Vol. 48 | No. 3 | 2010
Antibodies are among the most commonly
used research tools, routinely used for
Western blot (WB), immunoprecipitation
(IP), enzyme-linked immunosorbent assays
(ELISA), quantitative immunofluorescence
(QIF), and immunohistochemistry (IHC).
They are also important tools in clinical
management with extensive use in both
laboratory medicine (ELISA assays and
flow cytometry) and anatomic pathology
(IHC). In anatomic pathology, IHC serves
as a diagnostic, prognostic, and predictive
method and IHC readings directly influence
the management of patients in the clinical
setting. For example, the assessment of
estrogen receptor α (ER-α), and human
epidermal growth factor receptor 2 (HER2)
by IHC in breast cancer patients is the defin-
itive test to determine whether or not a
patient will receive therapies that can cost
as much as $100,000 per year. Thus, in the
clinic, as well as in the research laboratory,
careful accurate validation of antibody
reagents is critical for correct results.
The influence of antibody-based tests
on clinical decisions has led to a number
of publications that have highlighted
the unmet need for standardization of
such assays and development of antibody
validation guidelines (1–8). Although
many groups have enunciated the need,
there are no universally accepted guide-
lines for best practice in antibody-based
tests. There are a number of books on
the topics by world leaders such as Clive
Taylor and David Dabbs, and recently, an
ad hoc group published a set of “recom-
mendations” (2). However, these works
focus on the clinical aspects of IHC,
often using subjective criteria and often
not taking advantage of recent scientific
advances that allow more quantitative
evaluation of antibodies. Conversely, there
are other groups that have done biolog-
ically rigorous evaluation of antibodies
using surface plasmon resonance (9) and
even X-ray crystallization of antibodies
bound to their antigens (10), methods that
are unachievable in a routine research or
clinical setting. The wide range of rigor
and methodology in what is used for
validation is probably responsible for a
lack of consensus on a single method for
antibody validation. Here we present
an overview of antibody validation
approaches and the pitfalls associated with
the failures of validation. This work specif-
ically focuses on assessment of prognostic
and predictive cancer-related biomarkers
on formalin-fixed paraffin embedded
What is antibody validation?
The FDA defines validation as “the process
of demonstrating, through the use of specific
laboratory investigations, that the perfor-
mance characteristics of an analytical
method are suitable for its intended
analytical use” (www.fda.gov/downloads/
For antibodies, one must demonstrate that
they are specific, selective, and reproducible
in the context for which they are used.
When it comes to IHC, standardization
can be quite challenging due to the number
of pre-analytical, analytical, and post-
analytical factors known to influence
staining in FFPE tissue. Variable time to
fixation, inadequate fixation period, differ-
ences in fixative used, and tissue processing
can all affect tissue antigenicity (5,11).
Antibody clone and dilution, antigen
retrieval, detection system, and interpre-
tation of results using different cutoff points
are also important variables that regulate
IHC measurements (3,12) (V.K.A., unpub-
lished data). Here we focus on analytical
factors and highlight the importance of
proper antibody validation, especially for
IHC or QIF use.
Common pitfalls in
A recent editorial by Michel et al. (13)
emphasizes the lack of target specificity
for 49 antibodies against 19 subtypes of
G protein–coupled receptors calling for
more stringent antibody validation criteria.
Examples highlighted by the authors
included double-knockout mice lacking
the M2 and M3 subtypes of muscarinic
receptors still staining positive for M2 and
M3 receptor antibodies (14), and triple-
knockout mice for the three α1-adrenoceptor
Jennifer Bordeaux, Allison W. Welsh, Seema Agarwal, Elizabeth Killiam, Maria T. Baquero, Jason A. Hanna,
Valsamo K. Anagnostou, and David L. Rimm
Department of Pathology, Yale University School of Medicine, New Haven, CT, USA
BioTechniques 48:197-209 (March 2010) doi 10.2144/000113382
Keywords: antibody; validation; immunohistochemistry; immunofluorescence
Antibodies are among the most frequently used tools in basic science research and in clinical assays, but there are no
universally accepted guidelines or standardized methods for determining the validity of these reagents. Furthermore,
for commercially available antibodies, it is clear that what is on the label does not necessarily correspond to what is in
the tube. To validate an antibody, it must be shown to be specific, selective, and reproducible in the context for which
it is to be used. In this review, we highlight the common pitfalls when working with antibodies, common practices for
validating antibodies, and levels of commercial antibody validation for seven vendors. Finally, we share our algorithm
for antibody validation for immunohistochemistry and quantitative immunofluorescence.
Vol. 48 | No. 3 | 2010
1. Dowsett, M., W.M. Hanna, M. Kockx, F.
Penault-Llorca, J. Rüschoff, T. Gutjahr, K.
Habben, and M.J. van de Vijver. 2007. Standard-
ization of HER2 testing: results of an interna-
tional proficiency-testing ring study. Mod.
2. Goldstein, N.S., S.M. Hewitt, C.R. Taylor,
H. Yaziji, D.G. Hicks, and Members of the
Ad-Hoc Committee on Immunohistochemistry
Standardization. 2007. Recommendations for
improved standardization of immunohistochem-
istry. Appl. Immunohistochem. Mol. Morphol.
3. Shi, S.-R., C. Liu, and C.R. Taylor. 2007.
Standardization of immunohistochemistry
for formalin-fixed, paraffin-embedded tissue
sections based on the antigen-retrieval technique:
from experiments to hypothesis. J. Histochem.
4. Wolff, A.C., M.E.H. Hammond, J.N. Schwartz,
K.L. Hagerty, D.C. Allred, R.J. Cote, M.
Dowsett, P.L. Fitzgibbons, et al. 2007. American
Society of Clinical Oncology/College of
American Pathologists guideline recommenda-
tions for human epidermal growth factor receptor
2 testing in breast cancer. J. Clin. Oncol. 25:118-
5. Hicks, D.G. and S. Kulkarni. 2008. HER2+
breast cancer: review of biologic relevance and
optimal use of diagnostic tools. Am. J. Clin.
6. Yaziji, H., C.R. Taylor, N.S. Goldstein, D.J.
Dabbs, E.H. Hammond, B. Hewlett, A.D. Floyd,
T.S. Barry, et al. 2008. Consensus recommenda-
tions on estrogen receptor testing in breast cancer
by immunohistochemistry. Appl. Immunohis-
tochem. Mol. Morphol. 16:513-520.
7. Deutsch, E.W., C.A. Ball, J.J. Berman, G.S.
Bova, A. Brazma, R.E. Bumgarner, D. Campbell,
H.C. Causton, et al. 2008. Minimum infor-
mation specification for in situ hybridization
and immunohistochemistry experiments
(MISFISHIE). Nat. Biotechnol. 26:305-312.
8. McShane, L.M., D.G. Altman, W. Sauerbrei,
S.E. Taube, M. Gion, and G.M. Clark. 2006.
REporting recommendations for tumor MARKer
prognostic studies (REMARK). Breast Cancer
Res. Treat. 100:229-235.
9. Mullett, W.M., E.P. Lai, and J.M. Yeung. 2000.
Surface plasmon resonance-based immunoassays.
10. Franklin, M.C., K.D. Carey, F.F. Vajdos, D.J.
Leahy, A.M. de Vos, and M.X. Sliwkowski. 2004.
Insights into ErbB signaling from the structure
of the ErbB2-pertuzumab complex. Cancer Cell
11. Hsi, E.D. 2001. A practical approach for evalu-
ating new antibodies in the clinical immunohis-
tochemistry laboratory. Arch. Pathol. Lab. Med.
12. Leong, A.S. 2004. Quantitation in
immuno histology: fact or fiction? A discussion
of variables that influence results. Appl. Immuno-
histochem. Mol. Morphol. 12:1-7.
13. Michel, M.C., T. Wieland, and G. Tsujimoto.
2009. How reliable are G-protein-coupled
receptor antibodies? Naunyn Schmiedebergs
Arch. Pharmacol. 379:385-388.
14. Jositsch, G., T. Papadakis, R.V. Haberberger, M.
Wolff, J. Wess, and W. Kummer. 2009. Suitability
of muscarinic acetylcholine receptor antibodies
for immunohistochemistry evaluated on tissue
sections of receptor gene-deficient mice. Naunyn
Schmiedebergs Arch. Pharmacol. 379:389-395.
15. Jensen, B.C., P.M. Swigart, and P.C. Simpson.
2009. Ten commercial antibodies for alpha-1-
adrenergic receptor subtypes are nonspecific.
Naunyn Schmiedebergs Arch. Pharmacol.
16. Ramos-Vara, J.A. 2005. Technical
aspects of immunohistochemistry. Vet. Pathol.
17. Willingham, M.C. 1999. Conditional epitopes.
is your antibody always specific? J. Histochem.
18. Saper, C.B. and P.E. Sawchenko. 2003. Magic
peptides, magic antibodies: guidelines for appro-
priate controls for immunohistochemistry. J.
Comp. Neurol. 465:161-163.
19. Pezzella, F., A.G. Tse, J.L. Cordell, K.A. Pulford,
K.C. Gatter, and D.Y. Mason. 1990. Expression
of the bcl-2 oncogene protein is not specific for the
14;18 chromosomal translocation. Am. J. Pathol.
20. Willingham, M.C. and K. Bhalla.
1994. Transient mitotic phase localization of
bcl-2 oncoprotein in human carcinoma cells
and its possible role in prevention of apoptosis.
J. Histochem. Cytochem. 42:441-450.
21. Jacobs, T.W., A.M. Gown, H. Yaziji, M.J. Barnes,
and S.J. Schnitt. 1999. Specificity of HercepTest
in determining HER-2/neu status of breast
cancers using the United States Food and Drug
Administration-approved scoring system. J. Clin.
22. Spicer, S.S., M.A. Spivey, M. Ito, and
B.A. Schulte. 1994. Some ascites monoclonal
antibody preparations contain contaminants
that bind to selected Golgi zones or mast cells.
J. Histochem. Cytochem. 42:213-221.
23. Pozner-Moulis, S., M. Cregger, R.L. Camp,
and D.L. Rimm. 2007. Antibody validation by
quantitative analysis of protein expression using
expression of Met in breast cancer as a model.
Lab. Invest. 87:251-260.
24. Grimsey, N.L., C.E. Goodfellow, E.L.
Scotter, M.J. Dowie, M. Glass, and E.S. Graham.
2008. Specific detection of CB1 receptors;
cannabinoid CB1 receptor antibodies are not all
created equal! J. Neurosci. Methods 171:78-86.
25. Major, S.M., S. Nishizuka, D. Morita, R.
Rowland, M. Sunshine, U. Shankavaram, F.
Washburn, D. Asin, et al. 2006. AbMiner: a
bioinformatic resource on available monoclonal
antibodies and corresponding gene identifiers for
genomic, proteomic, and immunologic studies.
BMC Bioinformatics 7:192.
26. Björling, E. and M. Uhlén. 2008.
Antibodypedia, a portal for sharing antibody and
antigen validation data. Mol. Cell. Proteomics
27. Skliris, G.P., B.G. Rowan, M. Al-Dhaheri, C.
Williams, S. Troup, S. Begic, M. Parisien, P.H.
Watson, and L.C. Murphy. 2009. Immuno-
histochemical validation of multiple phospho-
specific epitopes for estrogen receptor alpha
(ERalpha) in tissue microarrays of ERalpha
positive human breast carcinomas. Breast Cancer
Res. Treat. 118:443-453.
28. Sompuram, S.R., V. Kodela, K. Zhang,
H. Ramanathan, G. Radcliffe, P. Falb, and S.A.
Bogen. 2002. A novel quality control slide for
quantitative immunohistochemistry testing. J.
Histochem. Cytochem. 50:1425-1434.
29. Saper, C.B. 2005. An open letter to our readers
on the use of antibodies. J. Comp. Neurol.
30. Gustavson, M.D., B. Bourke-Martin, D.
Reilly, M. Cregger, C. Williams, J. Mayotte, M.
Zerkowski, G. Tedeschi, et al. 2009. Standard-
ization of HER2 immunohistochemistry in
breast cancer by automated quantitative analysis.
Arch. Pathol. Lab. Med. 133:1413-1419.
31. van der Vegt, B., G.H. de Bock, J. Bart, N.G.
Zwartjes, and J. Wesseling. 2009. Validation of
the 4B5 rabbit monoclonal antibody in deter-
mining Her2/neu status in breast cancer. Mod.
32. Zafrani, B., M.H. Aubriot, E. Mouret,
P. De Crémoux, Y. De Rycke, A. Nicolas, E.
Boudou, A. Vincent-Salomon, et al. 2000. High
sensitivity and specificity of immunohistochem-
istry for the detection of hormone receptors in
breast carcinoma: comparison with biochemical
determination in a prospective study of 793 cases.
33. Sasano, H., T.J. Anderson, S.G. Silverberg, R.J.
Santen, M. Conway, D.P. Edwards, A. Krause,
A.S. Bhatnagar, et al. 2005. The validation of new
aromatase monoclonal antibodies for immuno-
histochemistry--a correlation with biochemical
activities in 46 cases of breast cancer. J. Steroid
Biochem. Mol. Biol. 95:35-39.
34. Cheang, M.C., D.O. Treaba, C.H. Speers,
I.A. Olivotto, C.D. Bajdik, S.K. Chia, L.C.
Goldstein, K.A. Gelmon, et al. 2006. Immuno-
histochemical detection using the new rabbit
monoclonal antibody SP1 of estrogen receptor
in breast cancer is superior to mouse monoclonal
antibody 1D5 in predicting survival. J. Clin.
35. Brock, J.E., J.L. Hornick, A.L.
Richardson, D.A. Dillon, and S.C. Lester.
2009. A comparison of estrogen receptor SP1
and 1D5 monoclonal antibodies in routine
clinical use reveals similar staining results.
Am. J. Clin. Pathol. 132:396-401.
36. Couchman, J.R. 2009. Commercial
antibodies: the good, bad, and really ugly. J.
Histochem. Cytochem. 57:7-8.
37. Mandell, J.W. 2003. Phosphorylation state-
specific antibodies: applications in investi-
gative and diagnostic pathology. Am. J. Pathol.
38. Mandell, J.W. 2008. Immunohis-
tochemical assessment of protein phosphory-
lation state: the dream and the reality. Histochem.
Cell Biol. 130:465-471.
39. Camp, R.L., V. Neumeister, and D.L. Rimm.
2008. A decade of tissue microarrays: progress
in the discovery and validation of cancer
biomarkers. J. Clin. Oncol. 26:5630-5637.
40. Kay, E., A. O’Grady, J.M. Morgan, S.
Wozniak, and B. Jasani. 2004. Use of tissue
microarray for interlaboratory validation of
HER2 immunocytochemical and FISH testing.
J. Clin. Pathol. 57:1140-1144.
41. Rhodes, K.J. and J.S. Trimmer. 2008. Antibody-
based validation of CNS ion channel drug targets.
J. Gen. Physiol. 131:407-413.
42. Kalyuzhny, A.E. 2009. The dark side
of the immunohistochemical moon: industry. J.
Histochem. Cytochem. 57:1099-1101.
Received 26 January 2010; accepted 28 January
Address correspondence to David L. Rimm,
Department of Pathology, Yale University
School of Medicine, 310 Cedar St., PO Box
208023, New Haven, CT, 06520 USA. e-mail: