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Antibody-Based Detection of 3-Methylglutaconic Acid
AdvisorRyan, Robert O.
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The organic acid, 3-methylglutaconic acid (3MGA), is known to accumulate in urine of patients with disorders associated with compromised mitochondrial energy metabolism. Although the biochemical basis for this excretion is currently under investigation, there is evidence this metabolite can serve as a biomarker of disease. With the intent of creating a biomarker assay that can be used for disease diagnosis, monitoring progression of disease, or treatment efficacy, the development of a simple antibody-based assay was explored. Using immune serum from the inoculation of rabbits with a 3MGA-conjugated keyhole limpet hemocyanin (KLH) immunogen, several enzyme-linked immunosorbent assay (ELISA) formats were investigated to find the most sensitive assay. From this study, a competitive ELISA was chosen and optimized. The assay was then tested for reproducibility, specificity, and matrix effects in urine. In seven of 11 assays analyzed for inter-assay precision, the limit of detection (LOD) for measurement of 3MGA averaged 1.51 mg/mL with a coefficient of variation (CV%) of 31%. Specificity testing of similar dicarboxylic acids and a non-similar amino acid, showed that the anti-3MGA antibody had notably reduced binding activity compared to 3MGA. There was a noted decrease in signal when urine was used as the matrix, but measurement of free 3MGA spiked in urine was possible. Taken together, these data indicate that a quick and easy immunoassay to measure 3MGA is feasible. Future experiments could further refine reproducibility and sensitivity.