Abstract 623: Robust and High-Throughput Cholesterol Efflux Assay
Background: The objective of this study was to develop a robust high-throughput assay for ABCA1 specific cholesterol efflux supporting its use in large clinical trials, which require consistent robust performance and cost-efficiency.
Methods: J774 cells were seeded on a 48-well plate and pulsed with 3H Cholesterol in a media with ACAT inhibitor. ABCA1 expression was induced by 8-bromo cAMP. Test serum (HDL) isolated by PEG precipitation was added to each well and efflux capacity determined as the ratio of radioactive counts in growth medium versus total counts when comparing cells expressing ABCA1 with those not expressing this transporter. Precision, linearity, limit of detection, specificity, reference range, robustness, and short-and long term stability were assessed.
Results: The overall intra-assay precision varied from 8.5% to 12%, whereas the inter-assay precision was about 20% based on fifteen individual runs on different days by three different analysts. The linear range of the assay was 0.81%-6.5% and 0.43%-13% cholesterol efflux (CE) for PEGylated and non-PEGylated serum, respectively. The LOD was 1.08% CE based on mean of negative control from all 16 runs plus 3 SD. Specificity was determined as the relative increase (11.3 fold) of cholesterol efflux by ApoA1 between cAMP and non-cAMP treated cells. Verification of reference range was determined as 2.1%-7.0% CE (mean±2SD). Consistency of the assay determined by %CV was not affected when different lots of cAMP (CV:6.4%) or ApoA1 (10.3%) were used. The CE values were not appreciably affected when identical samples were run by different analysts (CV:3.2%). Based on average of three data sets samples were stable for up to three days at 2-8°C (mean difference 9.6%), 10 days at -20°C (7.8%), and 7 months at -80°C (8.1%). About 3,000 samples were run over 4 weeks equaling 750 samples /week in a high-throughput format with consistent performance as assessed by each of the five QCs run in each run.
In conclusion, the CE assay described herein is the first high-throughput and robust assay fit for use in large clinical trials where consistent performance during the development of a drug program is critical.
Page 1 of 1
Author Disclosures: M.N. Dixit: None.
- © 2014 by American Heart Association, Inc.