title: Garbage in, garbage out: a critical evaluation of strategies used for validation of immunohistochemical biomarkers.
abstract: The use of immunohistochemistry (IHC) in clinical cohorts is of paramount importance in determining the utility of a biomarker in clinical practice. A major bottleneck in translating a biomarker from bench-to-bedside is the lack of well characterized, specific antibodies suitable for IHC. Despite the widespread use of IHC as a biomarker validation tool, no universally accepted standardization guidelines have been developed to determine the applicability of particular antibodies for IHC prior to its use. In this review, we discuss the technical challenges faced by the use of immunohistochemical biomarkers and rigorously explore classical and emerging antibody validation technologies. Based on our review of these technologies, we provide strict criteria for the pragmatic validation of antibodies for use in immunohistochemical assays.
authors.affiliation: UCD School of Biomolecular and Biomedical Science, UCD Conway Institute, University College Dublin, Belfield, Dublin 4, Ireland; Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Uppsala University, 751 85 Uppsala, Sweden; OncoMark Ltd, NovaUCD, Belfield Innovation Park, Belfield, Dublin 4, Ireland.
authors.forename: Gillian
authors.initials: G
authors.lastname: O'Hurley
authors.affiliation: Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Uppsala University, 751 85 Uppsala, Sweden.
authors.forename: Evelina
authors.initials: E
authors.lastname: Sjöstedt
authors.affiliation: OncoMark Ltd, NovaUCD, Belfield Innovation Park, Belfield, Dublin 4, Ireland.
authors.forename: Arman
authors.initials: A
authors.lastname: Rahman
authors.affiliation: UCD School of Biomolecular and Biomedical Science, UCD Conway Institute, University College Dublin, Belfield, Dublin 4, Ireland.
authors.forename: Bo
authors.initials: B
authors.lastname: Li
authors.affiliation: Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Uppsala University, 751 85 Uppsala, Sweden.
authors.forename: Caroline
authors.initials: C
authors.lastname: Kampf
authors.affiliation: Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Uppsala University, 751 85 Uppsala, Sweden. Electronic address: Fredrik.Ponten@igp.uu.se.
authors.forename: Fredrik
authors.initials: F
authors.lastname: Pontén
authors.affiliation: UCD School of Biomolecular and Biomedical Science, UCD Conway Institute, University College Dublin, Belfield, Dublin 4, Ireland; OncoMark Ltd, NovaUCD, Belfield Innovation Park, Belfield, Dublin 4, Ireland. Electronic address: William.Gallagher@ucd.ie.
authors.forename: William M
authors.initials: WM
authors.lastname: Gallagher
authors.affiliation: Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Uppsala University, 751 85 Uppsala, Sweden.
authors.forename: Cecilia
authors.initials: C
authors.lastname: Lindskog
mesh_terms: D000906:Antibodies / Q000378:metabolism; D014408:Biomarkers, Tumor / Q000032:analysis / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods* / Q000592:standards; D009369:Neoplasms / Q000175:diagnosis / Q000378:metabolism; D012015:Reference Standards; D013048:Specimen Handling / Q000379:methods / Q000592:standards; D014481:United States; D014486:United States Food and Drug Administration; D054928:Validation Studies as Topic
chemical_list: D000906:Antibodies; D014408:Biomarkers, Tumor
keywords: Antibody reliability; Antibody validation; Biomarker discovery; Immunohistochemistry; Workflow
references.citation: Brief Funct Genomic Proteomic. 2004 Nov;3(3):212-9
references.pmid: 15642185
references.citation: Biotechniques. 2010 Mar;48(3):197-209
references.pmid: 20359301
references.citation: Cancer Chemother Pharmacol. 2008 Jan;61(1):23-32
references.pmid: 17909809
references.citation: Adv Anat Pathol. 2007 Mar;14(2):129-31
references.pmid: 17471119
references.citation: Diagn Mol Pathol. 2003 Mar;12(1):1-13
references.pmid: 12605030
references.citation: Biotech Histochem. 2006 Jan-Feb;81(1):3-12
references.pmid: 16760122
references.citation: J Clin Pathol. 1994 Sep;47(9):816-22
references.pmid: 7962650
references.citation: Proteomics. 2012 Jul;12(13):2067-77
references.pmid: 22623277
references.citation: J Pathol. 2000 Aug;191(4):452-61
references.pmid: 10918222
references.citation: Nature. 1975 Aug 7;256(5517):495-7
references.pmid: 1172191
references.citation: Hum Pathol. 1994 Jan;25(1):2-11
references.pmid: 7508882
references.citation: J Pathol. 2014 Jan;232(2):190-8
references.pmid: 24030847
references.citation: Cancer Lett. 1991 Sep;59(3):251-6
references.pmid: 1680544
references.citation: Mol Cell Proteomics. 2014 Feb;13(2):397-406
references.pmid: 24309898
references.citation: Clin Cancer Res. 2005 Jun 15;11(12):4338-40
references.pmid: 15958615
references.citation: Histopathology. 2006 Oct;49(4):411-24
references.pmid: 16978205
references.citation: Pathology. 1992 Jan;24(1):1-4
references.pmid: 1579363
references.citation: Clin Pharmacol Ther. 2001 Mar;69(3):89-95
references.pmid: 11240971
references.citation: Mol Cell Proteomics. 2008 Oct;7(10):2028-37
references.pmid: 18667413
references.citation: Nat Methods. 2006 Dec;3(12):995-1000
references.pmid: 17072308
references.citation: Histopathology. 2014 Jan;64(2):293-305
references.pmid: 24330150
references.citation: Nature. 2007 Jan 11;445(7124):168-76
references.pmid: 17151600
references.citation: J Histochem Cytochem. 2005 Nov;53(11):1311-21
references.pmid: 16009962
references.citation: J Histochem Cytochem. 2007 Feb;55(2):105-9
references.pmid: 16982846
references.citation: J Clin Pathol. 1997 May;50(5):422-8
references.pmid: 9215127
references.citation: Nat Genet. 1998 Jul;19(3):225-32
references.pmid: 9662393
references.citation: Genome Biol. 2009;10(11):R130
references.pmid: 19919682
references.citation: Mol Syst Biol. 2009;5:337
references.pmid: 20029370
references.citation: J Clin Pathol. 2006 Jul;59(7):706-10
references.pmid: 16461563
references.citation: Cell Tissue Res. 2006 Jan;323(1):147-55
references.pmid: 16007506
references.citation: Histochem Cell Biol. 2008 Sep;130(3):447-63
references.pmid: 18679705
references.citation: FASEB J. 2014 Jul;28(7):2901-14
references.pmid: 24648543
references.citation: Nat Med. 1998 Jul;4(7):844-7
references.pmid: 9662379
references.citation: J Immunol Methods. 2009 Feb 28;341(1-2):1-18
references.pmid: 19063895
references.citation: J Histochem Cytochem. 2009 Jan;57(1):1-5
references.pmid: 18854594
references.citation: Environ Health Perspect. 1993 Dec;101 Suppl 5:207-9
references.pmid: 7912187
references.citation: Nat Biotechnol. 2010 Dec;28(12):1248-50
references.pmid: 21139605
references.citation: J Histochem Cytochem. 1997 Feb;45(2):155-8
references.pmid: 9016305
references.citation: Methods. 2014 Nov;70(1):34-8
references.pmid: 24525140
references.citation: PLoS One. 2012;7(3):e32927
references.pmid: 22412953
references.citation: Mol Cell Proteomics. 2005 Dec;4(12):1920-32
references.pmid: 16127175
references.citation: J Immunol Methods. 2004 Oct;293(1-2):143-51
references.pmid: 15541284
references.citation: J Vis Exp. 2012 May 31;(63):
references.pmid: 22688270
references.citation: J Comp Neurol. 2005 Dec 26;493(4):477-8
references.pmid: 16304632
references.citation: Nature. 2003 Oct 16;425(6959):737-41
references.pmid: 14562106
references.citation: Mol Syst Biol. 2008;4:222
references.pmid: 18854821
references.citation: Hum Pathol. 2007 Jul;38(7):1037-1046
references.pmid: 17442373
references.citation: J Clin Oncol. 2011 Nov 10;29(32):4273-8
references.pmid: 21990413
references.citation: J Mol Histol. 2008 Aug;39(4):389-99
references.pmid: 18584295
references.citation: Am J Pathol. 1998 Dec;153(6):1767-73
references.pmid: 9846967
references.citation: J Clin Pathol. 2012 Jun;65(6):496-502
references.pmid: 22447914
references.citation: J Intern Med. 2010 Sep;268(3):232-45
references.pmid: 20695973
references.citation: J Histochem Cytochem. 1969 Sep;17(9):563-9
references.pmid: 4186250
references.citation: N Engl J Med. 1995 Jun 1;332(22):1521-2
references.pmid: 7739705
references.citation: J Histochem Cytochem. 2004 Mar;52(3):347-54
references.pmid: 14966201
references.citation: J Mol Biol. 2004 May 14;338(5):1027-36
references.pmid: 15111065
references.citation: Nature. 2011 Oct 19;478(7369):343-8
references.pmid: 22012392
references.citation: Clin Chim Acta. 1998 Dec;278(2):177-84
references.pmid: 10023825
references.citation: Clin Chem. 2010 Jan;56(1):99-110
references.pmid: 19926775
references.citation: Methods. 2008 Jul;45(3):227-32
references.pmid: 18620061
references.citation: Mol Cell Proteomics. 2006 Apr;5(4):573-88
references.pmid: 16332733
references.citation: Am J Surg Pathol. 2002 Sep;26(9):1161-8
references.pmid: 12218572
references.citation: J Histochem Cytochem. 2011 Apr;59(4):356-65
references.pmid: 21411807
references.citation: Anal Biochem. 1982 Jan 1;119(1):142-7
references.pmid: 7072935
references.citation: Adv Drug Deliv Rev. 2009 Jul 25;61(9):746-59
references.pmid: 19389436
references.citation: Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2002 Jan;93(1):56-74
references.pmid: 11805778
references.citation: J Histochem Cytochem. 1991 Jun;39(6):741-8
references.pmid: 1709656
references.citation: Bioinformatics. 2012 Apr 15;28(8):1184-5
references.pmid: 22345621
references.citation: Nature. 2007 Aug 23;448(7156):959-63
references.pmid: 17713539
references.citation: Nat Rev Genet. 2009 Jan;10(1):57-63
references.pmid: 19015660
references.citation: Nat Biotechnol. 2013 Mar;31(3):230-2
references.pmid: 23360966
references.citation: J Pathol. 2004 Apr;202(4):430-8
references.pmid: 15095270
references.citation: Am J Surg Pathol. 2010 Oct;34(10):1472-9
references.pmid: 20871222
references.citation: APMIS. 1994 Sep;102(9):641-6
references.pmid: 7946267
references.citation: J Histochem Cytochem. 1968 Sep;16(9):557-60
references.pmid: 5717715
references.citation: Cancer Biol Ther. 2005 May;4(5):512-7
references.pmid: 15908802
references.citation: J Intern Med. 2011 Nov;270(5):428-46
references.pmid: 21752111
references.citation: Mol Cell Proteomics. 2007 Sep;6(9):1589-98
references.pmid: 17533221
references.citation: J Histochem Cytochem. 2011 Apr;59(4):366-81
references.pmid: 21411808
references.citation: J Clin Lab Anal. 1997;11(1):2-9
references.pmid: 9021518
references.citation: Proc Natl Acad Sci U S A. 1969 Jun;63(2):378-83
references.pmid: 4895535
references.citation: J Immunol Methods. 1983 Dec 16;65(1-2):217-23
references.pmid: 6606681
references.citation: Breast Cancer Res. 2010;12(4):R47
references.pmid: 20615243
references.citation: J Immunol Methods. 2004 Jul;290(1-2):81-92
references.pmid: 15261573
references.citation: Mol Oncol. 2014 Jun;8(4):783-98
references.pmid: 24725481
references.citation: J Clin Oncol. 2012 Dec 20;30(36):4477-84
references.pmid: 23045591
references.citation: J Biomed Opt. 2005 Jul-Aug;10(4):41207
references.pmid: 16178631
references.citation: Lab Invest. 1986 Aug;55(2):244-8
references.pmid: 3525985
references.citation: Curr Opin Neurobiol. 2002 Oct;12(5):562-5
references.pmid: 12367636
references.citation: Methods Mol Biol. 1999;115:405-14
references.pmid: 10098207
references.citation: Clin Proteomics. 2013 Oct 02;10(1):13
references.pmid: 24088261
references.citation: Proc Natl Acad Sci U S A. 2013 Jul 16;110(29):11982-7
references.pmid: 23818604
references.citation: Biotechnol J. 2014 Mar;9(3):435-45
references.pmid: 24403002
references.citation: Biotechniques. 2005 May;38(5):723-7
references.pmid: 15945371
references.citation: Proteomics. 2005 Nov;5(17):4327-37
references.pmid: 16237735
references.citation: Eur J Morphol. 1993 Mar-Jun;31(1-2):144-50
references.pmid: 8398549
references.citation: J Histochem Cytochem. 2009 Jan;57(1):7-8
references.pmid: 18854593
references.citation: Science. 2013 Feb 15;339(6121):823-6
references.pmid: 23287722
references.citation: Appl Immunohistochem Mol Morphol. 2000 Mar;8(1):61-70
references.pmid: 10937051
references.citation: Nature. 2011 May 19;473(7347):337-42
references.pmid: 21593866
references.citation: Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350-4
references.pmid: 388439
references.citation: Mod Pathol. 2002 Dec;15(12):1302-8
references.pmid: 12481011
references.citation: Rom J Morphol Embryol. 2012;53(2):233-42
references.pmid: 22732791
references.citation: Patholog Res Int. 2012;2012:947041
references.pmid: 22482085
references.citation: J Clin Pathol. 2008 Jan;61(1):79-83
references.pmid: 17412873
references.citation: Proteomics. 2010 Mar;10(6):1141-9
references.pmid: 20175080
references.citation: Bioinformatics. 2002 Apr;18(4):585-96
references.pmid: 12016056
references.citation: J Pathol. 1993 Oct;171(2):83-98
references.pmid: 7506771
references.citation: Appl Immunohistochem Mol Morphol. 2006 Jun;14(2):225-8
references.pmid: 16785795
references.citation: J Proteomics. 2012 May 17;75(9):2611-20
references.pmid: 22465717
references.citation: Br J Cancer. 2004 Apr 19;90(8):1583-90
references.pmid: 15083189
references.citation: BMC Cell Biol. 2008 Mar 19;9:13
references.pmid: 18366689
references.citation: Bioinformatics. 2008 Dec 15;24(24):2928-9
references.pmid: 18945683
references.citation: J Clin Oncol. 2010 Jun 1;28(16):2784-95
references.pmid: 20404251
references.citation: J Clin Pathol. 2002 Aug;55(8):575-6
references.pmid: 12147648
references.citation: Pathobiology. 2011;78(2):115-21
references.pmid: 21677474
references.citation: Nat Biotechnol. 2007 Jan;25(1):117-24
references.pmid: 17187058