Optimal detection of cholinesterase activity in biological samples: Modifications to the standard Ellman's assay

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F00179906%3A_____%2F14%3A10281198" target="_blank" >RIV/00179906:_____/14:10281198 - isvavai.cz</a>

Výsledek na webu

<a href="http://dx.doi.org/10.1016/j.ab.2014.05.031" target="_blank" >http://dx.doi.org/10.1016/j.ab.2014.05.031</a>

DOI - Digital Object Identifier

<a href="http://dx.doi.org/10.1016/j.ab.2014.05.031" target="_blank" >10.1016/j.ab.2014.05.031</a>

Jazyk výsledku

angličtina

Název v původním jazyce

Optimal detection of cholinesterase activity in biological samples: Modifications to the standard Ellman's assay

Popis výsledku v původním jazyce

Ellman's assay is the most commonly used method to measure cholinesterase activity. It is cheap, fast, and reliable, but it has limitations when used for biological samples. The problems arise from 5,5-dithiobis(2-nitrobenzoic acid) (DTNB), which is unstable, interacts with free sulfhydryl groups in the sample, and may affect cholinesterase activity. We report that DTNB is more stable in 0.09 M Hepes with 0.05 M sodium phosphate buffer than in 0.1 M sodium phosphate buffer, thereby notably reducing background. Using enzyme-linked immunosorbent assay (ELISA) to enrich tissue homogenates for cholinesterase while depleting the sample of sulfhydryl groups eliminates unwanted interactions with DTNB, making it possible to measure low cholinesterase activityin biological samples. To eliminate possible interference of DTNB with enzyme hydrolysis, we introduce a modification of the standard Ellman's assay. First, thioesters are hydrolyzed by cholinesterase to produce thiocholine in the absence

Název v anglickém jazyce

Optimal detection of cholinesterase activity in biological samples: Modifications to the standard Ellman's assay

Popis výsledku anglicky

Ellman's assay is the most commonly used method to measure cholinesterase activity. It is cheap, fast, and reliable, but it has limitations when used for biological samples. The problems arise from 5,5-dithiobis(2-nitrobenzoic acid) (DTNB), which is unstable, interacts with free sulfhydryl groups in the sample, and may affect cholinesterase activity. We report that DTNB is more stable in 0.09 M Hepes with 0.05 M sodium phosphate buffer than in 0.1 M sodium phosphate buffer, thereby notably reducing background. Using enzyme-linked immunosorbent assay (ELISA) to enrich tissue homogenates for cholinesterase while depleting the sample of sulfhydryl groups eliminates unwanted interactions with DTNB, making it possible to measure low cholinesterase activityin biological samples. To eliminate possible interference of DTNB with enzyme hydrolysis, we introduce a modification of the standard Ellman's assay. First, thioesters are hydrolyzed by cholinesterase to produce thiocholine in the absence

Druh

J_x - Nezařazeno - Článek v odborném periodiku (Jimp, Jsc a Jost)

CEP obor

FR - Farmakologie a lékárnická chemie

OECD FORD obor

—

Projekt

—

Návaznosti

I - Institucionalni podpora na dlouhodoby koncepcni rozvoj vyzkumne organizace

Rok uplatnění

2014

Kód důvěrnosti údajů

S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů

Název periodika

Analytical Biochemistry

ISSN

0003-2697

e-ISSN

—

Svazek periodika

462

Číslo periodika v rámci svazku

October

Stát vydavatele periodika

US - Spojené státy americké

Počet stran výsledku

9

Strana od-do

67-75

Kód UT WoS článku

000341803700012

EID výsledku v databázi Scopus

—