Glycation, or nonenzymatic glycation, is the nonenzymatic reaction of glucose and other reducing sugars with free amino groups of proteins, lipids and nucleic acids. The amino groups of the side chains of arginine and lysine are the primary targets for this type of modification. Over time, the initial glycation products may undergo intramolecular rearrangements and oxidation reactions (glycosidation) and ultimately transfrom in stable, so-called advanced glycation end products (AGEs). Several compounds, e.g. NƐ-carboxymethyl-lysine (CML), pentosidine, or methylglyoxal (MGO) derivates, serve as examples of well-characterized and widely studies AGEs.
AGEs have the potential to interact with a specific receptor (RAGE), a member of the immunoglobulin superfamily, initiating signal pathways that amplify infammation and oxidative stress, and thereby leading to cellular injury and death. High levels of circulating AGEs are associated with cardiovascular disease, diabetes, chronic kidney disease, and increased mortality.
MGO is a highly reactive dicarbonyl metabolite formed during glucose, protein and fatty acid metabolism. Several nonenzymatic as well as enzymatic reactions are involved in MGO formation. MGO can be formed by nonenzymatic elimination of phoshate from triose phosphates, glyceraldehyde-3-phosphate and glycerone phosphate, as well as enzymatically from dihydroxyacetone phosphate by MGO synthase. MGO reacts with protein by initial reversible reactions: with arginine and lysine residues forming glycosylamine, with cysteine forming hemithioacetal. The irreversible reaction of MGO with lysine residues of protein forms NƐ-carboxyethyl-lysine (CEL), NƐ-carboxymethyl-lysine (CML) and 1,3-di(N-lysino)-4-methyl-imidazolium (MOLD). MGO reacts with arginine residues of protein to form the non-fluorescent products argpyrimidine and hydroxyimidazolone.
MGO-induced AGEs have been linked to the aging process and age-related diseases such as cardiovascular complications of diabetes, neurodegenerative disease and connective tissue disorders. Furthermore, MGO levels are elevated in hyperglycemia and has been implicated in hypertension and atherosclerosis.
The MGO assay can be used to measure MGO-adducts as HSA-MGO arbitrary units. In normal citrate plasma ~200-400 AU/ml can be measured.
The competitive MGO ELISA kit is to be used for in vitro quantitative determination of MGO in plasma and faeces samples
The competitive MGO ELISA is a ready-to-use solid-phase enzyme-linked immunosorbent assay based on the inhibition principle with a working time of 3½ hours.
The efficient format of a plate with twelve disposable 8-well strips allows free choice of batch size for the assay.
Samples and standards are pre-incubated with biotinylated tracer antibody in U-shape microtiter plate.
Pre-incubated samples and standards with the biotinylated tracer antibodies are incubated on coated strips.
Streptavidin-peroxidase conjugate will bind to the biotinylated tracer antibody.
Streptavidin-peroxidase will react with the substrate, tetramethylbenzidine (TMB).
The enzyme reaction is stopped by the addition of oxalic acid.
The absorbance at 450 nm is measured with a spectrophotometer. A standard curve is obtained by plotting the absorbance (linear) versus the corresponding concentrations of the known standards of the HSA-MGO standards (log).
The MGO concentration of samples, which are run concurrently with the standards, can be determined from the standard curve.