Abstract: The present invention relates to a method for detecting the amount of a target polynucleotide in a sample. A combination is provided in a medium. The combination comprises (i) a sample suspected of containing the target polynucleotide, the target polynucleotide being in single stranded form, (ii) a reference polynucleotide comprising a sequence that is common with a sequence of the target polynucleotide, and (iii) a predetermined amount of an oligonucleotide probe that has a sequence that hybridizes with the sequence that is common. The combination is subjected to conditions for amplifying the target polynucleotide and the reference polynucleotide. The conditions permit formation of substantially non-dissociative complexes of the target polynucleotide and the reference polynucleotide, respectively, with the oligonucleotide probe. Furthermore, the predetermined amount of the oligonucleotide probe is less than the expected amount of the amplified target polynucleotide.

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: Compounds that are modified polysaccharides having pendant aldehyde functionalities are disclosed. Each of the aldehyde functionalities is attached through a linker to a position corresponding to a hydrogen atom of a different hydroxyl group of unmodified polysaccharide. Also disclosed is a method for introducing an amine-reactive functionality into a dextran. The method comprises (a) reacting the dextran with an alkylating agent having a functionality that reacts with an hydroxyl group of the dextran thereby forming an alkylated dextran wherein the alkylating agent has an olefin group and (b) treating the alkylated dextran to convert the olefin group to an amine-reactive functionality. A polysaccharide can be conjugated to a biomolecule by carrying out the above method and reacting the amine-reactive functionality with an amine functionality on the biomolecule to produce polysaccharide conjugated to the biomolecule.

Abstract: The present invention relates to 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+ 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 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: 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 relates to a method for detecting the amount of a target polynucleotide in a sample. A combination is provided in a medium. The combination comprises (i) a sample suspected of containing the target polynucleotide, the target polynucleotide being in single stranded form, (ii) a reference polynucleotide comprising a sequence that is common with a sequence of the target polynucleotide, and (iii) a predetermined amount of an oligonucleotide probe that has a sequence that hybridizes with the sequence that is common. The combination is subjected to conditions for amplifying the target polynucleotide and the reference polynucleotide. The conditions permit formation of substantially non-dissociative complexes of the target polynucleotide and the reference polynucleotide, respectively, with the oligonucleotide probe. Furthermore, the predetermined amount of the oligonucleotide probe is less than the expected amount of the amplified target polynucleotide.

Abstract: The current invention relates to the use of luminescent oxygen channeling immunoassay (“LOCI”) technology to monitor amplification reactions, especially polymerase chain reactions (“PCR”). More specifically, the current invention involves the use of LOCI to measure the kinetics of a PCR reaction in an all-in-one assay format in order to quantitatively and qualitatively detect a target polynucleotide.

Abstract: The invention relates to methods of determining the presence or amount of an analyte in a sample suspected of containing the analyte, said method comprising the steps of: (a) bringing together in an aqueous medium to form a mixture: (i) the sample; (ii) at least one specific binder for the analyte; (iii) a first binding agent coupled to either (1) exogenous analyte or (2) the specific binder for the analyte; (iv) a support comprising a second binding agent; b) adding an activator to the mixture, wherein the activator binds the first binding agent and the second binding agent of the support to immobilize the first binding agent; c)determining the amount of the analyte in the sample by detecting the immobilized first binding agent, the presence or amount thereof being related to the presence or amount of the analyte in the sample.

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 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: The present invention provides novel cyclosporine C (CsC) derivatives having improved protein conjugatibility and hydrolytic stability. The present invention further provides a CsC derivative conjugated to a carrier, e.g., a solid support. Preferably, the solid support is a latex or magnetic particle.

Abstract: The present invention relates to a method for selectively extending an oligonucleotide primer along a specific target polynucleotide sequence in a mixture of polynucleotides. A combination is provided comprising the mixture, an oligonucleotide primer having a modification, and a binding substance for the modification wherein the binding substance binds to the oligonucleotide and prevents the extension of the oligonucleotide along the target polynucleotide sequence. The temperature of the combination is adjusted to a level sufficient to irreversibly denature the binding substance and permit the extension of the oligonucleotide primer along the specific target polynucleotide sequence. The invention has particular application in the amplification of nucleic acids. Also disclosed are kits for carrying out a method in accordance with the present invention.

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 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 provides novel cyclosporine C (CsC) derivatives having improved protein conjugatibility and hydrolytic stability. The present invention further provides a CsC derivative conjugated to a carrier, e.g., a solid support. Preferably, the solid support is a latex or magnetic particle.

Abstract: Compounds that are modified polysaccharides having pendant aldehyde functionalities are disclosed. Each of the aldehyde functionalities is attached through a linker to a position corresponding to a hydrogen atom of a different hydroxyl group of unmodified polysaccharide. Also disclosed is a method for introducing an amine-reactive functionality into a dextran. The method comprises (a) reacting the dextran with an alkylating agent having a functionality that reacts with an hydroxyl group of the dextran thereby forming an alkylated dextran wherein the alkylating agent has an olefin group and (b) treating the alkylated dextran to convert the olefin group to an amine-reactive functionality. A polysaccharide can be conjugated to a biomolecule by carrying out the above method and reacting the amine-reactive functionality with an amine functionality on the biomolecule to produce polysaccharide conjugated to the biomolecule.