Non-standard AGE4 epitopes that predict polyneuropathy independently of obesity can be detected by slot dot-blot immunoassay

Abstract

Cite asBronowicka-Szydełko A, Krzystek-Korpacka M, Kuzan A, et al. Non-standard AGE4 epitopes that predict polyneuropathy independently of obesity can be detected by slot dot-blot immunoassay. AbstractBackground. Advanced glycation end-products (AGEs) are formed during cascade reactions between reducing sugars or reactive aldehydes and proteins, lipids or DNA molecules. They constitute a group of various stable compounds. Advanced glycation end-products are considered potential biomarkers of metabolic disorders. However, so far only a few methods to determine the level of individual AGEs have been developed.Objectives. The aim of the study was to compare the efficiency of the slot-dot blot method and direct enzyme-linked immunosorbent assay (ELISA) in detecting non-standard epitopes of methylglyoxal (MGO)modified proteins (AGE4) found in diabetes serum in trace amounts, and to assess AGE4 in diabetes and associated metabolic abnormalities.Material and methods. The presence of AGE4 was detected using 2 methods: direct ELISA and the slotdot blot method -a newly developed immunoassay based on monoclonal, commercially available antibody detection of non-standard AGE epitopes. AGE4 quantification, expressed as median AGE4 in arbitrary units (AU) and AGE4 positivity (the percent of samples with detectable AGE4) was related to diabetes-associated metabolic abnormalities, complications and treatment.Results. Slot-dot blot was significantly more efficient than ELISA in detecting non-standard AGE4 epitopes. AGE4 positivity was less frequent in patients with microangiopathy and in those with polyneuropathy. In patients with abnormal glucose metabolism, metformin treatment was associated with higher AGE4. AGE4 positivity was significantly lower in gliptin-treated patients. Multivariate analysis showed that polyneuropathy and obesity were independently associated with AGE4 positivity, with odds ratios (ORs) of 0.21 and 3.02, respectively. Moreover, logistic regression showed that AGE4 positivity and HbA1c are independent predictors of polyneuropathy. Considering both indicators allows correct classification of 70.4% of cases with a general accuracy of 76%. Conclusions.The slot dot-blot method detects compounds found in serum in trace amounts. Accumulation of AGE4 was associated with glucose metabolism abnormalities. A tendency toward AGE4 positivity was less frequent in patients with microangiopathy and in non-treated and gliptin-treated diabetes patients.

Is the Novel Slot Blot a Useful Method for Quantification of Intracellular Advanced Glycation End-Products?

“…Therefore, researchers cannot compare the AGE content of samples with that calculated using other methods. In addition, nitrocellulose membranes were selected for the other experiments, because PVDF membranes may have been unsuitable for use in slot blotting [14,52,53], although some studies still report their use [13,54]. Bronowicka-Szydełko et al blotted serum samples onto PVDF membranes and used anti-MGO-derived AGEs as primary and secondary antibodies or only secondary antibodies for probing the proteins [13].…”

“…In addition, nitrocellulose membranes were selected for the other experiments, because PVDF membranes may have been unsuitable for use in slot blotting [14,52,53], although some studies still report their use [13,54]. Bronowicka-Szydełko et al blotted serum samples onto PVDF membranes and used anti-MGO-derived AGEs as primary and secondary antibodies or only secondary antibodies for probing the proteins [13]. Furthermore, they quantified the luminance value of the MGO-AGEs per band of the secondary antibody.…”

“…Several studies have quantified AGEs using various methods, such as western blotting [3,4], immunostaining [5][6][7], enzyme-linked immunosorbent assay (ELISA) [5,8,9], gas chromatography-mass spectrometry (MS) (GC-MS) [10], matrix-associated laser desorption/ionization-MS (MALDI-MS) [11], liquid chromatography-electrospray ionization-MS analysis (LC-ESI-MS) [12], and slot blot analysis [13][14][15][16]. The data obtained in these studies were analyzed using statistics to evaluate significant increases or decreases in AGE levels.…”

Protocol for High-Throughput Screening of Neural Cell or Brain Tissue Protein Using a Dot-Blot Technique with Near-Infrared Imaging

“…Adapters are available for the application of either 96 “dots” in a 12×8 array, or as rectangular “slots” in a 6×8 array. The current protocol is designed for use with the 96 “dot” array, but could be modified for the slot array ( Bronowicka-Szydełko et al, 2020 ). Details on setup and cleaning of the apparatus can be found in the product manual ( https://www.bio-rad.com/webroot/web/pdf/lsr/literature/M1706545.pdf ).…”

Advanced Glycation End-Products in Common Non-Infectious Liver Diseases: Systematic Review and Meta-Analysis

“…Early research on AGEs focused mainly on the fluorescent fraction of AGEs; however, advances in biochemistry allowed detection of non-fluorescent fractions, with N-(carboxymethyl)lysine (CML) and pentosidine being the most widely studied. Novel approaches have allowed for the detection of even trace amounts of non-standard AGE subtypes [20].…”