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    Multiple enzyme assays
    1.
    发明授权
    Multiple enzyme assays 有权
    多重酶测定

    公开(公告)号：US06586196B1

    公开(公告)日：2003-07-01

    申请号：US09459982

    申请日：1999-12-14

    申请人： Irena Bronstein ,  Christopher Martin ,  Corinne Olesen ,  John Voyta ,  Yu-xin Yan

    发明人： Irena Bronstein ,  Christopher Martin ,  Corinne Olesen ,  John Voyta ,  Yu-xin Yan

    IPC分类号： C12Q142

    CPC分类号： C12Q1/6897 ,  C12Q1/00 ,  C12Q1/34 ,  C12Q1/42 ,  C12Q1/66 ,  G01N2333/916 ,  G01N2333/924 ,  G01N2333/95

    摘要： The present invention discloses multiple enzyme assays which measure the activity of at least one endogenous enzyme in a single aliquot and a method of measuring the activity of multiple enzymes in an aliquot of a cell extract, wherein at least one of the enzymes is an endogenous enzyme. In one embodiment of the invention the activity of a first enzyme is quantified by measuring the light signal produced by degradation of a first enzyme substrate by the first enzyme and the activity of the second enzyme is quantified by measuring the light signal produced by the degradation of a second substrate. In the method of the present invention, both quantifications are performed on the same aliquot of cell extract. Different embodiments of the present invention provide for the detection of more than one endogenous enzyme and for the detection of at least one reporter enzyme and at least one endogenous enzyme. The present invention also discloses kits for detecting the activity of multiple enzymes.

    摘要翻译： 本发明公开了测量单一等分试样中至少一种内源性酶的活性的多种酶测定法和测量细胞提取物等分试样中多种酶的活性的方法，其中至少一种酶是内源性酶 。 在本发明的一个实施方案中，通过测量由第一酶降解第一酶底物产生的光信号来定量第一酶的活性，并且通过测量由第一酶降解产生的光信号来量化第二酶的活性 第二基板。 在本发明的方法中，两种定量在相同等分的细胞提取物上进行。 本发明的不同实施方案提供了多于一种内源性酶的检测和用于检测至少一种报道酶和至少一种内源性酶。 本发明还公开了用于检测多种酶的活性的试剂盒。

    Dioxetane labeled probes and detection assays employing the same
    2.
    发明授权
    Dioxetane labeled probes and detection assays employing the same 有权
    二氧杂环丁烷标记的探针和使用其的检测测定

    公开(公告)号：US06451531B1

    公开(公告)日：2002-09-17

    申请号：US09340726

    申请日：1999-06-29

    申请人： Irena Bronstein ,  Brooks Edwards ,  Christopher Martin ,  John Voyta

    发明人： Irena Bronstein ,  Brooks Edwards ,  Christopher Martin ,  John Voyta

    IPC分类号： C12Q168

    CPC分类号： C07H21/00 ,  C07D321/00 ,  C12Q1/6816 ,  C12Q1/686 ,  Y02P20/55 ,  C12Q2561/101 ,  C12Q2565/113

    摘要： Probes labeled with 1,2-dioxetane precursors can be employed in a variety of assays. The probes may be nucleic acid, peptide nucleic acid, proteins including enzyme, antibody or antigen, steroid, carbohydrate, drug or non-drug hapten. The probe is provided with a 1,2-dioxetane precursor bound thereto, generally either covalently, or a strong ligand bond. The dioxetane precursor moiety is converted to a bound 1,2-dioxetane by exposure to singlet oxygen. These dioxetane (labels) either spontaneously decompose, or are induced to decompose by an appropriate trigger to release light. The trigger may be a change in pH temperature, or an agent which removes a protective group. Assay formats in which these 1,2-dioxetane labeled probes and referents may be used to include hybridization assays, immuno assays, gel-based assays and Capillary Zone Electrophoresis.

    摘要翻译： 用1,2-二氧杂环丁烷前体标记的探针可用于多种测定。 探针可以是核酸，肽核酸，蛋白质，包括酶，抗体或抗原，类固醇，碳水化合物，药物或非药物半抗原。 探针具有与其结合的1,2-二氧杂环丁烷前体，通常共价或强配体键。 通过暴露于单线态氧将二氧杂环丁烷前体部分转化为结合的1,2-二氧环乙烷。 这些二氧杂环丁烷（标记）会自发分解，或被适当的触发剂诱导分解以释放光。 触发可能是pH温度的变化，或者是除去保护基团的试剂。 这些1,2-二氧杂环丁烷标记的探针和指示物可用于包括杂交测定，免疫测定，基于凝胶的测定和毛细管区域电泳的测定形式。

    Dioxetane-precursor-labeled probes and detection assays employing the same
    3.
    发明授权
    Dioxetane-precursor-labeled probes and detection assays employing the same 失效
    二氧杂环丁烷 - 前体标记的探针和使用其的检测测定

    公开(公告)号：US5800999A

    公开(公告)日：1998-09-01

    申请号：US767479

    申请日：1996-12-16

    申请人： Irena Bronstein ,  Brooks Edwards ,  Christopher Martin ,  John Voyta

    发明人： Irena Bronstein ,  Brooks Edwards ,  Christopher Martin ,  John Voyta

    IPC分类号： G01N33/532 ,  C07D321/00 ,  C07H21/00 ,  C07H21/02 ,  C07H21/04 ,  C12N15/09 ,  C12Q1/68 ,  C12N15/11

    CPC分类号： C07H21/00 ,  C07D321/00 ,  C12Q1/6816 ,  C12Q1/686 ,  Y02P20/55

    摘要： Probes labeled with 1,2-dioxetane precursors can be employed in a variety of assays. The probes may be nucleic acid, peptide nucleic acid, proteins including enzyme, antibody or antigen, steroid, carbohydrate, drug or non-drug hapten. The probe is provided with a 1,2-dioxetane precursor bound thereto, generally either covalently, or a strong ligand bond. The dioxetane precursor moiety is converted to a bound 1,2-dioxetane by exposure to singlet oxygen. These dioxetane (labels) either spontaneously decompose, or are induced to decompose by an appropriate trigger to release light. The trigger may be a change in pH temperature, or an agent which removes a protective group. Assay formats in which these 1,2-dioxetane labeled probes and referents may be used to include hybridization assays, immuno assays, gel-based assays and Capillary Zone Electrophoresis.

    摘要翻译： 用1,2-二氧杂环丁烷前体标记的探针可用于多种测定。 探针可以是核酸，肽核酸，蛋白质，包括酶，抗体或抗原，类固醇，碳水化合物，药物或非药物半抗原。 探针具有与其结合的1,2-二氧杂环丁烷前体，通常共价或强配体键。 通过暴露于单线态氧将二氧杂环丁烷前体部分转化为结合的1,2-二氧环乙烷。 这些二氧杂环丁烷（标记）会自发分解，或被适当的触发剂诱导分解以释放光。 触发可能是pH温度的变化，或者是除去保护基团的试剂。 这些1,2-二氧杂环丁烷标记的探针和指示物可用于包括杂交测定，免疫测定，基于凝胶的测定和毛细管区域电泳的测定形式。

    Chemiluminescent energy transfer assays
    8.
    发明授权
    Chemiluminescent energy transfer assays 有权
    化学发光能量转移测定

    公开(公告)号：US06287767B1

    公开(公告)日：2001-09-11

    申请号：US09190181

    申请日：1998-11-12

    申请人： Irena Bronstein ,  Brooks Edwards ,  John Voyta

    发明人： Irena Bronstein ,  Brooks Edwards ,  John Voyta

    IPC分类号： G01N3353

    CPC分类号： G01N33/581 ,  C07D321/00 ,  C12Q1/34 ,  G01N33/535 ,  Y10S435/966 ,  Y10S435/968 ,  Y10S435/975

    摘要： A chemiluminescent assays for the determination of the presence or amount of a biopolymer in bound assays using 1,2-dioxetanes in connection with AttoPhos™ as chemiluminescent substrates for enzyme-labeled targets or probes is provided. Further disclosed is a kit for conducting a bioassay for the presence or concentration of a biopolymer comprising a) an enzyme complex; b) a 1,2-dioxetane; and c) AttoPhos™.

    摘要翻译： 提供了一种化学发光测定法，用于在结合测定中测定生物聚合物的存在或数量，使用与作为酶标记的靶标或探针的化学发光底物的AttoPhos TM相关的1,2-二氧杂环己烷。 还公开了用于进行生物聚合物的存在或浓缩的试剂盒，其包含a）酶复合物; b）1,2-二氧环乙烷; 和c）AttoPhos TM。

    Protease inhibitor assay
    9.
    发明授权
    Protease inhibitor assay 失效
    蛋白酶抑制剂测定

    公开(公告)号：US06243980B1

    公开(公告)日：2001-06-12

    申请号：US09035820

    申请日：1998-03-06

    申请人： Irena Bronstein ,  John Voyta ,  Michelle Palmer ,  Bonnie Tillotson

    发明人： Irena Bronstein ,  John Voyta ,  Michelle Palmer ,  Bonnie Tillotson

    IPC分类号： G01N33533

    CPC分类号： G01N33/582 ,  C12Q1/37 ,  G01N33/581 ,  G01N2333/81 ,  Y10S436/80 ,  Y10S436/805

    摘要： Heterogenous and homogenous assays are provided for the detection of protease inhibitory activity in a sample or target compound, taking advantage of the chemiluminescent characteristics of 1,2-dioxetanes. In the heterogenous assay, a peptide bearing a cleavage site for the protease of interest is provided with a first member of a first ligand binding pair at one end, and a first member of a second ligand binding pair at the other end. The other member of the first ligand binding pair is attached to a surface, which binds the peptide, or protease substrate, to the surface. The peptide substrate is combined with the protease and target compound or sample. Substrate cleavage, if not inhibited, is allowed to occur, and any unbound cleaved fragments are removed. An enzyme complexed with the second member of the second ligand binding pair is added, and allowed to bind to any of the (uncleaved) first member of the second ligand binding pair remaining. Unbound complex is removed, and a 1,2-dioxetane substrate for the enzyme is added. If any peptide substrate has not been cleaved, the dioxetane will chemiluminesce, indicating inhibitory activity. In a homogenous assay, the same substrate bears at one end a fluorescent energy accepting moiety, and at the other end a 1,2-dioxetane or precursor. If the substrate is cleaved by the protease, the dioxetane and the fluorescent moiety are not in close physical relationship, and no energy transfer occurs when the dioxetane is caused to decompose. If cleavage has not occurred, indicating inhibition, when the dioxetane is caused to decompose, energy is transferred to the fluorescing entity, which releases light of a wavelength recognizably distinct from that of the dioxetane.

    摘要翻译： 提供异源和均一的测定法，用于检测样品或目标化合物中蛋白酶抑制活性，利用1,2-二氧杂环己烷的化学发光特性。 在异源测定中，具有目的蛋白酶的切割位点的肽在一端具有第一配体结合对的第一个成员，另一端具有第二配体结合对的第一个成员。 第一配体结合对的另一个成员连接到表面，其将肽或蛋白酶底物结合到表面。 将肽底物与蛋白酶和目标化合物或样品组合。 如果不抑制底物裂解，则可以发生底物裂解，并且去除任何未结合的切割的片段。 加入与第二配体结合对的第二个成员配合的酶，并使其与残留的第二配体结合对的任何（未切割的）第一个成员结合。 去除未结合的络合物，并加入酶的1,2-二氧环乙烷底物。 如果任何肽底物未被切割，二氧杂环丁烷将化学发光，表明抑制活性。 在同质测定中，相同的底物在一端承载荧光能量接受部分，另一端在1,2-二氧杂环丁烷或前体上。 如果底物被蛋白酶切割，二氧杂环丁烷和荧光部分不是很密切的物理关系，当二氧杂环丁烷分解时，不会发生能量转移。 如果没有发生裂解，表明抑制，当二氧杂环丁烷分解时，能量被转移到荧光实体，其释放与二氧杂环丁烷的波长可辨别的波长的光。