Development of a three-plex single molecule immunoassay enabling measurement of the EGFR ligands amphiregulin, betacellulin and transforming growth factor α simultaneously in human serum samples

Dorte Aalund Olsen*, Ina Mathilde Kjaer, Ivan Brandslund

*Corresponding author for this work

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Abstract

Background: 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 serum samples. Method: The three-plex immunoassay was developed using single molecule array (Simoa) technology and requires only 20 μL of serum. Results: 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. Conclusion: 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.

Original languageEnglish
JournalJournal of Immunological Methods
Volume459
Pages (from-to)63-69
ISSN0022-1759
DOIs
Publication statusPublished - 2018

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Immunoassay
Ligands
Serum
Limit of Detection
Betacellulin
Amphiregulin

Keywords

  • Digital ELISA
  • EGFR ligands
  • Multiplexed immunoassays
  • Simoa
  • Single molecule array

Cite this

@article{e34fa0662dac4ac9954d1287b98c60cd,
title = "Development of a three-plex single molecule immunoassay enabling measurement of the EGFR ligands amphiregulin, betacellulin and transforming growth factor α simultaneously in human serum samples",
abstract = "Background: 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 serum samples. Method: The three-plex immunoassay was developed using single molecule array (Simoa) technology and requires only 20 μL of serum. Results: 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. Conclusion: 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.",
keywords = "Digital ELISA, EGFR ligands, Multiplexed immunoassays, Simoa, Single molecule array",
author = "Olsen, {Dorte Aalund} and Kjaer, {Ina Mathilde} and Ivan Brandslund",
year = "2018",
doi = "10.1016/j.jim.2018.05.002",
language = "English",
volume = "459",
pages = "63--69",
journal = "Journal of Immunological Methods",
issn = "0022-1759",
publisher = "Elsevier",

}

TY - JOUR

T1 - Development of a three-plex single molecule immunoassay enabling measurement of the EGFR ligands amphiregulin, betacellulin and transforming growth factor α simultaneously in human serum samples

AU - Olsen, Dorte Aalund

AU - Kjaer, Ina Mathilde

AU - Brandslund, Ivan

PY - 2018

Y1 - 2018

N2 - Background: 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 serum samples. Method: The three-plex immunoassay was developed using single molecule array (Simoa) technology and requires only 20 μL of serum. Results: 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. Conclusion: 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.

AB - Background: 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 serum samples. Method: The three-plex immunoassay was developed using single molecule array (Simoa) technology and requires only 20 μL of serum. Results: 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. Conclusion: 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.

KW - Digital ELISA

KW - EGFR ligands

KW - Multiplexed immunoassays

KW - Simoa

KW - Single molecule array

U2 - 10.1016/j.jim.2018.05.002

DO - 10.1016/j.jim.2018.05.002

M3 - Journal article

VL - 459

SP - 63

EP - 69

JO - Journal of Immunological Methods

JF - Journal of Immunological Methods

SN - 0022-1759

ER -