Monoclonal Antibody-Based ELISA Quantification of Serum Methylglyoxal-Derived Hydroimidazolone-1.

Background/

Objectives

Methylglyoxal-derived hydroimidazolone-1 (MG-H1), an advanced glycation end product, has implications in the pathogenesis of diabetic kidney disease (DKD). Although liquid chromatography-mass spectrometry is the current gold standard for quantifying MG-H1, its overall complexity limits its utility. We developed an ELISA to measure MG-H1 using a specific monoclonal antibody.

Methods

Competitive ELISA was used to quantify total, high-molecular-weight (HMW), and low-molecular-weight (LMW) MG-H1 in serum. The assay's specificity was validated against structurally related compounds. Spike-and-recovery experiments were conducted to assess accuracy and precision. Serum samples from healthy controls, diabetic patients without kidney disease, and patients with DKD were analyzed (n = 10, 23, and 19, respectively). MG-H1's correlation with renal biomarkers and diagnostic performance was assessed using receiver operating characteristic analyses.

Results

The ELISA exhibited preferential reactivity toward MG-H1 compared with structurally related compounds. Spike-and-recovery experiments resulted in recovery rates ranging 108-119%. MG-H1 levels were increased in patients with DKD, although the magnitude of the changes varied among the MG-H1 forms. All MG-H1 forms correlated positively with serum creatinine and blood urea nitrogen, and negatively with estimated glomerular filtration rate. No significant correlations were observed with glycoalbumin, and only a modest association was observed between LMW MG-H1 and HbA1c. Exploratory ROC analyses suggested that all MG-H1 forms could discriminate DKD from DM, with total and HMW MG-H1 showing performance comparable to that of conventional renal function markers.

Conclusions

This competitive ELISA enables high-throughput quantification of MG-H1 in serum and demonstrates analytical feasibility; further multicenter validation is required before clinical implementation.