Lambert Kristiansen Sørensen

ReactELISA method for quantifying methylglyoxal levels in plasma and cell cultures

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ReactELISA method for quantifying methylglyoxal levels in plasma and cell cultures. / Kold-Christensen, Rasmus; Jensen, Karina Kragh; Smedegård-Holmquist, Emil; Sørensen, Lambert Kristiansen; Hansen, Jakob; Jørgensen, Karl Anker; Kristensen, Peter; Johannsen, Mogens.

In: Redox Biology, Vol. 26, 101252, 2019.

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Kold-Christensen, Rasmus ; Jensen, Karina Kragh ; Smedegård-Holmquist, Emil ; Sørensen, Lambert Kristiansen ; Hansen, Jakob ; Jørgensen, Karl Anker ; Kristensen, Peter ; Johannsen, Mogens. / ReactELISA method for quantifying methylglyoxal levels in plasma and cell cultures. In: Redox Biology. 2019 ; Vol. 26.

Bibtex

@article{ffcb9259528e4b6397f8a1cdf957205b,
title = "ReactELISA method for quantifying methylglyoxal levels in plasma and cell cultures",
abstract = "Methylglyoxal (MG) is a toxic glycolytic by-product associated with increased levels of inflammation and oxidative stress and has been linked to ageing-related diseases, such as diabetes and Alzheimer's disease. As MG is a highly reactive dicarbonyl compound, forming both reversible and irreversible adducts with a range of endogenous nucleophiles, measuring endogenous levels of MG are quite troublesome. Furthermore, as MG is a small metabolite it is not very immunogenic, excluding conventional ELISA for detection purposes, thus only more instrumentally demanding LC-MS/MS-based methods have demonstrated convincing quantitative data. In the present work we develop a novel bifunctional MG capture probe as well as a high specificity monoclonal antibody to finally setup a robust reaction-based ELISA (ReactELISA) method for detecting the highly reactive and low-level (nM) metabolite MG in human biological specimens. The assay is tested and validated against the current golden standard LC-MS/MS method in human blood plasma and cell-culture media. Furthermore, we demonstrate the assays ability to measure small perturbations of MG levels in growth media caused by a small molecule drug buthionine sulfoximine (BSO) of current clinical relevance. Finally, the assay is converted into a homogenous (no-wash) AlphaLISA version (ReactAlphaLISA), which offers the potential for operationally simple screening of further small molecules capable of perturbing cellular MG. Such compounds could be of relevance as probes to gain insight into MG metabolism as well as drug-leads to alleviate ageing-related diseases.",
keywords = "Buthionine sulfoximine, Cell culture, ELISA, Glyoxalase, Methylglyoxal, Plasma",
author = "Rasmus Kold-Christensen and Jensen, {Karina Kragh} and Emil Smedeg{\aa}rd-Holmquist and S{\o}rensen, {Lambert Kristiansen} and Jakob Hansen and J{\o}rgensen, {Karl Anker} and Peter Kristensen and Mogens Johannsen",
year = "2019",
doi = "10.1016/j.redox.2019.101252",
language = "English",
volume = "26",
journal = "Redox Biology",
issn = "2213-2317",
publisher = "Elsevier BV",

}

RIS

TY - JOUR

T1 - ReactELISA method for quantifying methylglyoxal levels in plasma and cell cultures

AU - Kold-Christensen, Rasmus

AU - Jensen, Karina Kragh

AU - Smedegård-Holmquist, Emil

AU - Sørensen, Lambert Kristiansen

AU - Hansen, Jakob

AU - Jørgensen, Karl Anker

AU - Kristensen, Peter

AU - Johannsen, Mogens

PY - 2019

Y1 - 2019

N2 - Methylglyoxal (MG) is a toxic glycolytic by-product associated with increased levels of inflammation and oxidative stress and has been linked to ageing-related diseases, such as diabetes and Alzheimer's disease. As MG is a highly reactive dicarbonyl compound, forming both reversible and irreversible adducts with a range of endogenous nucleophiles, measuring endogenous levels of MG are quite troublesome. Furthermore, as MG is a small metabolite it is not very immunogenic, excluding conventional ELISA for detection purposes, thus only more instrumentally demanding LC-MS/MS-based methods have demonstrated convincing quantitative data. In the present work we develop a novel bifunctional MG capture probe as well as a high specificity monoclonal antibody to finally setup a robust reaction-based ELISA (ReactELISA) method for detecting the highly reactive and low-level (nM) metabolite MG in human biological specimens. The assay is tested and validated against the current golden standard LC-MS/MS method in human blood plasma and cell-culture media. Furthermore, we demonstrate the assays ability to measure small perturbations of MG levels in growth media caused by a small molecule drug buthionine sulfoximine (BSO) of current clinical relevance. Finally, the assay is converted into a homogenous (no-wash) AlphaLISA version (ReactAlphaLISA), which offers the potential for operationally simple screening of further small molecules capable of perturbing cellular MG. Such compounds could be of relevance as probes to gain insight into MG metabolism as well as drug-leads to alleviate ageing-related diseases.

AB - Methylglyoxal (MG) is a toxic glycolytic by-product associated with increased levels of inflammation and oxidative stress and has been linked to ageing-related diseases, such as diabetes and Alzheimer's disease. As MG is a highly reactive dicarbonyl compound, forming both reversible and irreversible adducts with a range of endogenous nucleophiles, measuring endogenous levels of MG are quite troublesome. Furthermore, as MG is a small metabolite it is not very immunogenic, excluding conventional ELISA for detection purposes, thus only more instrumentally demanding LC-MS/MS-based methods have demonstrated convincing quantitative data. In the present work we develop a novel bifunctional MG capture probe as well as a high specificity monoclonal antibody to finally setup a robust reaction-based ELISA (ReactELISA) method for detecting the highly reactive and low-level (nM) metabolite MG in human biological specimens. The assay is tested and validated against the current golden standard LC-MS/MS method in human blood plasma and cell-culture media. Furthermore, we demonstrate the assays ability to measure small perturbations of MG levels in growth media caused by a small molecule drug buthionine sulfoximine (BSO) of current clinical relevance. Finally, the assay is converted into a homogenous (no-wash) AlphaLISA version (ReactAlphaLISA), which offers the potential for operationally simple screening of further small molecules capable of perturbing cellular MG. Such compounds could be of relevance as probes to gain insight into MG metabolism as well as drug-leads to alleviate ageing-related diseases.

KW - Buthionine sulfoximine

KW - Cell culture

KW - ELISA

KW - Glyoxalase

KW - Methylglyoxal

KW - Plasma

UR - http://www.scopus.com/inward/record.url?scp=85067878306&partnerID=8YFLogxK

U2 - 10.1016/j.redox.2019.101252

DO - 10.1016/j.redox.2019.101252

M3 - Journal article

C2 - 31254735

AN - SCOPUS:85067878306

VL - 26

JO - Redox Biology

JF - Redox Biology

SN - 2213-2317

M1 - 101252

ER -