Citation:

Pleil, J., M. Angrish, AND M. Madden. Human Exhaled Breath Condensate (EBC) Media: Implementation of Automated Quanterix SIMOA Immunochemistry Instrumentation. IABR Meeting, Vienna, AUSTRIA, September 13 - 16, 2015.

Impact/Purpose:

The National Exposure Research Laboratory (NERL) Human Exposure and Atmospheric Sciences Division (HEASD) conducts research in support of EPA mission to protect human health and the environment. HEASD research program supports Goal 1 (Clean Air) and Goal 4 (Healthy People) of EPA strategic plan. More specifically, our division conducts research to characterize the movement of pollutants from the source to contact with humans. Our multidisciplinary research program produces Methods, Measurements, and Models to identify relationships between and characterize processes that link source emissions, environmental concentrations, human exposures, and target-tissue dose. The impact of these tools is improved regulatory programs and policies for EPA.

Description:

Immunochemistry is an important clinical tool for observing biological pathways leading to disease. Standard enzyme-linked immunosorbent assays (ELISA) for cytokines are typically labor intensive and lack sensitivity at sub pg/ml concentrations. Here we report on emerging technology implementing fully-automated ELISA capable of molecular level detection and describe its application towards exhaled breath condensate (EBC) samples. The Quanterix SIMOA HD-1 analyzer (Lexington, MA, USA) was evaluated for analytical performance for the inflammatory cytokines IL-6, TNF-α, IL-1β and IL-8. The system sensitivity was challenged with human EBC representing the most dilute and analytically difficult of the biological media. Calibrations from synthetic samples and spiked EBC showed excellent linearity at trace levels (r2 > 0.99). Sensitivities varied by analyte, but were robust from ~0.006 (IL-6) to ~0.01 (TNF-α) pg/ml. All analytes demonstrated response suppression when diluted with pure water. Analytical runs required ~45 minutes setup time (samples, reagents, calibrants, etc.), subsequently the instrument operates without further intervention for up to 288 separate samples in plate mode, or 96 samples in vial mode. Currently, available kits are limited to single-plex analyses and so sample volumes require dilutions that should be made with assay buffer solution to avoid response suppression. The internal 5-parameter logistic (pl) calibration model should be supplemented with a linear regression spline at the very lowest analyte levels, (below ~ 1.5 pg/ml). The implementation of the automated Quanterix platform was successfully demonstrated using EBC, which poses the greatest challenge to ELISA due to limited sample volumes and low protein levels.

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