Abstract: A method for performing a multiplexed diagnostic assay, such as for two or more different targets in a sample, is described. One embodiment comprised contacting the sample with two or more specific binding moieties that bind specifically to two or more different targets. The two or more specific binding moieties are conjugated to different haptens, and at least one of the haptens is an oxazole, a pyrazole, a thiazole, a nitroaryl compound other than dinitrophenyl, a benzofurazan, a triterpene, a urea, a thiourea, a rotenoid, a coumarin, a cyclolignan, a heterobiaryl, an azo aryl, or a benzodiazepine. The sample is contacted with two or more different anti-hapten antibodies that can be detected separately. The two or more different anti-hapten antibodies may be conjugated to different detectable labels.

Abstract: Disclosed embodiments concern quantifying a biomolecule conjugated to a nanoparticle. Quantifying typically comprises determining the number of biomolecules per nanoparticle. Any suitable biomolecule can be used, including but not limited to, amino acids, peptides, proteins, haptens, nucleic acids, oligonucleotides, DNA, RNA, and combinations thereof. A single type of biomolecule may be conjugated to the nanoparticle, more than one biomolecule of a particular class may be conjugated to the nanoparticle, or two or more classes of biomolecules may be conjugated to the nanoparticle. Certain disclosed embodiments comprise enzymatically or chemically digesting a biomolecule conjugated to the nanoparticle, or displacing a biomolecule using ligand-exchange chemistry.

Abstract: A method is disclosed for making a conjugate of two molecules using a hydrazide thiol linker. In a particular working embodiment, an Fc-specific antibody-enzyme conjugate is made using the method and demonstrated to provide exceptional staining sensitivity and specificity in immunohistochemical and in situ hybridization assays.

Abstract: The invention is directed to enhanced methods for detecting an analyte of interest in situ, by immunoassay, or by hybridization comprising binding an enzyme-labeled conjugate molecule to an analyte of interest in the presence of a redox-inactive reductive species and a soluble metal ion. The enzyme catalyzes the conversion of the inactive reductive species to an active reducing agent, which in turn reduces the metal ion to a metal atom thereby providing an enhanced means of detecting the analyte via metal deposition.

Abstract: A method for performing a multiplexed diagnostic assay, such as for two or more different targets in a sample, is described. One embodiment comprised contacting the sample with two or more specific binding moieties that bind specifically to two or more different targets. The two or more specific binding moieties are conjugated to different haptens, and at least one of the haptens is an oxazole, a pyrazole, a thiazole, a nitroaryl compound other than dinitrophenyl, a benzofurazan, a triterpene, a urea, a thiourea, a rotenoid, a coumarin, a cyclolignan, a heterobiaryl, an azo aryl, or a benzodiazepine. The sample is contacted with two or more different anti-hapten antibodies that can be detected separately. The two or more different anti-hapten antibodies may be conjugated to different detectable labels.

Abstract: Disclosed embodiments concern quantifying a biomolecule conjugated to a nanoparticle. Quantifying typically comprises determining the number of biomolecules per nanoparticle. Any suitable biomolecule can be used, including but not limited to, amino acids, peptides, proteins, haptens, nucleic acids, oligonucleotides, DNA, RNA, and combinations thereof. A single type of biomolecule may be conjugated to the nanoparticle, more than one biomolecule of a particular class may be conjugated to the nanoparticle, or two or more classes of biomolecules may be conjugated to the nanoparticle. Certain disclosed embodiments comprise enzymatically or chemically digesting a biomolecule conjugated to the nanoparticle, or displacing a biomolecule using ligand-exchange chemistry.

Abstract: The invention is directed to enhanced methods for detecting an analyte of interest in situ, by immunoassay, or by hybridization comprising binding an enzyme-labeled conjugate molecule to an analyte of interest in the presence of a redox-inactive reductive species and a soluble metal ion. The enzyme catalyzes the conversion of the inactive reductive species to an active reducing agent, which in turn reduces the metal ion to a metal atom thereby providing an enhanced means of detecting the analyte via metal deposition.

Abstract: The invention is directed to novel compositions of matter and methods of detecting in situ an immunohistochemical epitope or nucleic acid sequence of interest in a biological sample comprising binding an enzyme-labeled conjugate molecule to the epitope or sequence of interest in the presence of a redox-inactive reductive species and a soluble metal ion, thereby facilitating the reduction of the metal ion to a metal atom at or about the point where the enzyme is anchored. Novel phosphate derivatives of reducing agents are described that when exposed to a phosphatase are activated to their reducing form, thereby reducing metal ions to insoluble metal.

Abstract: Conjugate compositions are disclosed that include a specific-binding moiety covalently coupled to a nanoparticle through a heterobifunctional polyalkyleneglycol linker. In one embodiment, a conjugates is provided that includes a specific-binding moiety and a fluorescent nanoparticle coupled by a heterobifunctional PEG linker. Fluorescent conjugates according to the disclosure can provide exceptionally intense and stable signals for immunohistochemical and in situ hybridization assays on tissue sections and cytology samples, and enable multiplexing of such assays.

Abstract: Antibody/signal-generating moiety conjugates are disclosed that include an antibody covalently linked to a signal-generating moiety through a heterobifunctional polyalkyleneglycol linker. The disclosed conjugates show exceptional signal-generation in immunohistochemical and in situ hybridization assays on tissue sections and cytology samples. In one embodiment, enzyme-metallographic detection of nucleic acid sequences with hapten-labeled probes can be accomplished using the disclosed conjugates as a primary antibody without amplification.

Abstract: Solutions exhibiting little or no evaporative loss at elevated temperatures, i.e., in excess of 100° C., are employed in place of conventional aqueous-based antigen retrieval solutions.

Abstract: A method for preparing a nucleic acid probe is provided. The method comprises forming an activated cytosine or cytidine by a bisulfite catalyzed reaction; and covalently linking a reporter molecule to the activated cytosine or cytidine, wherein said activating step, said covalently linking step, or both are conducted in the presence of microwave energy. Also provided by the invention are nucleic acid probes.

Abstract: Solutions exhibiting little or no evaporative loss at elevated temperatures, i.e., in excess of 100° C., are employed in place of conventional aqueous-based antigen retrieval solutions.

Abstract: Certain disclosed embodiments of the present invention concern the synthesis, derivatization, conjugation to immunoglobulins and signal amplification based on discrete, relatively short polymers having plural reactive functional groups that react with plural molecules of interest. Reactive functional groups, such as hydrazides, may be derivatized with a variety of detectable labels, particularly haptens. The remaining reactive functional groups may be conjugated directly to a specific binding molecule, such as to the oxidized carbohydrate of the Fc region of the antibody. Disclosed conjugates display large signal amplification as compared to those based on molecules derivatized with single haptens, and are useful for assay methods, particularly multiplexed assays.

Abstract: Disclosed embodiments concern quantifying a biomolecule conjugated to a nanoparticle. Quantifying typically comprises determining the number of biomolecules per nanoparticle. Any suitable biomolecule can be used, including but not limited to, amino acids, peptides, proteins, haptens, nucleic acids, oligonucleotides, DNA, RNA, and combinations thereof. A single type of biomolecule may be conjugated to the nanoparticle, more than one biomolecule of a particular class may be conjugated to the nanoparticle, or two or more classes of biomolecules may be conjugated to the nanoparticle. Certain disclosed embodiments comprise enzymatically or chemically digesting a biomolecule conjugated to the nanoparticle, or displacing a biomolecule using ligand-exchange chemistry.

Abstract: A method for performing a multiplexed diagnostic assay, such as for two or more different targets in a sample, is described. One embodiment comprised contacting the sample with two or more specific binding moieties that bind specifically to two or more different targets. The two or more specific binding moieties are conjugated to different haptens, and at least one of the haptens is an oxazole, a pyrazole, a thiazole, a nitroaryl compound other than dinitrophenyl, a benzofurazan, a triterpene, a urea, a thiourea, a rotenoid, a coumarin, a cyclolignan, a heterobiaryl, an azo aryl, or a benzodiazepine. The sample is contacted with two or more different anti-hapten antibodies that can be detected separately. The two or more different anti-hapten antibodies may be conjugated to different detectable labels.

Abstract: The invention is directed to methods and compositions for deparaffinizing paraffin-embedded biological samples for subsequent tissue staining. The compositions are microemulsions that may include water/oil/surfactant microemulsions, and optionally a cosurfactant. The microemulsions enable deparaffinization without the use of xylene or toluene, and also enable solvent exchange without the use of intermediary alcohol dehydration or alcohol rehydration compositions.

Abstract: A stabilized hematoxylin composition is disclosed that includes one or both of a host compound and an antioxidant. The disclosed composition exhibits sufficient stability to be utilized in an automated staining process without undue degradation prior to use of the composition to stain a biological sample. Methods of using and making the stabilized composition also are disclosed.

Abstract: A method and composition are disclosed that are useful for processing biological samples. In one aspect, a biological sample such as a tissue section is treated using a histochemical technique and is contacted with a lipid compound during the process to enhance the definition of cellular and sub-cellular features that are observable in the sample when it is viewed microscopically. In other aspects, a coverslipping composition that includes a lipid compound and a method of coverslipping a sample using the coverslipping composition are disclosed.

Abstract: A method is disclosed for making a conjugate of two molecules using a hydrazide thiol linker. In a particular working embodiment, an Fc-specific antibody-enzyme conjugate is made using the method and demonstrated to provide exceptional staining sensitivity and specificity in immunohistochemical and in situ hybridization assays.