Abstract: Multiplexed determinations of large numbers of events are achieved in an accurate and simple manner by using a multitude of primer reagents in combination with different capture reagents that serve for sequestering all the reagents at a single site, followed by independent release of subsets of the primer reagents using differential release conditions. Also included as part of the primer reagents may be identifiers, which serve to identify a particular characteristic. The method is illustrated using primers with sequences for initiation of chain extension that are joined to or serve as a capture sequence, and where the extended primer has an identifier. After extending the primer, the extended primers are sequestered via the capture sequence onto a sequestering agent, sequentially released and the released extended primers analyzed to provide multiplexed determinations.

Abstract: A method is disclosed for modifying an oligonucleotide, which method has application to the detection of a polynucleotide analyte. An oligonucleotide is reversibly hybridized with a polynucleotide, for example, a polynucleotide analyte, in the presence of a 5′-nuclease under isothermal conditions. The polynucleotide analyte serves as a recognition element to enable a 5′-nuclease to cleave the oligonucleotide to provide (i) a first fragment that is substantially non-hybridizable to the polynucleotide analyte and (ii) a second fragment that lies 3′ of the first fragment (in the intact oligonucleotide) and is substantially hybridizable to the polynucleotide analyte. At least a 100-fold molar excess of the first fragment and/or the second fragment are obtained relative to the molar amount of the polynucleotide analyte. The presence of the first fragment and/or the second fragment is detected, the presence thereof indicating the presence of the polynucleotide analyte.

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: Receptors are disclosed that are antibodies that exhibit a binding affinity for an immune complex of a monoepitopic antigen and an antibody for such antigen that is substantially greater than the binding affinity for the monoepitopic antigen or the antibody for the monoepitopic antigen apart from the immune complex. Normally, the monoepitopic antigen has a molecular weight less than 1500 and is an organic compound. The antibodies of the present invention find use in a method for determining a monoepitopic antigen in a sample suspected of containing such antigen. The method comprises forming an immune sandwich complex comprising the monoepitopic antigen or an analog thereof, a first monoclonal antibody that binds to the monoepitopic antigen, and a second monoclonal antibody that is an antibody of the present invention and detecting the immune sandwich complex.

Abstract: A method is disclosed for producing at least one copy of a pair of complementary single stranded polynucleotides. The method comprises forming, in the presence of nucleoside triphosphates and template dependent polynucleotide polymerase along each of the complementary single stranded polynucleotides, an extension of a polynucleotide primer. The polynucleotide primer is comprised of at least a sequence of 16 nucleotides terminating at its 3′ end in a 2 to 9 nucleotide sequence (S1), which is complementary with the 3′ ends of both of the complementary single stranded polynucleotides. The polynucleotide primer has at least an 8 nucleotide sequence (S2) that is 5′ of S1, where S2 is 50 to 80% complementary to the nucleotide sequences contiguous with the 3′ ends of the complementary single stranded polynucleotides. The extended polynucleotide primer and the single stranded polynucleotides are then dissociated.

Abstract: The present invention provides methods and apparatus for mixing two or more liquids and detecting light emitted by the mixture. The method comprises forming a liquid droplet containing two or more liquids on a substantially planar surface in containerless containment on the surface and causing the droplet to deform in an essentially zero air flow environment thereby mixing the liquids and surrounding the planar surface with a reflective housing and photodetector. An apparatus of the invention comprises (a) a substantially planar support, (b) means for dispensing liquids onto the support to form a droplet, and (c) non-evaporative means for causing the droplet to deform without deforming the support thereby mixing the liquids and a two part reflective housing and photodetector which is moveable. The drop can be deformed by, for example, application of acoustic energy or a variable electrostatic field. The methods and apparatus have particular application to the determination of an analyte.

Abstract: A method is disclosed for detecting the presence of a difference between two related nucleic acid sequences. In the method a complex is formed comprising both strands of each sequence. Each member of at least one pair of non-complementary strands within the complex have labels. The association of the labels as part of the complex is determined as an indication of the presence of a difference between the two related sequences. The complex generally comprises a Holliday junction. In one aspect a medium suspected of containing said two related nucleic acid sequences is treated to provide partial duplexes having non-complementary tailed portions at one end. The double stranded portions of the partial duplexes are identical except for said difference.

Abstract: A non-immunochemical method is disclosed for determining homocysteine in a sample. A combination of the sample and a first compound that comprises a cis-1,4-dioxo-2-butene moiety or hydrolytically derived precursor thereto is provided in a liquid medium. The combination is subjected to conditions under which homocysteine reacts with the cis-1,4-dioxo-2-butene moiety or hydrolytically derived precursor thereto to form a product, which is then detected without separation of the product from the medium. A second compound such as a mercaptan containing reagent or an antibody for cysteine can optionally be included to facilitate discrimination of the product formed from homocysteine from products formed from other amines in the sample. The product from the reaction of homocysteine has a six-member ring fused to an unsaturated five-member ring. The junction of the rings is comprised of the nitrogen of homocysteine and an atom that comprises a double bond.

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

Abstract: 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 present invention relates to compounds that are bis-biotins. These compounds comprise two biotinyl radicals connected by a chain of atoms, usually at least 16 atoms in length. The bis-biotin is conjugated to a member of a specific binding pair ("sbp member") wherein the chain is not part of the sbp member. Also disclosed are compositions comprising a complex of avidin and a bis-biotin as described above. The compounds and compositions of the invention find use in an assay for an analyte wherein there is employed a reagent system comprising an avidin reagent and a biotin reagent. The improvement of the present invention comprises using as the biotin reagent a bis-biotin as described above. Also disclosed are kits comprising the present bis-biotins and methods of preparing a bis-biotinylated conjugate of a member of a specific binding pair ("sbp member") for use in a specific binding assay.

Abstract: Compositions, methods and kits are disclosed. The compositions comprise a matrix having incorporated therein a label capable of being modified by singlet oxygen. A catalyst capable of catalyzing the formation of singlet oxygen is bound to the matrix, which permits the diffusion of singlet oxygen therein. The compositions may be used in methods for detecting hydrogen peroxide or a compound capable of generating hydrogen peroxide. A sample suspected of containing such compound is combined with a composition in accordance with the present invention. The combination is subjected to conditions wherein such compound generates hydrogen peroxide. The reaction of singlet oxygen with the label is determined, the reaction thereof indicating the presence of the compound capable of generating hydrogen peroxide.

Abstract: A method is disclosed for determining the presence of a polynucleotide analyte in a sample suspected of containing the analyte. The method comprises (a) forming as a result of the presence of an analyte a single stranded polynucleotide comprising a target polynucleotide binding sequence flanked by first and second polynucleotide sequences that differ from the sequence of the analyte or a sequence complementary to the analyte sequence, (b) forming multiple copies of the single stranded polynucleotide, and (c) detecting the single stranded polynucleotide. Also disclosed is a method of producing at least one copy of a single stranded polynucleotide.

Abstract: A method is disclosed for modifying an oligonucleotide, which method has application to the detection of a polynucleotide analyte. An oligonucleotide is reversibly hybridized with a polynucleotide, for example, a polynucleotide analyte, in the presence of a 5'-nuclease under isothermal conditions. The polynucleotide analyte serves as a recognition element to enable a 5'-nuclease to cleave the oligonucleotide to provide (i) a first fragment that is substantially non-hybridizable to the polynucleotide analyte and (ii) a second fragment that lies 3' of the first fragment (in the intact oligonucleotide) and is substantially hybridizable to the polynucleotide analyte. At least a 100-fold molar excess of the first fragment and/or the second fragment are obtained relative to the molar amount of the polynucleotide analyte. The presence of the first fragment and/or the second fragment is detected, the presence thereof indicating the presence of the polynucleotide analyte.

Abstract: A method is disclosed for modifying an oligonucleotide, which method has application to the detection of a polynucleotide analyte. An oligonucleotide is reversibly hybridized with a polynucleotide, for example, a polynucleotide analyte, in the presence of a 5'-nuclease under isothermal conditions. The polynucleotide analyte serves as a recognition element to enable a 5'-nuclease to cleave the oligonucleotide to provide (i) a first fragment that is substantially non-hybridizable to the polynucleotide analyte and (ii) a second fragment that lies 3' of the first fragment (in the intact oligonucleotide) and is substantially hybridizable to the polynucleotide analyte. At least a 100-fold molar excess of the first fragment and/or the second fragment are obtained relative to the molar amount of the polynucleotide analyte. The presence of the first fragment and/or the second fragment is detected, the presence thereof indicating the presence of the polynucleotide analyte.

Abstract: The present invention concerns methods for masking inhomogeneity of a member of a specific binding pair (sbp) employed in a capillary electroseparation. The method comprises binding the sbp member to synthetic particles that become localized during capillary electroseparation. Also disclosed is one embodiment of the present invention, which is a method for conducting a capillary electroseparation specific binding assay. The method involves the electroseparation of a labeled first member of a specific binding pair that is bound in a complex from labeled first member that is not bound in the complex. The complex comprises the first member and a second member of a specific binding pair. A combination is provided comprising a sample suspected of containing an analyte, a labeled first member of a specific binding pair, and a second member of a specific binding pair under conditions for forming a complex between labeled first member and the second member.

Abstract: A method for the detection of a polynucleotide target sequence is described. The method involves the formation of a covalent or non-covalent bonded pair of nucleotide sequences formed in response to a target polynucleotide sequence, adding nucleotide sequence specific binding proteins each capable of binding one member of the pair of nucleotide sequences, and detecting the specific binding proteins complexed to the pair of nucleotide sequences.

Abstract: Methods for immunoassay of analytes employing mutant glucose-6-phosphate dehydrogenase (G6PDH) enzymes as labels. In particular, the invention relates to the use of conjugates of an analyte or analyte analog and a mutant NAD.sup.+ dependent G6PDH differing from any precursor G6PDH by the deletion, substitution, or insertion, or any combination thereof of at least one amino acid per subunit. The invention also involves the construction of several mutations in precursor glucose-6-phosphate dehydrogenase (G6PDH) enzymes. Typically, the mutations involve deletion or substitution of one or more lysine residues, or introduction of one or more cysteine residues by insertion of cysteine to precursor G6PDH or substitution of precursor G6PDH amino acids residues with cysteine.

Abstract: A method for the detection of a polynucleotide target sequence is described. The method involves the formation of a covalent or non-covalent bonded pair of nucleotide sequences formed in response to a target polynucleotide sequence, adding nucleotide sequence specific binding proteins each capable of binding one member of the pair of nucleotide sequences, and detecting the specific binding proteins completed to the pair of nucleotide sequences.