Publications & Posters

Development of a Three-plex Single-Molecule Immunoassay Enabling Measurement of The EGFR Ligands Amphiregulin, Betacellulin, and Transforming Growth Factor Alpha Simultaneously in Human Serum Samples


Olsen DA, Kjaer IM and Brandslund I

J Immunol Methods. 2018 Aug;459:63-69.

DOI: 10.1016/j.jim.2018.05.002



Prior to large studies in breast cancer patients and healthy individuals we established a sensitive three-plex immunoassay to measure the EGFR ligands amphiregulin (AR), betacellulin (BTC) and transforming growth factor α (TGF-α) simultaneously in human serumsamples.


The three-plex immunoassay was developed using single molecule array (Simoa) technology and requires only 20 μL of serum.


AR, BTC and TGF-α were first established as three single-plex assays. Multiplexing the three single-plex assays showed no significant cross reactivity between the reagents. The concentrations of the ligands in serum samples showed correlations r2 ≥ 0.84 between the single-plex and three-plex methods. The three-plex assay demonstrated limit of detection levels at 0.16 ng/L for AR, 0.23 ng/L for BTC and 0.22 ng/L for TGF-α. Total coefficients of variations were 8.5%-31% for AR, 11%-21.8% for BTC and 12.4%-16.2% for TGF-α. Spiking experiments showed a mean recovery of 97% for AR, 86% for BTC and 81% for TGF-α. The concentrations of the EGFR ligands did not change significantly after series of freeze thaw cycles or incubation at 22 °C for up to 24 h.


This robust three-plex assay with up to 40-fold increase in sensitivity relative to conventional ELISA is the first published method that has the required sensitivity to measure AR, BTC and TGF-α simultaneously in human blood samples.