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

Journal of Immunological Methods

Olsen DA, Kjaer IM and Brandslund I

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





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.