Abstract 2104 PETH ELISA FOR ASSESSMENT OF ALCOHOL CONSUMPTION

Objective Phosphatidylethanol (PEth) is a phospholipid whole-blood biomarker for alcohol consumption that is remarkably stable as an analyte when stored, extracted, and measured from dried blood spot (DBS) cards. There are multiple options for collection, extraction, and analysis of PEth by mass spectrometry (MS) but no standardized best practices for immunoassays which keep the field from advancing. We sought to expand the PEth analysis options with two goals. First, develop and apply a PEth immunoassay to quantitatively differentiate alcohol consumption levels in two types of samples. Second, to demonstrate the flexibility of the ELISA using multiple blood collection devices compared to standard DBS cards. Methods We built a quantitative ELISA format by incorporating several types of PEth Standards: (i) a synthetic analog of the 16:0/18:1 most abundant natural isoform and (ii) authentic whole-blood, ex-vivo samples to construct standard curves. These standard curves were used to interpolate PEth values. We used simple extraction and sample prep methods to test whole blood samples and specimens prepared on DBS cards and alternative blood collection devices. We grouped samples into four alcohol consumption categories based on literature MS values: abstainer ( 250 ng/mL). Data and Results We determined PEth concentrations from whole-blood DBS samples derived from drinking/abstaining volunteers with at least five subjects per category. We used 5-parameter non-linear regression analysis on GraphPad software to determine best-fit curves with r2 values > 95%. ELISA PEth values were compared with PEth values determined by established LC/MS methods. Using 120 samples from an HIV-positive drinking cohort in Uganda, we found that the ELISA method effectively discriminated abstainer, low, moderate, and high categories in a qualitative fashion; and the measured ELISA values have significant correlation with MS values (Pearson, r = 0.72) but are quantitatively different likely due to differential matrix effects. The PEth ELISA has an LOD below 50 ng/mL, is compatible with multiple blood collection devices, and demonstrates significant sensitivity and specificity useful for a clinical setting. Conclusions This 96-well immunoassay expands the availability of PEth analysis to differentiate between multiple categories of alcohol consumption; and is poised to become a valuable clinical tool in screening large numbers of samples in high-throughput workflows in conjunction with MS testing. Partial funding for Drs. Hahn and Muyindike provided by NIH R01AA024990.