Nonalcoholic fatty liver disease (NAFLD) is the most common liver disease, with a global prevalence of 32.4% (Riaz et al.). Among individuals with NAFLD, approximately 10¬ to 30% can develop nonalcoholic steatohepatitis (NASH) (Dyson et al.), which can progress to liver fibrosis or cirrhosis and eventually hepatocellular carcinoma if detected late. The standard practice for differentiating NASH from simple steatosis is liver biopsy, which is costly, invasive, limited by sampling errors, and unsuitable for some patients (Sumida et al.; Castera et al.). Therefore, it is critical to develop accurate and cost-effective non-invasive diagnostic strategies for the early detection of NASH among NAFLD patients. This study introduces a potential non-invasive ultra-sensitive diagnostic method using the Erenna Immunoassay System powered by Single-Molecule Counting (SMC) technology with CK-18 serum level as a biomarker. HepG2 cells were treated with oleic acid (OA) at concentrations ranging from 0 (control) to 1.6 mmol/L to induce lipid droplet accumulation and cell death to stimulate NAFLD progression in vitro. The Erenna Immunoassay System was used to determine CK-18 concentration in the HepG2 cell supernatant from each NAFLD model. The conventional ELISA was performed to compare method sensitivity. Results demonstrated that the ELISA could not accurately measure lactate dehydrogenase (LDH) concentrations when OA concentration was lower than 0.1 mmol/L, but the Erenna immunoassay yielded increasing CK-18 concentrations as the OA concentration increased, suggesting its superior sensitivity. For future studies, clinical serum samples should be tested in place of supernatant to evaluate method sensitivity in clinical scenarios. The study offers researchers unprecedented insight into developing a new non-invasive ultra-sensitive diagnostic method for the early detection of NASH.
Keywords: NAFLD,NASH, Erenna Immunoassay System, Single-Molecule Counting, CK-18
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