular disease. The assay can be a homogeneous assay using the ability of PLP to function as a co-enzyme for homocysteinase and related enzymes and measuring the products of the reaction preferably spectrophotometrically. The invention also includes improvements in sensitivity of assays for measuring hydrogen sulfide production by measuring fluorescence as opposed to absorbance of the oxidized product of H2S with N,N-dialkyl p-phenylene diamine.

Abstract: The invention provides a compound, useful as an optical probe or sensor of the activity of at least one cytochrome P450 enzyme, and methods of using the compound to screen candidate drugs, and candidate drugs identified by these methods.

Abstract: Novel fluorescent substrates of human cytochrome P450 enzymes are provided. Also provided are methods for their manufacture and use. These substrates are useful in assessing cytochrome P450 enzyme activity and in selecting compounds which inhibit cytochrome P450 enzyme activity and, in particular, for identifying potential adverse drug interactions which are mediated by inhibition of cytochrome P450 enzyme activity.

Abstract: The invention provides compounds of formula (I) in which R1, R2, R3, R4 and R5 are independently selected from hydrogen and chromogenic substituents, and X is selected from the group consisting of hydroxyl; OR6 wherein R6 is selected from the group consisting of C1-C4 alkyl; and O−Me+ wherein Me+ is a cation derived from an organic or inorganic base. A preferred species of formula (I) is 4-Methylumbelliferyl myo-inositol-1-phosphate and salts thereof with an organic or inorganic base. The invention also provides fluorogenic methods for detecting various pathogenic bacteria, e.g., Listeria, Staphylococcus and Clostridium species, using substrates containing at least one formula (I) compound. A kit for detecting a phosphatidyl-inositol-specific phospholipase C enzyme as an indication of bacterial activity is disclosed.

Abstract: A method for judging effectiveness of a drug having protease inhibitory activity, which comprises the steps of: (1) bringing a biosample isolated or collected from a patient with a disease in which participation of a protease is suspected into contact with a thin membrane containing a protease substrate and formed on a surface of a support; (2) detecting a trace of digestion formed on the thin membrane by action of a protease; and (3) judging that a drug having protease inhibitory activity is effective for the patient when a trace of digestion is formed on the thin membrane. The method enables accurate judgment as to whether or not a drug having protease inhibitory activity is effective for a patient with a disease in which participation of a protease is suspected such as rheumatoid arthritis and cancer before administration of the drug.

Abstract: Methods are disclosed for determining an analyte in a medium suspected of containing the analyte. One method comprises treating a medium suspected of containing an analyte under conditions such that the analyte, if present, causes a photosensitizer and a chemiluminescent compound to come into close proximity. The photosensitizer generates singlet oxygen and activates the chemiluminescent compound when it is in close proximity. The activated chemiluminescent compound subsequently produces light. The amount of light produced is related to the amount of analyte in the medium. Preferably, at least one of the photosensitizer and chemiluminescent compound is associated with a surface which is usually a suspendible particle, and a specific binding pair member is bound thereto. Compositions and kits are also disclosed.

Abstract: A system and method for monitoring cellular activity in a cellular specimen. According to one embodiment, a plurality of excitable markers are applied to the specimen. A multi-photon laser microscope is provided to excite a region of the specimen and cause fluorescence to be radiated from the region. The radiating fluorescence is processed by a spectral analyzer to separate the fluorescence into respective wavelength bands. The respective bands of fluorescence are then collected by an array of detectors, with each detector receiving a corresponding one of the wavelength bands.

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: Fungi and bacteria can be detected and rapidly quantified by using the nucleotide sequences taught here that are specific to the particular species or group of species of fungi or bacteria. Use of the sequences can be made with fluorescent labeled probes, such as in the TaqMan™ system which produces real time detection of polymerase chain reaction (PCR) products. Other methods of detection and quantification based on these sequences include hybridization, conventional PCR or other molecular techniques.

Abstract: Methods and kits for simultaneously measuring both members of a binding pair are described.

Abstract: T2K kinase activity is detected by forming a mixture of a T2K kinase and a substrate; incubating the mixture under conditions whereby the kinase phosphorylates the substrate at a first rate; and detecting the first rate as an indication of the kinase activity. The substrate comprises SX1X2X3SX4 (SEQ ID NO:1) wherein X1 and X4 are aliphatic residues and both of the S residues are targets of the kinase, and especially, IKK&agr; or IKK&bgr;. In another embodiment, the mixture substrate comprises a particular IL-1 or TNF signaling cascade component. The mixture may be used to screen for agents which modulate the activity of the kinase, e.g. as an immuno-chemiluminescent assay.

Abstract: ADMA is determined in a physiological sample by first removing interfering components: proteins by precipitation; and amines and citrulline, by means of a cation exchange column, followed by enzymatic hydrolysis of the ADMA to citrulline with DDAH. The citrulline is then determined spectrophotometrically.

Abstract: A fluorescence polarization assay for Equine Infectious Anemia Virus utilizes a short peptide reagent probe derived from a conserved immunodominant region of gp45. The probe is N-terminally labeled, preferably with 6-carboxyfluorescein, and purified by HPLC, which reacts in a homogenous assay with anti-EIAV antibodies contained in the serum of field infected horses and ponies. The assay has a sensitivity of about 90 percent with a specificity approaching 100 percent.

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.

Abstract: A method for detecting the binding of a test compound to a probe molecule comprises providing a test compound, the test compound having a first fluorophore bound thereto, and providing a screening substrate. The screening substrate comprises a solid support, a probe molecule bound to the solid support, and a second fluorophore bound to the solid support adjacent the probe molecule. An advantage of the invention is that this obviates the need for binding the second fluorophore directly to the probe molecule. Preferably, the second fluorophore is bound to the solid support by a flexible linker group. This enables the second fluorophore to interogate different positions on the probe molecule, which is also bound to the solid support adjacent the linker group, enhancing the ability of the method of the invention to detect positive binding events (specific binding of the test compound to the probe molecule.

Abstract: A biphase immunoassay method is provided in which a water immiscible liquid is qualitatively or quantitatively measured by mixing a sample of the water immiscible liquid with an aqueous solution containing a specific binding partner of the analyte. Binding occurs at or across the interface between the respective liquids and the degree of association of the analyte with its binding partner is dependent upon the concentration ratio rather than absolute quantities. The degree of association may be determined and used to determine the presence and/or concentration of analyte in the sample.

Abstract: A method is disclosed for determining the viability of sporocyst-forming protozoa. The method involves treating a sample of protozoa with at least one vital dye and determining the viability of the protozoa in the sample by differential staining. The viability of protozoa in the sample can then be correlated with the viability of protozoa in the population from which the sample was obtained.

Abstract: A method for the rapid detection of actively respiring microorganisms comprises the steps of detecting the presence of microorganisms utilizing microbial enzymatic conversion of tetrazolium salts to formazan products, detecting the presence of formazan product.

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: The invention provides monoclonal antibodies and other binding agents to human cytochrome P450 1A2 having advantageous properties, including capacity substantially to inhibit enzyme activity of human cytochrome P450 1A2 and lack of specific binding to other human cytochromes P450. The binding agents of the invention are useful inter alia in methods for screening drugs for metabolism by cytochrome P450 1A2, and in methods of measuring p450 1A2 levels in individuals relative to p450 1A2 levels in a control population.

Abstract: This invention relates to a novel method of screening for inhibitors of beta-amyloid production, and thereby identifying such inhibitors as therapeutics for neurological and other disorders involving APP processing and beta-amyloid production. This invention also relates to identifying macromolecules involved in APP processing and beta-amyloid production. Furthermore, inhibitors identified by the screening method of the present invention are useful in the treatment of neurological disorders, such as Alzheimer's disease, which involve elevated levels of A&bgr; peptides.

Abstract: The presence or absence of a predetermined target first generation environmental sourced microbe in an environmentally derived sample is determined by adding a testing medium to the sample, or vice versa. The testing medium provides a selective growth medium for the target microbe and includes a specific nutrient which only the target microbe can metabolize. This specific nutrient is modified by attaching a sample-altering moiety thereto, thereby converting the nutrient to a nutrient-indicator. The sample-altering moiety is activated to alter the sample only if the specific nutrient is metabolized by the target microbe. The sample-altering moiety can be a material which changes the color of the sample (visible or non-visible) or changes an electrical characteristic of the sample, or alters some other detectable characteristic of the sample. The testing media does not have to be kept sterile, and the testing procedure does not have to be performed in a sterile environment.

Abstract: A method for monitoring both the planktonic and sessile microbial populations in an industrial water system by the addition of a fluorogenic dye compound is described and claimed.

Abstract: The present invention relates to a quick method for the qualitative and quantitative medical-diagnostic analysis on the protein level of the substitution of single amino acids with pathogenic and non-pathogenic effects on the organism. The medical-diagnostic analysis is performed by a combination of enzymatic or chemical cleavage of the isolated peptide, chromatographical separation of the fragments and analysis by mass spectrometry, both direct LC/MS and indirect MALDI-MS, and analysis by capillary electrophoresis. By comparing protein samples from healthy humans with those of ill humans, the method described is suitable for establishing new, as yet unknown mutations and quantifying the expression and incorporation of wild type to mutant.

Abstract: The invention provides monoclonal antibodies and other binding agents to human cytochrome P450 2A6 having advantageous properties, including capacity substantially to inhibit enzyme activity of human cytochrome P450 2A6 and lack of specific binding to other human cytochromes P450. The binding agents of the invention are useful inter alia in methods for screening drugs for metabolism by cytochrome P450 2A6, and in methods of measuring p450 2A6 levels in an individual relative to p450 2A6 levels in a control population.

Abstract: The invention concerns a method and reagent for the determination of lipase in the presence of a colour substrate and at least one N-substituted carboxylic acid amide derivative or a compound of the general formula (I) or (Ia) in which R1 and R2 independently of one another represent hydrogen or a saturated or unsaturated, substituted or unsubstituted hydrocarbon residue with 2 to 24 carbon atoms, Z denotes a saturated or unsaturated, substituted, cyclic or straight-chained hydrocarbon residue with 1 to 10 carbon atoms, X represents an atom or a group of atoms with positive charge and n is a number from 1 to 3. Tetra-sodium-N-(1,2-dicarboxylethyl)-N-alkylsulfosuccinamide or a mixture containing such a compound has proven to be particularly advantageous for the elimination of unspecific reactions in the determination of lipase in a biological sample.

Abstract: The methods of cancer screening allow for a safe, reliable, inexpensive and minimally invasive diagnosis. Cells are first collected through non-invasive or minimally invasive means. The collected cells are stored in a cell culture media. A chemical compound such as 5-ALA is introduced to the cultured cells. The cells are incubated a period of time to allow interaction between the introduced chemical compound and the collected cells. The cells are then studied under a fluorescence microscope which emits a specific frequency of light matching the excitation frequency of fluorescing abnormal cells. Pre-malignant and malignant cells will fluoresce while normal healthy cells generally will not fluoresce. In lieu of or in addition to observing the cells by the fluorescence microscope, an imager may be used to observe the cells wherein the imager includes selectable and variable charge integration capability. Observable fluorescence can be maximized by selecting the appropriate integration period.

Abstract: Methods of measuring carboxypeptidase A levels and total carboxypeptidase A levels, wherein procarboxypeptidase A is converted to carboxypeptidase A by addition of clostripain, in a biological fluid with a carboxypeptidase A substrate, specificity of which is enhanced by addition of a carboxypeptidase A specific inhibitor are provided. In addition, methods of diagnosing acute pancreatitis by measurement of carboxypeptidase A levels and pancreatic cancer by measurement of total carboxypeptidase A levels are also provided.