Antibody Validation- A Review.

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Review   Antibody val validatio idation n  Jennier Bordeaux, Allison Al lison W. Welsh, Welsh, Seema Se ema Agarwal, Agar wal, Elizabeth Eli zabeth Killiam, Kil liam, Maria . Baquero, Jason A. Hanna, Valsamo K. Anagnostou, and David L. Rimm  Department of Pathology, Pathology, Yale Yale University School School of Medicine, Medicine, New New Haven, CT, CT, USA   BioT  Bio Techniques 48:197-209 (March 2010) doi 10.2144/000113382

Keywords: antibody; validation; immunohistochemistry; immunoluorescence

Antibodies are among the most requently used tools in basic science research and in clinical assays, but there are no universally accepted guidelines guidel ines or standardized methods or determining the validity o these reagents. Furthermore, or commercially available antibodies, it is clear that what is on the label does not necessarily correspond to what is in the tube. o o validate an antibody, it must be shown to be specific, spe cific, selective, and reproducible in the context or which it is to be used. In this review, we highlight highlig ht the common pitalls when working with antibodies, common practices or  validating  valida ting ant antibodies, ibodies, and levels o comm commerci ercial al antibody antibody valida validation tion or seven ven vendors. dors. Fin Finally ally,, we we share share our algorith algorithm m or antibody validation or immu immunohistochemistry nohistochemistry and quantita quantitative tive immunofluorescence. immunofluorescence.

Introduction 

Antibodies are among the most commonly used research tools, routinely used or  Western  W estern blot blot (WB), immunop immunoprecip recipitati itation on (IP), enzyme-linked immunosorbent assays (ELISA), quantitative immunofluorescence (QIF), and immunohistochemistry (IHC). Tey 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 i nfluence the management o patients in the clinical setting. For example, the assessment o estrogen receptor α (ER-α), and human epidermal growth actor receptor 2 (HER2) by IHC in breast cancer patients is the definitive test to determine whether or not a  patient  patie nt will receive therapies therapies that can cost as much as $100,000 per year. Tus, in the clinic, as well as in the research laboratory, careul accurate validation o antibody reagents is critical or correct results. Te influence o antibody-based tests on clinical decisions has led to a number o publications that have highlighted the unmet need or standardization o such assays and development development o antibody  validat  val idation ion gu idel ideline iness (1–8). A ltho lthoug ughh many groups have enunciated theguideneed, there are no universally accepted lines or best practice in antibody-based tests. Tere are a number o books on the topics by world leaders such as Clive

aylor and David Dabbs, and recently recently,, an ad hoc group published a set o “recommendations” (2). However, these works ocus on the clinical aspects o IHC, ofen using subjective criteria and ofen not taking advantage o recent scientific advances that allow more quantitative evaluation o antibodies. antibodies . Conversely, there there are other groups that have done biologically rigorous evaluatio e valuationn o antibodies using surace plasmon resonance (9) and even X-ray crystallization o antibodies bound to their antigens anti gens (10), (10), methods that are unachievable in a routine research or clinical setting. Te wide range o rigor and methodology in what is used or  validat  val idation ion is proba probably bly resp responsi onsible ble or a lack o consensus on a single method or antibody validation. Here we present an overview o antibody validation approaches and the pitalls pitall s associated with the ailures o validation. val idation. Tis work speciically ocuses  ocuses on assessment o prognostic and predictive cancercancer-related related biomarkers on ormalin-ixed parain embedded (FFPE) tissue.

 What is antibody a ntibody validation? Te FDA defines validation as “the process o demonstrating, demonstrating, through t hrough the use o specific laboratory investigations, that the t he perormance o an methodcharacteristics are suitable or its analytical intended analytical use” (www.da.gov/downloads/ Drugs/GuidanceComplianceRegulatoryInormation/Guidances/UCM070107.pd).

For antibodies, one must demonstrate that they are specific, selective, and reproducible in the context or which they are used.  When it comes to IHC, standardization can be quite challenging due to the number o pre-analytical, analytical, and postanalytical actors known to inluence staining in FFPE tissue. Variable time to fixation, inadequate fixation period, differ di ffer-ences in fixative used, and a nd tissue processing can all aect tissue antigenicity (5,11). Antibody clone and dilution, antigen retrieval, detection system, and interpretation o results using different di fferent cutoff points are also important variables that regulate IHC measurements (3,12) (V.K. (V.K.A., A., unpublished data). Here we ocus on analytical actors and highlight the importance o  proper antibody validation, especially especial ly or IHC or QIF use.

Common pitfalls in antibody studies Nonspecific antibodies

A recent editorial by Michel et al. (13) emphasizes the lack o target specificity or 49 antibodies against 19 subtypes o G protein–coupled receptors calling or more stringent antibody validation criteria. Examples highlighted by the authors included double-knockout lacking the M2 and M 3  subtypes omice muscarinic receptors still staining positive or M 2 and M3  receptor antibodies (14), and tripleknockout mice or the three α1-adrenoceptor

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subtypes demonstrating staining patterns similar simil ar to wild-type wi ld-type mice m ice (15). (15). Determining the speciicity o an antibody is in part dependent on the type o the immunogens: synthetic peptides or purified proteins. Synthetic peptides  provi  pro vide de th thee ad adva vant ntag ag e o kn know owin ingg the amino acid sequence to which the antibody binds; however, these peptides do not necessarily structure or post-translational recapitulatemodificathe 3-D tions o the native protein (16). (16). As a result, antibodies generated against a synthetic  peptide may not work work well when a protein protein is in its native conormation with intact 3-D structure. Such antibodies may not be useul or IP or IHC experiments, but may bind the protein o interest afer it is ully denatured when running SDS WB. Te opposite could also be the case, especially i the immunogen was the purified protein,  where the antibody antibody works works well well or prote proteins ins in their native conormation, but not when denatured. Tus WB cannot be an absolute standardization or antibody binding in IHC or other assays where the antigen is in its native conormation. Te native versus denatured confirmation is urther ur ther complicated by methods used to fix tissue. Epitopes that are not exposed in the native proteins can be exposed in fixed tissue and vice versa, even though they may not be truly denatured. Tus, an antibody could recognize one epitope in resh tissue, but when applied to fixed tissue recognize another epitope (17,18). A representative example is an epitope on BCL-2 (41–54 amino acids) that is exposed when BCL-2 is present in the cytoplasm but is inaccessible in the nuclear compartment most likely due to interaction  with other proteins (19). (19). However, However, when BCL-2 is phosphorylated at sitesavailable, close to the epitope, the epitope becomes as seen when the protein is extracted extract ed rom cells or denatured by SDS (20). he issue o epitope speciicity is urther complicated by the choice o monoclonal versus polyclonal antibodies. Polyclonal antibodies represent a pool o antibodies against the immunogen and typically show a higher probability or detection in a range o different conditions, whereas a monoclonal antibody is more likely to work in only one set o conditions. Polyclonal antibodies might nevertheless contain superior ainity clones. Perhaps the best example o this is the FDA-approved FDA-approved Herceptest antibody rom Dako, which is the industry standard sta ndard or HER2 testing in breast cancer (21). Monoclonal antibodies are more pure, since they are produced rom single clones o used cells producing immune

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Figure 1. Examples of non-specific antibodies f or HoxA1 and phospho-4EBP1. (A) Cell lysates were denatured and separated by SDS PAGE, transferred to nitrocellulose and blotted with a mouse polyclonal antibody against HoxA1. A band of the expected molecular weight is seen in CaCo2 lysate (marked by the ar row). Note the numerous bands in all cell lines at unexpected molecular weights. (B) A representative example of IHC staining on FFPE breas t carcinoma tissue for HoxA1. Note the cytoplasmic staining for a nuclear transcription factor. (C) Western blot for phospho-4EBP1. Arrow denotes band at the expected molecular weight; again, all lysates show numerous bands at unexpected molecular weights. (D) A representative example of IHC staining on FFPE lung carcinoma tissue for p-4EBP1. Note the predominantly nuclear localization for a protein expected to localize to the cytoplasm and nucleus.

globulins. However, these clones may be grown in host animals where the ascites

lysates (denoted by the arrow). All o the lysates show several bands above and below

fluid containing secreted antibody is collected (16,18); the thereore, these antibody  preparatio  prepa rations ns may be conta contamin minated ated by antibodies other than the monoclonal antibody o interest. A study by Spicer et al. demonstrated that seven o 20 monoclonal antibody preparations (35%) they analyzed had staining patterns localized to the Golgi cisternae unrelated to the antigenic specificity o the antibody, and that five o these cross-reactive antibodies ailed to even stain the antigen o interest (22). Antibody validation efforts in our lab have shown similar lack o specificity, even in monoclonal antibodies. Figure 1 provides two examples o nonspecific antibodies tested in our lab. In Figure 1A, a mouse  polyclonal  poly clonal antibody against HoxA1 HoxA1 (B0 (B01P; 1P; Abnova, aipei City, aiwan) aiwan) was used to  probe  pro be lysates rom ten cell lines by WB. Te Te expected molecular weight is 37 kD and a band o this size was seen in the CaCo2

this expected molecular weight at a lower signal level, such that i this a ntibody antibody were  validated only against a cell line transected to overexpress HoxA1, HoxA1, these bands ba nds would have been missed. Tis level o noise as seen by WB raises concerns about nonspecificity. Figure 1B provides a representative example o the staining pattern seen with this HoxA1 antibody a ntibody on breast carcinoma tissue. Te predominately cytoplasmic staining or a homeobox transcription actor indicates that this antibody should not be used or IHC or IF methods. Figure 1C provides an example o a monoclonal antibody or phospho-4EBP1 (rabbit MAb, clone 236B4; Cell Signaling echnology, Beverley, MA, USA)  showing a band o the expected molecular weight (denoted by the arrow) as well as numerous additional bands at higher molecular weights. Figure 1D is a representative example o the nuclear staining observed when using this

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could represent represent the same sa me target at different  post-tra nslationa  post-translat ionall mod modiic iication ation stat status, us, breakdown products, or splice variants. However, such observations should raise concerns or using this antibody or urther experiments. Major et al. (2006) published their database AbMiner as a resource or inormation including the immunogen, im munogen,  vendor,, and antigen on over 600 commer vendor commer-

ciallythe that available group validated monoclonal via WB antibodies against

 pooledd cell  poole cell lysates lysates rom rom each each o the NCI-60

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Figure 2. Lack of reproducibility is seen with the VEGF VG-1 clone antibody.  (A) IHC on FFPE FF PE lung carcinoma tissue with VG-1 (1:50 dilution) demonstrates specific staining of expected localization. (B) Serial sections of our lung TMA stained with VG-1 (1:50) do not correlate with each other. (C,D) IHC staining of serial sections of the same patient tissue showing a high level of positivity in Run 1 (C) is negative for VEGF in Run 2 (D). Inset represents cytokeratin staining of the tumor for reference.

antibody or IHC on FFPE lung carcinoma

tissue when the expected localization or  p-4E BP1 is cytoplasmi  p-4EBP1 cy toplasmic. c. Non-reproducible antibodies

Poor correlations between antibody lots are cause or concern, as demonstrated by a recent study rom our lab on antibodies against the Met tyrosine kinase receptor (23). Strikingly, different lotsstained o the monoclonal 3D4two Met antibody on an array o 688 breast cancer cases showed opposite staining patterns—one nuclear and one membranous membranous and cytoplascy toplasmic—with a regression between the two lots having an R2 value o 0.038 (23 (23). ). Another example o non-reproducible antibodies  was demonstrat demonstrated ed by by Grimsey et al. (2008 (2008))  with antibodies a ntibodies supposedly targeting the cannabinoid CB1 receptor. Te authors tested multiple lots o antibodies rom  various commercial sources by both WB WB and IF against HEK cells transected  with HA-tagged H A-tagged CB1 receptor. Only two o the six antibodies tested showed specific membranous staining that co-localized  with detection detection using an anti-HA antibody antibody.. Additionally, WB analysis on HEK cell lysates demonstrated that the antibodies  with poor IF specificity specificit y detected proteins o incorrect molecular weight, no proteins at all, or proteins present in wild-type

HEK cells which do not even express the CB1 receptor (24). Some examples o non-reproducibility can be very subtle. Figure 2 shows two examples o what appears to be specific staining with expected localization. However, staining with the same conditions on a serial section s ection o the MA (representing the same patient tissues) shows extremely pooractor correlation. is aansecreted growth and as VEGF such, we expected cytoplasmic staining on patient tissues with the VG-1 clone (Figure 2A). Staining on a serial section o the same tissue microarray with the same lot o antibody was not reprodu reproducible, cible, with an  R2 value o 0.016 (Figure 2B).

Current practice in antibody validation  Antibody specificity  speci ficity 

 WB is wid widely ely use usedd to det deter ermi mine ne an antibody’s speciicity and is an appro priate first validat validation ion step i the t he antibody recognizes the denatured antigen. Te first indication that the antibody is specific or the selected target would be observing a single band at the known molecular weight or the target. Presence o multiple bands or bands not at the proper molecular weight

cell lines. An antibody was considered  validated i it produced a band (or bands) o the expected molecular weight(s) or the target protein (25). (25).  While  Whi le this type o antibody validation is a useul first step, it only guarantees that a given antibody will provide accurate results or WB analysis. I the goal is to use the antibody or IHC or IF, then the user must demonstrate that the antibody is also able to specifically recognize its target when used or those applications. Antibody pedia is also an a n online onli ne portal or sharing antibody validation data or WB, IHC, and IF. Only antibodies commercially available are submitted, and in addition a ddition to a validation score, the inclusion o original experimental data is required. (26). Antibody speciicity has also been evaluated by using blocking peptides especially or IHC (27). Tese peptides are the sequences used to generate the antibody and are incubated with the antibody in great excess. Te antibody with and without the blocking peptide is then used to stain tissue known to express the target o interest. I the antibody is specific, the addition o the blocking peptide will result in loss o staining on the tissue. An example using this technique was recently published or the validation o phosphophospho-specific specific(27) antibodies or ERα. Although this method demonstrates that the antibody is specific or the immunogen rom which it was generated, it does not  prove sele selectiv ctivity ity o the antib antibody ody sinc sincee off-target binding activity o the antibody  willl also  wil a lso be inhibited i nhibited by pre-adsorption pre-ad sorption  withh the blo  wit block ckin ingg pept peptide ide.. So whi le blocking peptides can prove that an antibody is bad, when nonspecific staining is seen in the presence o the peptide, they cannot prove that an antibody is good. Blocking peptides had been used with a number o the G protein–coupled protein– coupled receptor antibodies and have been ound not to be selective upon more stringent validation (13). Tus, we do not typically include blocking peptides as part o our antibody  validation  validat ion process. Te key to proving antibody specificity is ofen the correct use o controls. A negative control, including no primary

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Figure 3. The Rimm L ab Algorithm for antibody validation of IHC/QIF. Step 1 of antibody validation is using cell lines in vitro to test antibody spe cificity. Step 2 (below the dotted line) line) involves further validation of antibody antibody on tissue microarray (TMA) for expected tar get localization and reproducibility between assay runs and different antibody lots.

antibody in an IHC I HC or QIF experiment, is valuable but insuicient. A better negative control is a cell line or tissue that is known not to express the protein o interest. Knockout cells thus provide the best negative controls. Similarly, non-expressing cells, transected with the protein o interest, provide the best  positive controls. controls . Since these reagents reagent s are ofen beyond the reachthat o many labs, there are other approaches can be used to obtain comparable control results. Ofen

there are readily available cell lines that have been biologically proven not to express a specific speci fic protein o interest. For example, H1650 H1650 cells are PEN-null and a nd thereby make a good negative control or PEN antibodies. Similarly, overex pression  pres sion ca cann be va valuab luable le as a pos positive itive control.l. For example, contro ex ample, A431 cells overex overex- press wild-type EGFR and can be used as a  positive contro controll or anti-EGFR antibodies. An alternative alternat ive to knockout cell controls controls is siRNA or shRNA knockdown controls, as will be discussed with our Rimm Lab Algorithm o antibody validation. Finally, another type o quality control slide has been developed by Sompuram et al. that couples peptides to glass slides that mimic the epitope o the native antigen and are thereore specific controls or a given

monoclonal antibody (28). Te drawback to this type ty pe o control is that it only works or monoclonal antibodies, as the antibody must interact with a known epitope in order to generate the appropriate peptide controls.  Antibody reproducibi lity  An important criterion or validation and standardization standardizat ion is antibody reproducibility. reproducibility.

et al. compared HER2 IHC staining o 283 breast adenocarcinomas on tissue microarrays with the 4B5 rabbit monoclonal antibody to the previously established CB11 mouse monoclonal antibody. Additionally, both antibodies were also compared to each other and to the corresponding FISH scores or each case. Te authors ound no significant differences in sensitivity, speci-

Tis staining o thelots same over means time with different on antibody different days, as well as comparison o a new antibody to either a previously validated antibody or to a second independent means o measuring the target that would yield similar results (29). Tere are not many studies on reproducibility o antibodies, since this is ofen assumed a ssumed when using new lots o the same antibody or new aliquots o an antibody used previously by the same sa me lab. Our lab published one such example o this type o evaluation (23), and a similar  work to show assay reproducibi lity has been done or HER2 by Gustavson and colleagues (30). A second issue related to reproducibility is when a new reagent is introduced and there is a desire to compare the new antibody to previously validated standards. Tis sort o study is seen more commonly in the literature. For example, van der Vegt

ficity, or predictive values between the two antibodies, validating that 4B5 could be used to assess HER2 expression (31). A second example example by Zarani Za rani et al. a l. compared antibodies or ER and PR with IHC to biochemical determination using enzyme immunoassayy and ound significant links immunoassa between the two methods (32). A similar study by Sasano et al. validated the use o an aromatase monoclonal antibody by correlating the IHC score with biochemical activity as a s determined by product isolation assays (33). Sometimes this is done in an attempt to prove superiority superiority o a new reagent. In those cases, investigators must compare the reagents in the setting o intended use  with ou outco tcome me or res respon ponse se to ther therapy apy,, not the  previous  previo us assay, as the criterio criterionn standard. A good example o this is i s the work o Cheang et al. that showed the ER antibody SP-1 to be more prognostic and predictive than the current standard 1D5 (34), although this

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site-directed mutants. It also strived to demonstrate lot-to-lot consistency aided by combination o two or more pre-qualified individual lots to create each batch to minimize batch-to-batch variability. Company 7 describes their stringent  validation  validat ion protocol protoco l or al alll antibodies a ntibodies to include testing o each antibody by WB, IP, IHC, IF, flow cytometry, and ELISA. ELISA . It

It is estimated that there are over 180 antibody companies that produce over 350,000 antibodies or the research and clinical markets (www.antibodyresource.com/onlinecomp.html). So it is not surprising that there are commercially available antibodies against a huge collection o target proteins. When one buys a kit to isolate DNA, it is generally sae to assume it will not end up puriying  protein. However, However, the same is not true or antibodies. Just because the manuacturer claims an antibody is specific or protein Z does not necessarily mean that it will only bind protein Z and not a range o other proteins (29,36). In 2005, RamosVara noted that the inormation about antibodies targeting the same antigen  varied  var ied sig signi ni ica icantly ntly depe dependi nding ng on the manuacturer (16). In act, it is now clear

observed or Company 1’s phosphoAMPKβ1 polyclonal antibody. No inormation was readily available on its web site or datasheet describing any level o antibody validation criteria. Te datasheet itsel contained minimal inormation about the antibody including a brie background on the target with corresponding reerences, recommended applications and starting dilutions or WB, IF, and ELISA. However, no examples o successul use in any o these applications are provided and none o the reerences provided had utilized this antibody. Te datasheet also includes the animal host and immunogen source as a synthetic peptide, but does not include exact  peptide  pepti de sequence, sequence, although the peptide is available or purchase or use as a blocking  peptide. Te datasheet also cautions that this antibody “may” cross-react with correspondingly phosphorylated phosphorylated AMPK β2 . A moderate level o validation was observed or Companies 2–5. hese companies also did not provide any in-depth descriptions o antibody validation procedures. Datasheets all included background on the target, as well as inormation i normation on the

 verifies the specificity specificit reproducibility o antibodies by usingy and appropriate kinasespeciic activators or inhibitors when available; testing against a large panel o cell lines with known target expression;  phosphatase treatment or phospho-speciicity; comparison to isotype control;  verification in transected cells, cel ls, knockout cells, and siRNA-treated cells; utilizing blocking peptides to eliminate all signal;  veriying  veriyi ng correct subcellular localization or treatment-induced translocation; comparing new antibody lots to previous lots; providing optimal dilutions and buffers, as well as a s speciying both positive positive and negative control cell lines. For IHC, Company 7’s antibodies are tested on  paraffin-embedded cell pellets including cell pellets created afer the cell lines are subjected to treatments known to induce signaling changes or treated with siRNA to block expression o the target. issue is treated with phosphatase to additionally test phospho-specificity phospho-specificity on FFPE tissues.  Xenografs with cell lines o known target expression or treatments to modulate expression are parain-embedded and then stained. Te datasheets or these companies include everything seen with Companies 2–5 plus additional examples o the successul use o their antibody. For example, Company 6 includes represen-

that responsibility or not proo specificitythe is with the purchaser, theovendor. Different vendors provide different levels o validation, depending on their approach to the balance between making a profit and  providing high quality. For For this review, review, we sought to compare the level o validation and amount o inormation provided by seven dierent companies (reerred to herein simply as Companies 1–7) to the randomly selected molecule AMPKβ1. he companies are not revealed since only 7 o over 180 were chosen at random; ra ndom; urthermore, the level o validation at some companies varies based on the  product. When availa available, ble, we compared the validation protocol on the datasheet or phospho-specific antibodies, as these require another level o specificity and  validation  validat ion (37 (37,38). In general, we ound three levels o validation. he least validation was

immunogen. out o sequence our companies included theTree complete used and the ourth identiied the region surrounding the phosphorylation site as the sequence used. Tese companies also provided recommended applications  with starting star ting dilutions, and a nd all al l provided at least one example o the antibody successully identiying its target in one o the recommended applications. applications. WBs— W Bs— either with transected cells expressing the target or pre-incubation o the antibody  with block blocking ing peptide peptide—were —were the most commonly shown antibody validation examples. Te highest levels o validation were seen or Companies 6 and 7. Company 6 describes its validation procedures or  phospho-speci  phosphospeci fic antib antibodie odiess to includ includee  WB analysis in (i) multiple cell lines, (ii)  peptide  pept ide and phosp phospho-p ho-pepti eptide de comp compeetition experiments, and (iii) analysis o

tative data or all anded ll validated va lidated applicati applications ons  wit  with h rec recomme ommended antibo ant ibody dy conc concenentrations and includes low cytometry, immunocytochemistry on a positive control control cell line li ne (including (including incubation with either the phospho-peptide or the non–phospho peptide  pept ide demon demonstra stratin tingg sig signa nall abs absence ence only with the phospho-peptide), IHC o a positive control tissue, and a WB o a positive control cell line lysate also including detection afer pre-incubation  withh the phos  wit phosphopho- or non– non–phos phospho pho-specific peptides demonstrating a single band o the expected molecular weight  with signal si gnal absent only on ly afer a fer incubation  with the phospho-specific peptide. Antibodies purchased rom Company 1 (which provided the minimal amount o inormation and no examples o successul use o the product) would would require extensive ext ensive  validation by a researcher to demonstrate demonstrate that the results describe only the target

Figure 4. Stathmin expression is reduced by siRNA. (A) WB of BT-20 lysates 24 h post-mock; control scrambled siRNA or Stathmin siRNA transfection demonstrates specific reduction in Stathmin levels. GAPDH serves as a loading control. (B) BT-20 cells were fixed in 4% PFA 24 h post-transfe ction with scrambled control or Stathmin siRNA, and IF with Stathmin demonstrates a reduction in Stathmin expression.

conclusion was later disputed by Brock and colleagues (35).

 Vendor-based  Vendor -based validation valid ation of commercial antibodies

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o interest; the company provides little in the way o assurances that it will work. Companies 2–5 all provide more inormation on their respective antibodies and include at least one example o the product successully being used or at least one o their recommended applications with corresponding positive controls. While this is clearly preerable to Company 1’s

A

approach, antibodies still need to bethese validated by thewould researcher or target specificity in the application o interest and or lot-to-lot reproducibility. Company 6 describes the validation validat ion steps it uses to determine that its phospho-specific antibodies are both specific speci fic and reproducible. Furthermore, or each recommended application, an example o successul use is provided with corresponding inhibition by phospho-speciic blocking peptides. Company 7 provides what we consider the gold standard or antibody validation. Te extensive in-house testing described  provides  pro vides a high level o o confidence confidence that the antibodies it provides provides will work or all the applications that are recommended or the  particular  parti cular antib antibody ody.. It also pr provid ovides es a grea greater ter level o confidence that results obtained will be specific or the described target and will be reproducible among antibody lots. Even though Companies 6 and a nd 7 provide extensive validation, the researcher is still obliged to confirm that the product gives specific and reproducible reproducible results in the cell lines or tissues o interest in the lab. Indeed, even the best companies cannot control what happens afer the product leaves their door. Issues during shipping, shipping , inappropriate storage storage on or afer arrival in the laboratory, and antibody contamination contamination during usage are all  potential  pote ntial sour sources ces o o error error in antibod antibody-b y-based ased testing. Tus vigilant and comprehensive

B

C

Figure 5. ER- α antibody Clone 1D5 as an example of strong correlation between quantitative WB and IHC methods. (A) FFPE MCF7 cells stained with 1D5 (1:50 dilution) demonstrate expected nuclear localization for ER- α. (B) WB with 1D5 of a panel of breast cancer cell lines demonstrating varying expression of ER-a. Puro cells are MCF7 cells stably expressing an inducible ER- α. β-tubulin serves as a loading control. (C) ER- α  expression by WB was quantified using ImageJ and correlated to AQUA scores corresponding to the cell lysates demonstrating a strong correlation.

association, and thus we are not so bold as to call this a “recommendation.” Instead,  we provide what we eel is a compr compromise omise between rigor and lab economics that results in a level o evidence sufficient or data dissemination. Our algorithm or

siRNA were also ixed and decreased Stathmin expression was visualized  with IF I F (Figure 4B). Additionally, when known target activators and inhibitors are available, lysates rom treated cells can also be included when validating antibodies

antibodyor validation (Figure 3) is especially ocused the end use application applicatio n o IHC

specific orIdentification a particular state (e.g., phosphorylation). o a single band (or multiple bands i more than one isoorm o the protein is expected to interact with the t he

here are no uniorm or enorceable standards or antibody validation. Unlike drugs—whose sale is prohibited without FDA approval—there is no ederal agency governing what can be sold into the antibody-based antibody-based assay market. In the uture, we may see urther FDA clearance o antibodies or more rigorous labeling and regulation o reagents to be used in

or QIF on paraffin-embedded tissues, but could be equally valid va lid or modified or other antibody-based assays. Our irst line o evidence that an antibody is specific or the target o interest is done by WB. A variety o cell line l ine lysates (orr tissue homogenates) are selected, ideally (o  with known levels o target expressio expression n such such that both positive and negative cell lines are analyzed. Practically speaking, we ofen have no idea o target levels and select cell line series at random. Lines are ofen selected in such a way that we suspect some o the lines to be b e completely negative or the target protein (e.g., using fibroblast lines or epithelial targets). When true negative lines are not available, or when levels o target protein are highly dependent on growth conditions, we produce lysates o cell lines li nes where the target protein has been knocked down using RNAi or lysates rom cell lines typically null or the target that have been transected or overexpression constructs. In a recent example, we have used inducible overexpression (A.W.W., unpublished data). An example o using siRNA to aid in validating an antibody is shown in Figure 4 or Stathmin (rabbit monoclonal; Epitomics, Burlingame, CA, USA), a microtubule-destabilizing microtubule-destabilizing  protein. Lysates rom B B-20 -20 cells cel ls either e ither

antibody) o the correct molecular weight only in cell lines expressing the target encourages urther validation or use in IHC/QIF. IHC/Q IF. An antibody demonstrating no binding on WB may still be specific or its intended target when in its native conormation and an IP experiment can be the next step in determining the specificity o the antibody i the goal is or use  with IHC/QIF. Te second step in the validation o an antibody or IHC/QIF is to titer the antibody on a tissue microarray (MA) comprised o FFPE cell line pellets corresponding to the identical cell lines li nes used to first validate valid ate by WB in addition to patient tissue expected to express the target (or more inormation on the useulness o MAs, MA s, see Reerences 39 and 40). A good antibody will have the ollowing charactercharacteristics: (i) it will stain only the cell pellets expressing the target, (ii) the level o staining  willl decrease with increasing  wil i ncreasing dilutions di lutions o the antibody, and (iii) it will demonstrate an expression pattern that is consistent  with biological biological and mechanistic mechanistic data in the  published literature. Te target expression expression seen with these first two steps should be quantified—we use ImageJ or quantifi-

clinical testing. standard. However, We to date, there is no universal thereore  present our lab’s approach (the Rim Rimm m Lab Algorithm) to the validation issue. Our approach is not sanctioned or approved by any governing body or by any trade

mock-transected transected with scrambled siRNA orcontrol or siRNA specific or Stathmin were analyzed by  WB dem demons onstra tratin tingg los losss o Stat Stathm hmin in expression (Figure 4A). B-20 B-20 cells transtrans ected with either scrambled or Stathmin

cation o WBs and A󰁑UA or measuring the level o protein expression in each MA spot—and then the results should show a strong correlation to each other. An example o an antibody meeting these conditions is shown in Figure 5 or ER-α

controls should be done with each assay.

Te Rimm Lab  Algorithm  Al gorithm for antibody  validation for IHC/QIF IHC/QIF

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A

B

Figure 6. Examples of reproducible antibodies between assay repeats and antibody lots.  (A) Lot-to-lot reproducibility is shown using a MAP-tau monoclonal antibody on a breast index TMA. (B) MENA demonstr ating a high level has an R 2 value of 0.932 when stained on serial sections of our lung TMA demonstrating of reproducibility among assays run on different days using the same lot and dilution of antibody.

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(1D5 clone; Dako, Glostrup, Denmark). As expected rom  rom published literature, the staining on patient breast tissue is predominately nuclear (Figure 5A). Breast cell lines  with varying var ying ER expression ex pression levels (Figure 5B), 5B ), as well as a calibration curve o recom recom-binant ER protein, were analyzed by WB and the expression o ER protein (pg per μg o loaded lysate protein) against again st thecell expression level o was ER asplotted determined by IHC and quantified by A󰁑UA demonstrating a strong correlation ( R 2   value o 0.91 0.91)) between bet ween the two methods met hods (Figure 5C). he inal step in validation o an antibody or any application is to demonstrate that the antibody is reproducible between assay runs and between lots. We accomplish this by using a MA containing ~120 spots that is stained with each lot o the antibody. Te target expression is quantified and a regression between the t he two scores or each spot is perormed. A high correlation among multiple lots o

(13,18,36,41). hese authors point out that one o the best controls or antibody  validation is tissue tissue rom rom a knockout knockout animal lacking the antigen altogether. When available, this tissue tis sue would be an excellent addition to any control control MA, MA , but this is not available or every protein o interest and  would be a time-consuming time-consuming and expensive expensive investmentt or the sole purpose o validating investmen an antibody. Ideally, as suggested sugge sted in a recent  perspective by Alexander A lexander Kalyuzhny, Ka lyuzhny, we  will one day have have guidelines on the gener gener-ation and use o antibodies (42) where all companies comp anies will wil l be held to high standards or antibody validation and researchers can have greater confidence that their precious grant or clinical revenue dollars are not wasted on inaccurately labeled “vials o PBS” (36). For the meantime, however, the responsibility ultimately lies with the researcher or laboratory director to ensure that the antibodies used in their labs are validated or specificity and reproducibility.

the antibody our finalorequirement  validation. Anisexample this is shown shown or or

 Acknowledgments

an antibody to MAP-tau (United States Biological, Swampscott, MA, USA), where two different lots o the antibody were quantified with A󰁑UA demonstrating a high correlation (Figure 6A). Each time the antibody is used on a different MA, sections rom this control MA are stained in parallel as an additional staining control. control. Te control control MA M A slide should always a lways have a high correlation to previous experiments experi ments or a specific and reproducible antibody Te example in Figure 6B shows inter-assay reproducibility o MENA (mouse MAb, clone 21; BD ransduction Laboratories, Franklin Lakes, NJ, N J, USA). USA). Tese validation steps are relatively ast and inexpensive, but most importantly, they are comprehensive. Tis algorithm or antibody validation is somewhat similar to many other previously published approaches

Te authors thank Elaine Alarid o the University o Wisconsin or provision o cells lines with inducible expression o ER-α. Tis work was supported by the National Institutes o Health (NIH; grant nos. CA139431, CA 114277, CA 110511, and CA 106709, to D.L.R.); the Susan G. Komen Foundation (grant no. KG090562, to D.L.R.); D.L.R .); and the Department o Deense Breast Cancer Research Program [grant nos. 1W81XWH-06-1-0746 1W81XW H-06-1-0746 (to M. M ..B.), .B.), 1W81XWH-08-1-0404 (to J.B.), and 1W81XWH-08-1-0784 (to A.W.W.)]. Tis paper is subject to the NIH Public Access Policy.

Competing interests he authors declare no competing interests.

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