ES2645733T3 - Inmunoensayo para la detección de miARNs - Google Patents

Inmunoensayo para la detección de miARNs Download PDF

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ES2645733T3
ES2645733T3 ES12159196.0T ES12159196T ES2645733T3 ES 2645733 T3 ES2645733 T3 ES 2645733T3 ES 12159196 T ES12159196 T ES 12159196T ES 2645733 T3 ES2645733 T3 ES 2645733T3
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hybrid
nucleic acid
oligo
interest
antibody
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Andreas Kappel
Andreas Keller
Cord Friedrich Stähler
Ian Wright
Linda Marie Anderson-Mauser
William Bedzyk
Herbert Schwarz
Michaela Wicke
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Siemens Healthcare Diagnostics Products GmbH
Siemens Healthcare GmbH
Siemens Healthcare Diagnostics Inc
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Siemens Healthcare Diagnostics Products GmbH
Siemens Healthcare GmbH
Siemens Healthcare Diagnostics Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6804—Nucleic acid analysis using immunogens
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites

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  • Health & Medical Sciences (AREA)
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  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

Un método para detectar una molécula de ácido nucleico de interés en una muestra, en el que la molécula de ácido nucleico de interés es un microARN, que comprende las siguientes etapas: - proporcionar una sonda de ácido nucleico en solución; - hibridación de la molécula de ácido nucleico de interés a la sonda de ácido nucleico para obtener un híbrido de dicha molécula de ácido nucleico de interés y dicha sonda de ácido nucleico; - enlazar dicho híbrido a un anticuerpo capaz de enlazar específicamente a híbridos de ácido nucleico, en el que el anticuerpo está enlazado a una fase sólida; y - detectar el híbrido enlazado a anticuerpo, en el que el anticuerpo está enlazado a un antígeno competitivo antes del enlace de dicho primer híbrido y dicho híbrido es detectado por desplazamiento del antígeno competitivo, en el que el antígeno competitivo es un híbrido competitivo de ácidos nucleicos, portando dicho híbrido competitivo una unidad estructural marcadora.

Description

imagen1
imagen2
imagen3
imagen4
imagen5
imagen6
SEQ ID NO
Ácido Nucleico Secuencia (5'-3')
5
miR-380 UAUGUAAUAUGGUCCACAUCUU
6
miR380 Oligo 1 AGATGTGGACCATATTACATA
7
miR380 Oligo 2 TGTGGACCATATTACATA
8
miR380 Oligo 3 GGACCATATTACATA
imagen7
imagen8 imagen9
9
miR-1254 AGCCUGGAAGCUGGAGCCUGCAGU
10
miR1254 Oligo 1 GCAGGCTCCAGCTTCCAGGCT
11
miR1254 Oligo 2 GGCTCCAGCTTCCAGGCT
12
miR1254 Oligo 3 TCCAGCTTCCAGGCT
Las placas de ELISA se prerecubrieron con un anticuerpo anti-ADN:ARN-Híbrido bajo condiciones estándar (5 µg/ml en solución salina regulada con fosfato (PBS)). Después del recubrimiento, las placas de ELISA se lavaron con una solución de lavado comercialmente disponible o con un regulador TRIS/ácido cítrico.
5 Los híbridos se diluyeron 1:100, 1:1.000, 1:10.000 y 1:100.000 en
-PBS,
-PBS con 1 µg/ml de BSA y 0,1 µg/ml de Tween 20,
-PBS con 0,5 µg/ml de BSA y 0,05 µg/ml de Tween 20, o
-PBS con 0,25 µg/ml de BSA y 0,025 µg/ml de Tween 20 respectivamente y se incubaron sobre placas ELISA
10 recubiertas durante 1 hora a 37 ºC. Después de la incubación, las placas se lavaron e incubaron con un conjugado de Estreptavidina POD comercialmente disponible. Las placas se lavaron y se añadió una solución de sustrato cromogénico (tetrametilbencidina) a las placas. El desarrollo del color se paró mediante la adición de una solución de parada de ácido sulfúrico y se leyó la densidad óptica (OD) a 450 nm. Los resultados para las diluciones 1:100.000 de los híbridos 1 a 12 se dan en la tabla 2, a continuación.
15 Tabla 2: Valores de OD
Composición del Híbrido
Híbrido No. Diluyente
imagen10
1xPBS 1xPBS, 1g/l BSA, 0,1g/l Tween 20 1xPBS 0,5g/l BSA, 0,05g/l Tween 20 1xPBS 0,25g/l BSA, 0,025g/l Tween 20
1. ARN miR-7 con ADN miR7 Oligo 1, temperatura de hibridación 52,0 °C
Híbrido 1 3,094 3,289 3,275 3,325
8
Composición del Híbrido
Híbrido No. Diluyente
imagen11
1xPBS 1xPBS, 1g/l BSA, 0,1g/l Tween 20 1xPBS 0,5g/l BSA, 0,05g/l Tween 20 1xPBS 0,25g/l BSA, 0,025g/l Tween 20
2. ARN miR-7 con ADN miR7Oligo 2, temperatura de hibridación 46,9 °C
Híbrido 2 3,079 3,306 3,259 3,259
3. ARN miR-7 con ADN miR7 Oligo 3, temperatura de hibridación 42,4 °C
Híbrido 3 2,517 3,164 3,004 2,980
4. ARN miR-7 con ADN miR380 Oligo 1, temperatura de hibridación 52,0 °C
Híbrido 4 (no específico) 0,021 0,028 0,022 0,022
5. ARN miR-380 con ADN miR380 Oligo 1, temperatura de hibridación 52,0 °C
Híbrido 5 2,952 3,104 3,104 3,074
6. ARN miR-380 con ADN miR380 Oligo 2, temperatura de hibridación 46,9 °C
Híbrido 6 2,912 3,254 3,173 3,138
7. ARN miR-380 con ADN miR380 Oligo 3, temperatura de hibridación 39,6 °C
Híbrido 7 1,046 2,109 2,128 1,817
8. ARN miR-380 con ADN miR7 Oligo 1, temperatura de hibridación 52,0 °C
Híbrido 8 (noespecífico) 0,023 0,029 0,031 0,030
9. ARN miR-380 con ADN miR1254 Oligo 1, temperatura de hibridación 65,7 °C
Híbrido 9 3,139 3,244 3,270 3,215
10.ARN miR-1254 con ADN miR1254 Oligo 2, temperatura de hibridación 60,5 °C
Híbrido 10 3,003 3,230 3,259 3,217
11. ARN miR-1254 con ADN miR1254 Oligo 3, temperatura de hibridación 50,6 °C
Híbrido 11 2,554 3,164 3,106 3,067
9

Claims (1)

  1. imagen1
ES12159196.0T 2012-03-13 2012-03-13 Inmunoensayo para la detección de miARNs Active ES2645733T3 (es)

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EP12159196.0A EP2639312B1 (en) 2012-03-13 2012-03-13 Immunoassay for detection of miRNAs

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EP (2) EP2639312B1 (es)
KR (1) KR101862439B1 (es)
CN (1) CN104271766A (es)
DK (1) DK2639312T3 (es)
ES (1) ES2645733T3 (es)
PL (1) PL2639312T3 (es)
PT (1) PT2639312T (es)
SI (1) SI2639312T1 (es)
WO (1) WO2013135581A1 (es)

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EP2657348B1 (en) 2012-04-27 2017-11-15 Siemens Aktiengesellschaft Diagnostic miRNA profiles in multiple sclerosis
CN104962555B (zh) * 2015-06-10 2019-04-05 中国科学技术大学 利用级联的dna链替换反应的细胞内非编码rna检测方法
CN111763713B (zh) * 2020-03-23 2023-07-04 天津大学 非诊断目的基于靶标等温循环扩增及核酸试纸条技术检测miRNA-21的方法及试剂盒
CN113655108B (zh) * 2021-08-17 2023-04-07 湖北大学 一种栅电极修饰方法、栅控石墨烯晶体管生物传感器及miRNA浓度检测方法
CN115112883B (zh) * 2022-06-29 2025-05-09 深圳市德大科技有限公司 一种miR-Chr8:96检测试剂盒及其检测方法
CN118010990A (zh) * 2024-01-31 2024-05-10 广东工业大学 一种快速检测hpv16 dna-rna杂合体的免疫层析试纸条

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JP2008533167A (ja) * 2005-03-15 2008-08-21 アプレラ コーポレイション 分析物の検出のための、抗体の代替抗原系の使用
US20120269728A1 (en) * 2006-08-02 2012-10-25 Antara Biosciences Inc. Methods and compositions for detecting one or more target agents using tracking components
DE102007010252B4 (de) * 2007-03-02 2013-07-04 Sirs-Lab Gmbh Kontrollgene zur Normalisierung von Genexpressionsanalysedaten
CA2787924A1 (en) * 2010-01-29 2011-08-04 Qiagen Gaithersburg, Inc. Methods and compositions for sequence-specific purification and multiplex analysis of nucleic acids
WO2011097528A1 (en) * 2010-02-05 2011-08-11 Institute For Systems Biology Methods and compositions for profiling rna molecules
CA2838562A1 (en) * 2011-08-08 2013-02-14 Eugenia Wang Biomarker for alzheimer's disease and/or mild cognitively impairment, and use thereof

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PT2639312T (pt) 2017-10-23
CN104271766A (zh) 2015-01-07
PL2639312T3 (pl) 2018-01-31
US20150037797A1 (en) 2015-02-05
EP2825666A1 (en) 2015-01-21
KR20140137360A (ko) 2014-12-02
DK2639312T3 (da) 2017-11-06
EP2825666B1 (en) 2018-12-05
EP2639312A1 (en) 2013-09-18
EP2639312B1 (en) 2017-08-02
SI2639312T1 (sl) 2017-12-29
WO2013135581A1 (en) 2013-09-19
KR101862439B1 (ko) 2018-05-29

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