ES2645733T3 - Inmunoensayo para la detección de miARNs - Google Patents
Inmunoensayo para la detección de miARNs Download PDFInfo
- Publication number
- 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
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- antibody
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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
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- 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
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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
-
- 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)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Organic Chemistry (AREA)
- Molecular Biology (AREA)
- Analytical Chemistry (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biotechnology (AREA)
- Genetics & Genomics (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Biomedical Technology (AREA)
- General Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Pathology (AREA)
- Biophysics (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Cell Biology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Tropical Medicine & Parasitology (AREA)
- Plant Pathology (AREA)
- 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
- 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)
-
imagen1
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP12159196.0A EP2639312B1 (en) | 2012-03-13 | 2012-03-13 | Immunoassay for detection of miRNAs |
Publications (1)
Publication Number | Publication Date |
---|---|
ES2645733T3 true ES2645733T3 (es) | 2017-12-07 |
Family
ID=47901041
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
ES12159196.0T Active ES2645733T3 (es) | 2012-03-13 | 2012-03-13 | Inmunoensayo para la detección de miARNs |
Country Status (10)
Country | Link |
---|---|
US (1) | US20150037797A1 (es) |
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) |
Families Citing this family (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
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 |
Family Cites Families (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CA2600851A1 (en) * | 2005-03-15 | 2006-09-21 | Applera Corporation | The use of antibody-surrogate antigen systems for detection of analytes |
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 |
EP2528932B1 (en) * | 2010-01-29 | 2016-11-30 | QIAGEN Gaithersburg, Inc. | Methods and compositions for sequence-specific purification and multiplex analysis of nucleic acids |
EP2531612A1 (en) * | 2010-02-05 | 2012-12-12 | Institute for Systems Biology | Methods and compositions for profiling rna molecules |
US20130040303A1 (en) * | 2011-08-08 | 2013-02-14 | Eugenia Wang | Biomarker for Alzheimer's Disease and/or Mild Cognitive Impairment, and Use Thereof |
-
2012
- 2012-03-13 DK DK12159196.0T patent/DK2639312T3/da active
- 2012-03-13 EP EP12159196.0A patent/EP2639312B1/en active Active
- 2012-03-13 ES ES12159196.0T patent/ES2645733T3/es active Active
- 2012-03-13 PL PL12159196T patent/PL2639312T3/pl unknown
- 2012-03-13 SI SI201231105T patent/SI2639312T1/sl unknown
- 2012-03-13 PT PT121591960T patent/PT2639312T/pt unknown
-
2013
- 2013-03-08 CN CN201380013862.1A patent/CN104271766A/zh active Pending
- 2013-03-08 WO PCT/EP2013/054745 patent/WO2013135581A1/en active Application Filing
- 2013-03-08 EP EP13710334.7A patent/EP2825666B1/en active Active
- 2013-03-08 KR KR1020147025563A patent/KR101862439B1/ko active IP Right Grant
- 2013-03-08 US US14/384,577 patent/US20150037797A1/en not_active Abandoned
Also Published As
Publication number | Publication date |
---|---|
EP2825666A1 (en) | 2015-01-21 |
EP2639312B1 (en) | 2017-08-02 |
SI2639312T1 (sl) | 2017-12-29 |
CN104271766A (zh) | 2015-01-07 |
US20150037797A1 (en) | 2015-02-05 |
WO2013135581A1 (en) | 2013-09-19 |
PL2639312T3 (pl) | 2018-01-31 |
EP2825666B1 (en) | 2018-12-05 |
PT2639312T (pt) | 2017-10-23 |
DK2639312T3 (da) | 2017-11-06 |
KR101862439B1 (ko) | 2018-05-29 |
EP2639312A1 (en) | 2013-09-18 |
KR20140137360A (ko) | 2014-12-02 |
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