ES2527796B1 - Method for fluorimetric quantification of LDH enzyme in solution - Google Patents

Method for fluorimetric quantification of LDH enzyme in solution Download PDF

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Publication number
ES2527796B1
ES2527796B1 ES201300775A ES201300775A ES2527796B1 ES 2527796 B1 ES2527796 B1 ES 2527796B1 ES 201300775 A ES201300775 A ES 201300775A ES 201300775 A ES201300775 A ES 201300775A ES 2527796 B1 ES2527796 B1 ES 2527796B1
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ldh
kit
enzyme
quantification
solution
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ES201300775A
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Spanish (es)
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ES2527796A1 (en
Inventor
Francisco GARCÍA CAMACHO
Asterio SÁNCHEZ MIRÓN
Juan José GALLARDO RODRÍGUEZ
Lorenzo LÓPEZ ROSALES
Emilio Molina Grima
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Universidad de Almeria
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Universidad de Almeria
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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
    • C12Q1/26—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

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Organic Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biochemistry (AREA)
  • Hematology (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Analytical Chemistry (AREA)
  • Toxicology (AREA)
  • Zoology (AREA)
  • Physics & Mathematics (AREA)
  • Wood Science & Technology (AREA)
  • Urology & Nephrology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Biophysics (AREA)
  • Cell Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Tropical Medicine & Parasitology (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 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 (ie lighting, temperature, turbulence, dynamic fluid, etc.) that may cause cell membrane rupture. The kit measures the release of the cytoplasmic lactate dehydrogenase (LDH) enzyme from dead and / or lysed cells. This enzyme catalyzes the oxidation of lactate, present in the kit, to pyruvate. During the reaction, the 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 made in the cell culture supernatant. The method is applicable in both inorganic media for microalgae and commercial media for animal and insect cells.

Description

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Claims (1)

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ES201300775A 2013-07-29 2013-07-29 Method for fluorimetric quantification of LDH enzyme in solution Active ES2527796B1 (en)

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 ES2527796A1 (en) 2015-01-29
ES2527796B1 true ES2527796B1 (en) 2015-11-10

Family

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Family Applications (1)

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ES201300775A Active ES2527796B1 (en) 2013-07-29 2013-07-29 Method for fluorimetric quantification of LDH enzyme in solution

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023229819A1 (en) * 2022-05-24 2023-11-30 Bluerock Therapeutics Lp Methods of making oligodendrocyte progenitor cells

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6982152B2 (en) * 2002-04-17 2006-01-03 Promega Corporation Cytotoxicity assay
FR2944529B1 (en) * 2009-04-20 2013-09-06 Commissariat Energie Atomique METHOD FOR DETERMINING PLASMA ENZYMES IN WHOLE BLOOD

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023229819A1 (en) * 2022-05-24 2023-11-30 Bluerock Therapeutics Lp Methods of making oligodendrocyte progenitor cells

Also Published As

Publication number Publication date
ES2527796A1 (en) 2015-01-29

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