A-213 Assessing the Function of High-Density Lipoprotein and the Risk of Cardiovascular Disease Using Fully Automated Immunoassay Analyzer, HISCLTM

Abstract Background High-density lipoprotein (HDL) function has earned an attention as a way of assessing the risk of cardiovascular disease. Conventionally, cholesterol efflux capacity (CEC) has been used to measure the function of the HDL, but because of its time-consuming and complex procedure it has been withheld from being widely used in clinical fields. We have previously introduced an immunoassay-based technique that measures cholesterol uptake capacity (CUC) as a novel measure of HDL functionality. There we have demonstrated that CUC is capable of risk stratification in patients with coronary artery disease, in which coronary lesions was inversely associated with CUC (Fujimoto et al., Atherosclerosis 2022; Murakami et al., Scientific Reports 2023). However, since this method uses a specialized washing solution with mild surfactant to prevent delipidation from HDL rather than using the conventional washing solution, it had to be operated as a dedicated device. In other words, CUC could not be measured with automatic analyzer along with the existing immunoassay items. Here we propose an upgraded version that intends for IVD use. Method Automated immunoassay system, HISCL™ was used to measure CUC in serum. This rapid and sensitive immunoassay is based on anti-ApoA1 antibody coated magnetic beads that captures HDL particles from the sample, while HDL simultaneously takes in the PEGylated biotin-labeled cholesterol. After the cholesterol uptake reaction, magnetic beads are washed with the HISCL™ washing solution, a commercially available reagent used in conventional immunoassay. Then, using alkaline phosphatase labeled streptavidin and specific substrate, signals are detected. As for the samples, myeloperoxidase-mediated oxidized HDL, recombinant Lecithin-cholesterol acyltransferase (rLCAT) added HDL, and commercially available serum (as control) were measured using above mentioned method to evaluate functional change in HDL. Results Despite using the conventional washing solution, we were able to maintain specific signals corresponding to the dynamics of cholesterol probe by optimizing the reaction sequence and reducing the number of washing process. In this condition, the assay was linear between 0 and 50 nL of serum (R2 > 0.99), and CUC correlated with CEC (r = 0.897, P < 0.01). Furthermore, statistically significant difference (P < 0.05) in CUC level was seen with HDL in different functional states. In specific, CUC dropped with HDL oxidation while an opposite movement was found in HDL that’s been treated with rLCAT, an enzyme that esterifies cholesterol thereby improves cholesterol uptake of HDL. This result suggests that this assay is capable of accurately depicting the function of HDL. In our previous measuring system, CUC demonstrated its utility in coronary risk stratification. Here, we were able to see a significant correlation (r = 0.973, P < 0.01) between our previous data and our current data with high reproducibility (Coefficient of Variation < 10%). Conclusion Our results suggest that we are inching ever closer to expanding the research of HDL functionality to the clinical settings and diagnosing patients with high risk of cardiovascular disease.