title: Aqueous two-phase systems enable multiplexing of homogeneous immunoassays.
abstract: Quantitative measurement of protein biomarkers is critical for biomarker validation and early disease detection. Current multiplex immunoassays are time consuming costly and can suffer from low accuracy. For example, multiplex ELISAs require multiple, tedious, washing and blocking steps. Moreover, they suffer from nonspecific antibody cross-reactions, leading to high background and false-positive signals. Here, we show that co-localizing antibody-bead pairs in an aqueous two-phase system (ATPS) enables multiplexing of sensitive, no-wash, homogeneous assays, while preventing nonspecific antibody cross-reactions. Our cross-reaction-free, multiplex assay can simultaneously detect picomolar concentrations of four protein biomarkers ((C-X-C motif) ligand 10 (CXCL10), CXCL9, interleukin (IL)-8 and IL-6) in cell supernatants using a single assay well. The potential clinical utility of the assay is demonstrated by detecting diagnostic biomarkers (CXCL10 and CXCL9) in plasma from 88 patients at the onset of the clinical symptoms of chronic graft-versus-host disease (GVHD).
authors.forename: Arlyne B
authors.initials: AB
authors.lastname: Simon
authors.forename: John P
authors.initials: JP
authors.lastname: Frampton
authors.forename: Nien-Tsu
authors.initials: NT
authors.lastname: Huang
authors.forename: Katsuo
authors.initials: K
authors.lastname: Kurabayashi
authors.forename: Sophie
authors.initials: S
authors.lastname: Paczesny
authors.forename: Shuichi
authors.initials: S
authors.lastname: Takayama
references.citation: Curr Opin Chem Biol. 2014 Feb;18:29-37
references.pmid: 24534750
references.citation: Mol Cell Proteomics. 2012 Apr;11(4):M111.011460
references.pmid: 22171321
references.citation: Nat Biotechnol. 2002 Apr;20(4):359-65
references.pmid: 11923841
references.citation: Lab Chip. 2010 Dec 21;10(24):3335-40
references.pmid: 20959907
references.citation: Clin Cancer Res. 2008 Oct 1;14(19):5967-76
references.pmid: 18829475
references.citation: Sci Rep. 2014 May 02;4:4878
references.pmid: 24786974
references.citation: Clin Chem. 2005 Jul;51(7):1102-9
references.pmid: 15976097
references.citation: Mol Cell Proteomics. 2008 Oct;7(10):1863-75
references.pmid: 18622029
references.citation: Bone Marrow Transplant. 2010 Aug;45(8):1340-6
references.pmid: 20140022
references.citation: J Immunol. 1999 Mar 15;162(6):3549-58
references.pmid: 10092813
references.citation: Am J Respir Cell Mol Biol. 2006 Aug;35(2):211-9
references.pmid: 16543606
references.citation: J Proteome Res. 2008 Jun;7(6):2406-14
references.pmid: 18422355
references.citation: J Clin Invest. 1994 Aug;94(2):731-40
references.pmid: 8040328
references.citation: Clin Biochem Rev. 2008 Aug;29 Suppl 1:S49-52
references.pmid: 18852857
references.citation: Nano Lett. 2011 Jul 13;11(7):2579-83
references.pmid: 20804215
references.citation: Blood. 2009 Jan 8;113(2):273-8
references.pmid: 18832652
references.citation: Curr Chem Genomics. 2008 Feb 25;1:2-10
references.pmid: 20161822
references.citation: BMC Cancer. 2008 Sep 18;8:265
references.pmid: 18801189
references.citation: Blood. 2012 Nov 15;120(20):4246-55
references.pmid: 23012327
references.citation: Nat Med. 2004 Dec;10(12):1390-6
references.pmid: 15558056
references.citation: J R Soc Interface. 2011 Aug 7;8(61):1104-13
references.pmid: 21247950
references.citation: Respir Res. 2006 Oct 11;7:125
references.pmid: 17034639
references.citation: Nat Mater. 2009 Sep;8(9):736-41
references.pmid: 19684584
references.citation: Respir Res. 2005 Jul 20;6:75
references.pmid: 16033640
references.citation: Clin Cancer Res. 2011 Jan 1;17(1):77-88
references.pmid: 21047980
references.citation: J Immunol Methods. 2008 Dec 31;339(2):245-58
references.pmid: 18822292
references.citation: J Biomed Biotechnol. 2003;2003(5):299-307
references.pmid: 14688416
references.citation: J Immunol. 1994 Apr 15;152(8):4087-94
references.pmid: 8144974
references.citation: Clin Chem. 2010 Feb;56(2):314-8
references.pmid: 20022982
references.citation: Blood. 2009 Apr 9;113(15):3620-30
references.pmid: 19168793
references.citation: Nat Rev Drug Discov. 2006 Apr;5(4):310-20
references.pmid: 16582876