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Basic Research in Plasma Medicine - A Throughput Approach from Liquids to Cells
Published on: November 17, 2017
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ReactELISA method for quantifying methylglyoxal levels in plasma and cell cultures.
Rasmus Kold-Christensen1, Karina Kragh Jensen2, Emil Smedegård-Holmquist1
1Department of Forensic Medicine, Aarhus University, Palle Juul-Jensens Boulevard 99, 8200 Aarhus N, Denmark; Department of Chemistry, Aarhus University, Langelandsgade 140, 8000 Aarhus C, Denmark.
Redox Biology
|June 30, 2019
Summary
This study introduces a new ELISA assay for detecting methylglyoxal (MG), a toxic metabolite linked to aging diseases. The assay accurately measures MG levels in biological samples, offering a simpler alternative to current methods.
Area of Science:
- Biochemistry
- Metabolomics
- Assay Development
Background:
- Methylglyoxal (MG) is a toxic byproduct of glycolysis linked to inflammation, oxidative stress, and aging diseases like diabetes and Alzheimer's.
- Measuring low endogenous MG levels is challenging due to its reactivity and small size, limiting traditional immunoassay use.
- Current quantification relies on complex LC-MS/MS methods, necessitating simpler, robust alternatives.
Purpose of the Study:
- To develop a novel, highly specific assay for quantifying methylglyoxal (MG) in human biological specimens.
- To establish a reaction-based ELISA (ReactELISA) and a homogenous AlphaLISA assay for MG detection.
- To validate the new assay against LC-MS/MS and demonstrate its utility in measuring MG perturbations.
Main Methods:
- Development of a bifunctional MG capture probe and a high-specificity monoclonal antibody.
- Establishment and validation of a reaction-based ELISA (ReactELISA) for MG detection.
- Conversion of the assay to a homogenous (no-wash) AlphaLISA format for high-throughput screening.
Main Results:
- A robust ReactELISA method was successfully developed for detecting low (nM) levels of MG.
- The ReactELISA demonstrated comparable performance to the gold standard LC-MS/MS method in human plasma and cell culture media.
- The assay successfully measured MG level changes induced by buthionine sulfoximine (BSO) and was adapted to a homogenous AlphaLISA format.
Conclusions:
- The developed ReactELISA and ReactAlphaLISA assays provide sensitive and specific tools for measuring methylglyoxal.
- These assays offer simpler and more accessible alternatives to LC-MS/MS for MG quantification.
- The new assays can aid in understanding MG metabolism, identifying disease biomarkers, and screening for drugs targeting aging-related diseases.
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