Abstract: Chromogenic enzyme substrate compounds comprising a dibenz[b,e][1,4]oxazepinone or dibenzo[b,e][1,4]thiazepinone nucleus having an enzyme-cleavable group such as a radical of a sugar, carboxylic acid, amino acid, peptide, phosphoric acid, or sulfuric acid. The substrate compounds are, in general, highly soluble in aqueous media and only slightly colored, and produce, upon enzyme cleavage, a chromogen exhibiting a large change in absorbance and a pKa below 7. Such substrates find use as indicators for the determination of enzyme analytes and enzymes used as markers in a variety of assays, including immunoassays.

Abstract: A method and structure are provided for controlled deposition of liquid analytical reagents on one or more reagent zones disposed in a reaction channel used for performing analytical assay procedures and the like. The substrate surface on which the reaction channel is defined is provided with a unique surface geometry wherein each reagent zone is defined in the form of a sharp, substantially flat, raised portion or mesa-shaped node. The raised node provides a discontinuity in the surface of the reaction channel which is sufficient to prevent a liquid reagent deposited thereon from spreading to adjacent surfaces and, thereby, provides a discrete or localized area to serve as a reagent zone. Controlled deposition is realized since drops of deposited reagent are prevented from spreading uncontrollably over the surrounding substrate surface while, at the same time, allowing the deposited reagent to be completely contacted and removed by the action of a liquid reaction mixture washing the reagent zone.

Abstract: Optical indicator chalcogen (selenide or sulfide) compounds responsive to oxidants, e.g., hydrogen peroxide, and a method for using such indicators. Upon oxidation the resulting intermediate undergoes spontaneous elimination of the chalcogen residue to yield a signal compound which provides an optical signal such as fluorescence. Preferred fluorogenic indicator compounds are 3-chalcogen-3,4-dihydrocoumarin derivatives and 3-chalcogen-3,4-dihydro-2-quinolone derivatives. Such indicator compounds provide highly fluorescent products upon oxidation by hydrogen peroxide and are useful in analytical systems which generate hydrogen peroxide in response to the analyte under determination.

Abstract: A method for obtaining a substantially pure preparation of an enzyme-antibody conjugate having an enzyme component covalently coupled to a predetermined number of an antibody component where the molecular weight of the enzyme component is substantially greater than the molecular weight of the antibody component. The method involves the electrophoretic separation of the desired enzyme-antibody conjugate from an enzyme-antibody conjugate reaction mixture comprising, as separately migratable species, at least a free enzyme component and a poplulation of enzyme-antibody conjugates comprising one or more of the enzyme component convalently coupled to one or more of the antibody component. The resulting conjugate preparations are substantially pure and are therefore particularly useful as labeled reagents in immunometric assays.

Abstract: An immobilized hapten reagent for use in a specific binding assay for the determination of a hapten or binding analog thereof in a liquid test sample and methods for preparing and using the immobilized hapten reagent. The immobilized hapten reagent comprises a hapten moiety covalenty linked substantially only to the external surface of a gel particle. The immobilized hapten reagent is prepared by forming a reaction mixture comprising the hapten moiety and the carrier material in a nonswelling solvent wherein the gel particle is substantially impervious to the hapten moiety and wherein an analytically insignificant amount of the hapten moiety is nonspecifically bound to the gel particle. The immobilized hapten reagent is characterized by being substantially stable in aqueous solutions and exhibits an insignificant amount of leakage of the hapten moiety into a liquid test medium.

Abstract: Analytical reagent reaction vessel and method for performing sequential analytical assay procedures to determine an analyte in a liquid test sample. The reaction vessel is in the form of a closed container having a substantially horizontal axis of rotation and incorporated with one or more analytical reagents for performing a desired analytical assay procedure. The reaction vessel is designed to provide free gravitational movement of a liquid test sample disposed therein. The analytical reagents are incorporated into the reaction vessel such that they are contacted with a liquid test sample in a desired order or sequence. A liquid test sample disposed in the reaction vessel is capable of being manipulated therein by rotating the reaction vessel about the horizontal axis wherein the liquid test sample is transported by gravity.

Abstract: An analytical method for determining the relative amount of a particular hemoglobin derivative, e.g., glycated hemoglobin, in a blood sample wherein the amounts of both total hemoglobin and the hemoglobin derivative are measured and related mathematically, e.g., as a percentage. The blood sample is treated with the combination of a thiocyanate salt and an oxidant to denature the hemoglobin in the sample and to convert hemoglobin to met-hemoglobin. Met-hemoglobin is measured spectrophotometrically to give the amount of total hemoglobin present, and the denatured hemoglobin derivative can be distinguished and measured by immunoassay. The presence of the oxidant in the denaturant solution has been found to increase the rate of enaturation of hemoglobin thereby reducing the overall assay time and improving the reliability of the determination.

Abstract: A method for chemically coupling a controllable number of ligands, e.g., haptens, to a polymeric material by derivatization of the polymer with an activating agent to introduce a couplable functional group. The derivatization is performed in the presence of a blocking agent which is reactive with the same functionality on the polymer as the activating agent. By reacting the polymer with a mixture of a predetermined ratio of excess amounts of the activating and blocking agents, a controllable number of couplable functional groups are introduced to the polymer for subsequent linkage to the desired ligand. The resulting polymer-ligand conjugates are useful as reagents in immunoassays, particularly immunoturbidimetric assays.

Abstract: A multizone test device for the determination of analyte in a liquid test medium. The test device preferably comprises multilayers including a first layer comprising a solid, porous matrix incorporated with a reversibly immobilized first reagent and a second layer comprising a solid, porous matrix incorporated with a second reagent which interacts with the first reagent and analyte from the liquid test medium to provide a detectable signal. A reversible binding interaction between the first reagent and the matrix of the first layer prevents the first reagent from prematurely migrating into the second layer during manufacture of the device and prior to application of the liquid test medium to the test device. The reversible binding interaction is sufficiently disruptable by contact of the first layer with the liquid test medium to release and thereby render diffusible an analytically effective amount of the first reagent within the test device.

Abstract: A latex agglutination immunoassay method for determining an analyte in a blood sample in which the pH of the reaction mixture is maintained at about 8.5 or greater in order to overcome nonspecific agglutination of latex particles by hemoglobin present in the sample. The method is particularly applicable to the determination of glycated hemoglobin, e.g., Hb Alc. In another embodiment, native hemoglobin (Ao) can be determined based on its ability to cause agglutination of latex particles suspended in an aqueous solution having a pH of about 8 or below.

Abstract: A monoclonal antibody specific for DNA.multidot.RNA duplexes, particularly DNA.multidot.RNA heteropolymer duplexes, characterized by having cross-reactivity for binding to single- or double-stranded DNA or RNA as measured by competitive immunoassay of less than about 1:1000, and preferably less than 1:10,000, and an affinity for DNA.multidot.RNA heteropolymer duplexes greater than 10.sup.9 L/mole. The monoclonal antibody is prepared by conventional somatic cell hybridization techniques wherein the host animal is preferably immunized with an immunogen comprising a random DNA.multidot.RNA heteropolymer. The antibody, particularly in a labeled form, is useful in the specific detection of DNA.multidot.RNA duplexes in a test medium such as a nucleic acid hybridization assay mixture.

Abstract: Enzyme-labeled antibody reagents wherein the enzyme and an antibody reagent, e.g., whole polyclonal or monoclonal antibody or a fragment thereof, are covalently linked through a bis-maleimidopolyalkyleneglycol bridge group. The reagents are useful in immunoassay and other methods for detecting an antigen or hapten that can be bound by the antibody reagent. The conjugates are highly stable and water soluble, and are characterized by a high degree of immunoreactivity and enzyme activity.

Abstract: A specific binding assay, e.g., immunoassay, method and reagent system wherein a structural analog of the label portion of the labeled reagent is included in the reaction mixture to interact with potentially interfering substances in the test sample. The label analog is selected or designed to be substantially inactive in the assay detection system. The invention improves the precision of the assays, particularly homogeneous immunoassays, by significantly reducing or eliminating the variable interaction, e.g., binding, of the labeled reagent by interfering substances in the test sample.

Abstract: Immobilization of nucleic acids, e.g., DNA and RNA, by contact with a solid support comprising nylon whose amide groups have been partially solvolyzed. Solvolysis of the nylon support can be accomplished by treatment with an alkylating agent such as a trialkyloxonium salt under anhydrous conditions followed by addition of water. The immobilized nucleic acid is particularly useful as an immobilized probe in hybridization assays to detect specific polynucleotide sequences in a test sample.

Abstract: Immobilization of nucleic acids, e.g., DNA and RNA, by contact with a solid support comprising nylon having amide groups that have been derivatized to amidine residues. Derivatization of the nylon support can be accomplished by treatment with an alkylating agent such as a trialkyloxonium salt under anhydrous conditions followed by reaction with an amine. The immobilized nucleic acid is particularly useful as an immobilized probe in hybridization assays to detect specific polynucleotide sequences in a test sample.

Abstract: The reactant advantageously is an enzymatic reactant such as an enzyme substrate or coenzyme. The activity of the conjugated reactant as a constituent of a predetermined reaction is affected by reaction between the specific binding substance in the conjugate and a specific binding counterpart thereto. The presence of a ligand in a liquid medium may be determined using competitive or displacement binding or sequential saturation techniques wherein the specific binding substance in the conjugate is the ligand or a specific binding analog thereof, or using a direct binding technique wherein the specific binding substance is a specific binding partner of the ligand. The effect of the specific binding reaction on the activity of the conjugated reactant is related to the presence or amount of the ligand in the liquid medium tested.

Abstract: Optical indicator chalcogen (selenide or sulfide) compounds responsive to oxidants, e.g., hydrogen peroxide, and methods for preparing and using such indicators. Upon oxidation the resulting intermediate undergoes spontaneous elimination of the chalcogen residue to yield a signal compound which provides an optical signal such as fluorescence. Preferred fluorogenic indicator compounds are 3-chalcogen-3,4-dihydrocoumarin derivatives and 3-chalcogen-3,4-dihydro-2-quinolone derivatives. Such indicator compounds provide highly fluorescent products upon oxidation by hydrogen peroxide and are useful in analytical systems which generate hydrogen peroxide in response to the analyte under determination.

Abstract: A nucleic acid hybridization assay involving a labeled probe and formation of a hybrid having epitopes for an antibody reagent. The label provides a detectable response which is measurably different when the labeled probe is comprised in a hybrid that is bound by the antibody reagent compared to when not comprised in such a hybrid. Particularly useful antibody reagents are antibodies such as anti-DNA.RNA, anti-RNA.RNA and antibodies to intercalated duplexes which do not bind substantially to single stranded nucleic acids. Modulation of the label response can be accomplished in a variety of ways such as by steric inactivation or hindrance or by labeling the antibody reagent with a second label which interacts with the first label. The assay is particularly advantageous because no separation of hybridized and unhybridized probe is required.

Abstract: The rate of hybridization between two complementary polynucleotide segments in an aqueous medium is increased by the presence of the anionic polymers polyacrylate and polymethacrylate. The acceleration effect is particularly useful in nucleic acid hybridization assays involving immobilization of sample nucleic acids and the use of labeled probes. Nonspecific binding of probe to nitrocellulose supports is substantially lower in the presence of the present polymers than in the presence of the prior art accelerator dextran sulfate. Polyacrylate is particularly advantageous since it has been found to be effective at low concentrations and is significantly less expensive than the prior art compound.

Abstract: A specific binding assay method and reagent system based on the use of an inhibitory anti-enzyme, e.g., antibody or fragment thereof, as the label component. Such method and reagent system have been improved by selection of anti-(glucose-6-phosphate dehydrogenase) [anti-G6PDH] as the anti-enzyme label. The improved label is monitored by its ability to inhibit G6PDH. The resulting assay is more sensitive, requires lesser quantities of reagents, is less susceptible to sample interferences, and employs a reagent system having greater stability than the published prior art method employing an anti-peroxidase label. The present invention is particularly applicable to homogeneous immunoassays for determining substances appearing at low concentrations in biological fluids such as urine and serum.