ES2527796A1 - Method for fluorimetric quantification of LDH enzyme in solution - Google Patents
Method for fluorimetric quantification of LDH enzyme in solution Download PDFInfo
- Publication number
- ES2527796A1 ES2527796A1 ES201300775A ES201300775A ES2527796A1 ES 2527796 A1 ES2527796 A1 ES 2527796A1 ES 201300775 A ES201300775 A ES 201300775A ES 201300775 A ES201300775 A ES 201300775A ES 2527796 A1 ES2527796 A1 ES 2527796A1
- Authority
- ES
- Spain
- Prior art keywords
- ldh
- kit
- enzyme
- quantification
- solution
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 238000000034 method Methods 0.000 title abstract 4
- 238000011002 quantification Methods 0.000 title abstract 3
- 102000004190 Enzymes Human genes 0.000 title abstract 2
- 108090000790 Enzymes Proteins 0.000 title abstract 2
- 102000003855 L-lactate dehydrogenase Human genes 0.000 abstract 5
- 108700023483 L-lactate dehydrogenases Proteins 0.000 abstract 5
- 210000004027 cell Anatomy 0.000 abstract 2
- BOPGDPNILDQYTO-NNYOXOHSSA-N nicotinamide-adenine dinucleotide Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 BOPGDPNILDQYTO-NNYOXOHSSA-N 0.000 abstract 2
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 abstract 2
- 241000238631 Hexapoda Species 0.000 abstract 1
- JVTAAEKCZFNVCJ-UHFFFAOYSA-M Lactate Chemical compound CC(O)C([O-])=O JVTAAEKCZFNVCJ-UHFFFAOYSA-M 0.000 abstract 1
- 241001465754 Metazoa Species 0.000 abstract 1
- BAWFJGJZGIEFAR-NNYOXOHSSA-O NAD(+) Chemical compound NC(=O)C1=CC=C[N+]([C@H]2[C@@H]([C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 BAWFJGJZGIEFAR-NNYOXOHSSA-O 0.000 abstract 1
- LCTONWCANYUPML-UHFFFAOYSA-M Pyruvate Chemical compound CC(=O)C([O-])=O LCTONWCANYUPML-UHFFFAOYSA-M 0.000 abstract 1
- 238000004113 cell culture Methods 0.000 abstract 1
- 210000000170 cell membrane Anatomy 0.000 abstract 1
- 230000003833 cell viability Effects 0.000 abstract 1
- 239000013043 chemical agent Substances 0.000 abstract 1
- 238000006243 chemical reaction Methods 0.000 abstract 1
- 150000001875 compounds Chemical class 0.000 abstract 1
- 230000001086 cytosolic effect Effects 0.000 abstract 1
- 231100000433 cytotoxic Toxicity 0.000 abstract 1
- 230000001472 cytotoxic effect Effects 0.000 abstract 1
- 239000003814 drug Substances 0.000 abstract 1
- 229940079593 drug Drugs 0.000 abstract 1
- 239000012530 fluid Substances 0.000 abstract 1
- 238000005259 measurement Methods 0.000 abstract 1
- 239000000203 mixture Substances 0.000 abstract 1
- 230000003647 oxidation Effects 0.000 abstract 1
- 238000007254 oxidation reaction Methods 0.000 abstract 1
- 239000006228 supernatant Substances 0.000 abstract 1
Classifications
-
- 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
- C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
- C12Q1/32—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving dehydrogenase
-
- 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
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5014—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing toxicity
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biochemistry (AREA)
- Hematology (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Urology & Nephrology (AREA)
- Toxicology (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- General Health & Medical Sciences (AREA)
- Cell Biology (AREA)
- Pathology (AREA)
- Biophysics (AREA)
- Medicinal Chemistry (AREA)
- Tropical Medicine & Parasitology (AREA)
- General Physics & Mathematics (AREA)
- Food Science & Technology (AREA)
- General Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
Método para la cuantificación fluorimétrica de la enzima LDH en disolución. La invención está dirigida al desarrollo de un método y creación de un kit para la cuantificación del efecto citotóxico y/o viabilidad celular ante agentes químicos (elemento, compuesto, mezcla o fármaco en cualquier de sus estados) y/o físicos (i.e. iluminación, temperatura, turbulencia, fluidodinámica, etc.) que puedan producir rotura de la membrana celular. El kit mide la liberación de la enzima citoplasmática lactato deshidrogenasa (LDH) proveniente de células muertas y/o lisadas. Esta enzima cataliza la oxidación del lactato, presente en el kit, a piruvato. Durante la reacción, el NAD+, también presente en el kit, es reducido a NADH. La concentración de LDH, se cuantifica a través de la fluorescencia del NADH formado. Las mediciones indirectas de la enzima LDH se realizan en el sobrenadante de cultivos celulares. El método es aplicable tanto en medios inorgánicos para microalgas como en medios comerciales para células animales y de insecto.Method for the fluorimetric quantification of the LDH enzyme in solution. The invention is directed to the development of a method and creation of a kit for the quantification of the cytotoxic effect and/or cell viability before chemical agents (element, compound, mixture or drug in any of its states) and/or physical (i.e. lighting, temperature, turbulence, fluid dynamics, etc.) that can cause rupture of the cell membrane. The kit measures the release of the cytoplasmic enzyme lactate dehydrogenase (LDH) from dead and/or lysed cells. This enzyme catalyzes the oxidation of lactate, present in the kit, to pyruvate. During the reaction, NAD+, also present in the kit, is reduced to NADH. The LDH concentration is quantified through the fluorescence of the NADH formed. Indirect measurements of the LDH enzyme are performed on the supernatant of cell cultures. The method is applicable both in inorganic media for microalgae and in commercial media for animal and insect cells.
Description
Claims (1)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| ES201300775A ES2527796B1 (en) | 2013-07-29 | 2013-07-29 | Method for fluorimetric quantification of LDH enzyme in solution |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| ES201300775A ES2527796B1 (en) | 2013-07-29 | 2013-07-29 | Method for fluorimetric quantification of LDH enzyme in solution |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| ES2527796A1 true ES2527796A1 (en) | 2015-01-29 |
| ES2527796B1 ES2527796B1 (en) | 2015-11-10 |
Family
ID=52392475
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| ES201300775A Active ES2527796B1 (en) | 2013-07-29 | 2013-07-29 | Method for fluorimetric quantification of LDH enzyme in solution |
Country Status (1)
| Country | Link |
|---|---|
| ES (1) | ES2527796B1 (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA3256595A1 (en) * | 2022-05-24 | 2023-11-30 | Bluerock Therapeutics Lp | Methods of making oligodendrocyte progenitor cells |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003089635A1 (en) * | 2002-04-17 | 2003-10-30 | Promega Corporation | Cytotoxicity assay |
| EP2249158A1 (en) * | 2009-04-20 | 2010-11-10 | Commissariat à l'Énergie Atomique et aux Énergies Alternatives | Dosage of plasmatic enzymes in blood |
-
2013
- 2013-07-29 ES ES201300775A patent/ES2527796B1/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003089635A1 (en) * | 2002-04-17 | 2003-10-30 | Promega Corporation | Cytotoxicity assay |
| EP2249158A1 (en) * | 2009-04-20 | 2010-11-10 | Commissariat à l'Énergie Atomique et aux Énergies Alternatives | Dosage of plasmatic enzymes in blood |
Non-Patent Citations (1)
| Title |
|---|
| RAMAN, R.N. et al. "Quantification of in vivo autofluorescence dynamics during renal ischemia and reperfusion under 355 nm excitation". OPTICS EXPRESS. 31.03.2008, todo el documento. * |
Also Published As
| Publication number | Publication date |
|---|---|
| ES2527796B1 (en) | 2015-11-10 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| AR117697A2 (en) | CELLULAR CULTURE MEDIA | |
| ES2525411T3 (en) | Differentiated anucleated cells and method to prepare them | |
| EA201101141A1 (en) | OBTAINING CLOSED LINEAR DNA | |
| BR112012019396A2 (en) | method and kit for amplifying a nucleic acid. | |
| MX382080B (en) | METHOD FOR CULTIVATING EUKARYONTIC CELLS. | |
| AR092998A1 (en) | CELL OF ELECTROLYSIS OF ALKALINE SOLUTIONS | |
| BRPI0922572A2 (en) | method for culturing pluripotent cells, pluripotent mammalian cell culture, cell culture medium, isolated pluripotent animal cell, and method for increasing pluripotence of a mammalian cell. | |
| MX2016008251A (en) | Media for cell culture. | |
| BR112013025086A2 (en) | multifunctional bioreactor system and methods for cell classification and culture | |
| EP2344632A4 (en) | METHOD FOR INDUCING PLURIPOTENCE IN CELLS | |
| AR077369A1 (en) | METODOPARA PRODUCE Erythrocytes WITHOUT FEEDING CELLS | |
| JP2014513555A5 (en) | ||
| BR112013020217A2 (en) | method and bioreactor for the cultivation of microorganisms | |
| BR112017023011A2 (en) | anaerobic microorganism culture device | |
| MX378896B (en) | CONTROL OF CARBON DIOXIDE AND PH LEVELS IN SMALL VOLUME REACTORS, | |
| ES2527796B1 (en) | Method for fluorimetric quantification of LDH enzyme in solution | |
| BRPI0607194A2 (en) | method for quantitatively analyzing a microorganism by RNA labeling | |
| BR112017002791A2 (en) | METHODS FOR THE PRODUCTION OF OIL IN MICRO-ORGANISMS | |
| ES2660763T3 (en) | Genetically encoded probe for the quantification of pyruvate concentration and methods of use thereof | |
| BRPI1008762B8 (en) | methods for selecting mammalian cells expressing a heterologous protein to produce a product of interest and use of a selective culture medium | |
| ES2542257B1 (en) | PROCEDURE FOR THE DEPURATION OF GASES CONTAINING VOLATILE ORGANIC COMPOUNDS | |
| Zhdanov et al. | Low energy costs of F1Fo ATP synthase reversal in colon carcinoma cells deficient in mitochondrial complex IV | |
| BRPI1014141A2 (en) | method and culture medium for increased detection of mycobacteria. | |
| AR075808A1 (en) | METHOD FOR DETECTING GLYPHOSATE IN VEGETABLE FABRICS | |
| EP2586874A4 (en) | METHOD FOR SCREENING S1P LYASE INHIBITOR USING CELL CULTURE |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| FG2A | Definitive protection |
Ref document number: 2527796 Country of ref document: ES Kind code of ref document: B1 Effective date: 20151110 |