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ReviewACBDFigure 2. Lack of reproducibility is seen with the VEGF VG-1 clone antibody. (A) IHC on FFPE 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) IHCstaining of serial sections of the same patient tissue showing a high level of positivity in Run 1 (C) isnegative for VEGF in Run 2 (D). Inset represents cytokeratin staining of the tumor for reference.antibody for IHC on FFPE lung carcinomatissue when the expected localization forp-4EBP1 is cytoplasmic.Non-reproducible antibodiesPoor correlations between antibody lotsare cause for concern, as demonstrated bya recent study from our lab on antibodiesagainst the Met tyrosine kinase receptor(23). Strikingly, two different lots of themonoclonal 3D4 Met antibody stainedon an array of 688 breast cancer casesshowed opposite staining patterns—onenuclear and one membranous and cytoplasmic—with a regression between the two lotshaving an R 2 value of 0.038 (23). Anotherexample of non-reproducible antibodieswas demonstrated by Grimsey et al. (2008)with antibodies supposedly targeting thecannabinoid CB1 receptor. The authorstested multiple lots of antibodies fromvarious commercial sources by both WBand IF against HEK cells transfectedwith HA-tagged CB1 receptor. Only twoof the six antibodies tested showed specificmembranous staining that co-localizedwith detection using an anti-HA antibody.Additionally, WB analysis on HEK celllysates demonstrated that the antibodieswith poor IF specificity detected proteinsof incorrect molecular weight, no proteinsat all, or proteins present in wild-typeVol. 48 No. 3 2010HEK cells which do not even express theCB1 receptor (24).Some examples of non-reproducibilitycan be very subtle. Figure 2 shows twoexamples of what appears to be specificstaining with expected localization.However, staining with the same conditions on a serial section of the TMA (representing the same patient tissues) showsan extremely poor correlation. VEGF isa secreted growth factor and as such, weexpected cytoplasmic staining on patienttissues with the VG-1 clone (Figure 2A).Staining on a serial section of the sametissue microarray with the same lot ofantibody was not reproducible, with anR 2 value of 0.016 (Figure 2B).Current practice inantibody validationAntibody specificityWB is widely used to determine anantibody’s specificity and is an appropriate first validation step if the antibodyrecognizes the denatured antigen. The firstindication that the antibody is specific forthe selected target would be observing asingle band at the known molecular weightfor the target. Presence of multiple bands orbands not at the proper molecular weight200could represent the same target at differentpost-translational modification status,breakdown products, or splice variants.However, such observations should raiseconcerns for using this antibody for furtherexperiments. Major et al. (2006) publishedtheir database AbMiner as a resource forinformation including the immunogen,vendor, and antigen on over 600 commercially available monoclonal antibodiesthat the group validated via WB againstpooled cell lysates from each of the NCI-60cell lines. An antibody was consideredvalidated if it produced a band (or bands)of the expected molecular weight(s) for thetarget protein (25).While this type of antibody validationis a useful first step, it only guarantees that agiven antibody will provide accurate resultsfor WB analysis. If the goal is to use theantibody for IHC or IF, then the user mustdemonstrate that the antibody is also ableto specifically recognize its target whenused for those applications. Antibodypedia is also an online portal for sharingantibody validation data for WB, IHC,and IF. Only antibodies commerciallyavailable are submitted, and in addition toa validation score, the inclusion of originalexperimental data is required. (26).Antibody specificity has also beenevaluated by using blocking peptidesespecially for IHC (27). These peptidesare the sequences used to generate theantibody and are incubated with theantibody in great excess. The antibody withand without the blocking peptide is thenused to stain tissue known to express thetarget of interest. If the antibody is specific,the addition of the blocking peptide willresult in loss of staining on the tissue.An example using this technique wasrecently published (27) for the validationof phospho-specific antibodies for ERα.Although this method demonstrates thatthe antibody is specific for the immunogenfrom which it was generated, it does notprove selectivity of the antibody sinceoff-target binding activity of the antibodywill also be inhibited by pre-adsorptionwith the blocking peptide. So whileblocking peptides can prove that anantibody is bad, when nonspecific stainingis seen in the presence of the peptide, theycannot prove that an antibody is good.Blocking peptides had been used with anumber of the G protein–coupled receptorantibodies and have been found not to beselective upon more stringent validation(13). Thus, we do not typically includeblocking peptides as part of our antibodyvalidation process.The key to proving antibody specificity is often the correct use of controls.A negative control, including no primarywww.BioTechniques.com

ReviewFigure 3. The Rimm Lab Algorithm for antibody validation of IHC/QIF. Step 1 of antibody validation is using cell lines in vitro to test antibody specificity. Step 2 (below the dotted line) involves further validation of antibody on tissue microarray (TMA) for expected target localization and reproducibility between assay runs and different antibody lots.antibody in an IHC or QIF experiment,is valuable but insufficient. A betternegative control is a cell line or tissuethat is known not to express the proteinof interest. Knockout cells thus providethe best negative controls. Similarly,non-expressing cells, transfected withthe protein of interest, provide the bestpositive controls. Since these reagents areoften beyond the reach of many labs, thereare other approaches that can be used toobtain comparable control results. Oftenthere are readily available cell lines thathave been biologically proven not toexpress a specific protein of interest. Forexample, H1650 cells are PTEN-null andthereby make a good negative control forPTEN antibodies. Similarly, overexpression can be valuable as a positivecontrol. For example, A431 cells overexpress wild-type EGFR and can be used as apositive control for anti-EGFR antibodies.An alternative to knockout cell controls issiRNA or shRNA knockdown controls,as will be discussed with our Rimm LabAlgorithm of antibody validation.Finally, another type of quality controlslide has been developed by Sompuram etal. that couples peptides to glass slides thatmimic the epitope of the native antigen andare therefore specific controls for a givenVol. 48 No. 3 2010monoclonal antibody (28). The drawbackto this type of control is that it only works formonoclonal antibodies, as the antibody mustinteract with a known epitope in order togenerate the appropriate peptide controls.Antibody reproducibilityAn important criterion for validation andstandardization is antibody reproducibility.This means staining of the same antibodyover time with different lots on differentdays, as well as comparison of a newantibody to either a previously validatedantibody or to a second independent meansof measuring the target that would yieldsimilar results (29). There are not manystudies on reproducibility of antibodies,since this is often assumed when using newlots of the same antibody or new aliquotsof an antibody used previously by the samelab. Our lab published one such example ofthis type of evaluation (23), and a similarwork to show assay reproducibility hasbeen done for HER2 by Gustavson andcolleagues (30).A second issue related to reproducibility is when a new reagent is introducedand there is a desire to compare the newantibody to previously validated standards.This sort of study is seen more commonlyin the literature. For example, van der Vegt202et al. compared HER2 IHC staining of283 breast adenocarcinomas on tissuemicroarrays with the 4B5 rabbit monoclonalantibody to the previously established CB11mouse monoclonal antibody. Additionally,both antibodies were also compared toeach other and to the corresponding FISHscores for each case. The authors found nosignificant differences in sensitivity, specificity, or predictive values between the twoantibodies, validating that 4B5 could beused to assess HER2 expression (31). Asecond example by Zafrani et al. comparedantibodies for ER and PR with IHC tobiochemical determination using enzymeimmunoassay and found significant linksbetween the two methods (32). A similarstudy by Sasano et al. validated the useof an aromatase monoclonal antibody bycorrelating the IHC score with biochemicalactivity as determined by product isolationassays (33). Sometimes this is done in anattempt to prove superiority of a new reagent.In those cases, investigators must comparethe reagents in the setting of intended usewith outcome or response to therapy, not theprevious assay, as the criterion standard. Agood example of this is the work of Cheanget al. that showed the ER antibody SP-1 tobe more prognostic and predictive than thecurrent standard 1D5 (34), although thiswww.BioTechniques.com

ReviewABFigure 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 inStathmin levels. GAPDH serves as a loading control. (B) BT-20 cells were fixed in 4% PFA 24 hpost-transfection with scrambled control or Stathmin siRNA, and IF with Stathmin demonstratesa reduction in Stathmin expression.conclusion was later disputed by Brock andcolleagues (35).Vendor-based validationof commercial antibodiesIt is estimated that there are over 180antibody companies that produce over350,000 antibodies for the researchand clinical markets (www.antibodyresource.com/onlinecomp.html). So it isnot surprising that there are commercially available antibodies against a hugecollection of target proteins. When onebuys a kit to isolate DNA, it is generallysafe to assume it will not end up purifyingprotein. However, the same is not true forantibodies. Just because the manufacturerclaims an antibody is specific for proteinZ does not necessarily mean that it willonly bind protein Z and not a range ofother proteins (29,36). In 2005, RamosVara noted that the information aboutantibodies targeting the same antigenvaried significantly depending on themanufacturer (16). In fact, it is now clearthat the responsibility for proof of specificity is with the purchaser, not the vendor.Different vendors provide different levelsof validation, depending on their approachto the balance between making a profit andproviding high quality. For this review, wesought to compare the level of validationand amount of information provided byseven different companies (referred toherein simply as Companies 1–7) to therandomly selected molecule AMPKβ1.The companies are not revealed sinceonly 7 of over 180 were chosen at random;furthermore, the level of validation atsome companies varies based on theproduct. When available, we comparedthe validation protocol on the datasheetfor phospho-specific antibodies, as theserequire another level of specificity andvalidation (37,38).In general, we found three levelsof validation. The least validation wasVol. 48 No. 3 2010observed for Company 1’s phosphoAMPKβ1 polyclonal antibody. No information was readily available on its website or datasheet describing any level ofantibody validation criteria. The datasheetitself contained minimal information aboutthe antibody including a brief backgroundon the target with corresponding references, recommended applications andstarting dilutions for WB, IF, and ELISA.However, no examples of successful use inany of these applications are provided andnone of the references provided had utilizedthis antibody. The datasheet also includesthe animal host and immunogen source as asynthetic peptide, but does not include exactpeptide sequence, although the peptide isavailable for purchase for use as a blockingpeptide. The datasheet also cautions thatthis antibody “may” cross-react with correspondingly phosphorylated AMPK β2 .A moderate level of validation wasobserved for Companies 2–5. Thesecompanies also did not provide any in-depthdescriptions of antibody validation procedures. Datasheets all included backgroundon the target, as well as information on theimmunogen. Three out of four companiesincluded the complete sequence usedand the fourth identified the regionsurrounding the phosphorylation siteas the sequence used. These companiesalso provided recommended applicationswith starting dilutions, and all providedat least one example of the antibodysuccessfully identifying its target in oneof the recommended applications. WBs—either with transfected cells expressing thetarget or pre-incubation of the antibodywith blocking peptide—were the mostcommonly shown antibody validationexamples.The highest levels of validation wereseen for Companies 6 and 7. Company6 describes its validation procedures forphospho-specific antibodies to includeWB analysis in (i) multiple cell lines, (ii)peptide and phospho-peptide competition experiments, and (iii) analysis of204site-directed mutants. It also strived todemonstrate lot-to-lot consistency aidedby combination of two or more pre-qualified individual lots to create each batchto minimize batch-to-batch variability.Company 7 describes their stringentvalidation protocol for all antibodies toinclude testing of each antibody by WB,IP, IHC, IF, flow cytometry, and ELISA. Itverifies the specificity and reproducibilityof antibodies by using appropriate kinasespecific activators or inhibitors whenavailable; testing against a large panel ofcell lines with known target expression;phosphatase treatment for phospho-specificity; comparison to isotype control;verification in transfected cells, knockoutcells, and siRNA-treated cells; utilizingblocking peptides to eliminate all signal;verifying correct subcellular localizationor treatment-induced translocation;comparing new antibody lots to previouslots; providing optimal dilutions andbuffers, as well as specifying both positiveand negative control cell lines. For IHC,Company 7’s antibodies are tested onparaffin-embedded cell pellets includingcell pellets created after the cell lines aresubjected to treatments known to inducesignaling changes or treated with siRNAto block expression of the target. Tissue istreated with phosphatase to additionallytest phospho-specificity on FFPE tissues.Xenografts with cell lines of known targetexpression or treatments to modulateexpression are paraffin-embedded andthen stained.The datasheets for these companiesinclude everything seen with Companies2–5 plus additional examples of thesuccessful use of their antibody. Forexample, Company 6 includes representative data for all validated applicationswith recommended antibody concentrations and includes flow cytometry,immunocytochemistry on a positive controlcell line (including incubation with eitherthe phospho-peptide or the non–phosphopeptide demonstrating signal absenceonly with the phospho-peptide), IHCof a positive control tissue, and a WBof a positive control cell line lysate alsoincluding detection after pre-incubationwith the phospho- or non–phosphospecific peptides demonstrating a singleband of the expected molecular weightwith signal absent only after incubationwith the phospho-specific peptide.Antibodies purchased from Company1 (which provided the minimal amount ofinformation and no examples of successfuluse of the product) would require extensivevalidation by a researcher to demonstratethat the results describe only the targetwww.BioTechniques.com

Reviewof interest; the company provides little inthe way of assurances that it will work.Companies 2–5 all provide more information on their respective antibodies andinclude at least one example of the productsuccessfully being used for at least one oftheir recommended applications withcorresponding positive controls. Whilethis is clearly preferable to Company 1’sapproach, these antibodies would stillneed to be validated by the researcherfor target specificity in the application ofinterest and for lot-to-lot reproducibility.Company 6 describes the validation steps ituses to determine that its phospho-specificantibodies are both specific and reproducible.Furthermore, for each recommendedapplication, an example of successful useis provided with corresponding inhibitionby phospho-specific blocking peptides.Company 7 provides what we considerthe gold standard for antibody validation.The extensive in-house testing describedprovides a high level of confidence that theantibodies it provides will work for all theapplications that are recommended for theparticular antibody. It also provides a greaterlevel of confidence that results obtained willbe specific for the described target and willbe reproducible among antibody lots.Even though Companies 6 and 7 provideextensive validation, the researcher is stillobliged to confirm that the product givesspecific and reproducible results in the celllines or tissues of interest in the lab. Indeed,even the best companies cannot control whathappens after the product leaves their door.Issues during shipping, inappropriate storageon or after arrival in the laboratory, andantibody contamination during usage are allpotential sources of error in antibody-basedtesting. Thus vigilant and comprehensivecontrols should be done with each assay.The Rimm LabAlgorithm for antibodyvalidation for IHC/QIFThere are no uniform or enforceablestandards for antibody validation. Unlikedrugs—whose sale is prohibited withoutFDA approval—there is no federalagency governing what can be sold intothe antibody-based assay market. In thefuture, we may see further FDA clearanceof antibodies or more rigorous labelingand regulation of reagents to be used inclinical testing. However, to date, thereis no universal standard. We thereforepresent our lab’s approach (the Rimm LabAlgorithm) to the validation issue. Ourapproach is not sanctioned or approvedby any governing body or by any tradeVol. 48 No. 3 2010ACBFigure 5. ER-α antibody Clone 1D5 as an example of strong correlation between quantitative WB andIHC methods. (A) FFPE MCF7 cells stained with 1D5 (1:50 dilution) demonstrate expected nuclearlocalization for ER-α. (B) WB with 1D5 of a panel of breast cancer cell lines demonstrating varyingexpression of ER-a. Puro cells are MCF7 cells stably expressing an inducible ER-α. β-tubulin servesas a loading control. (C) ER-α expression by WB was quantified using ImageJ and correlated toAQUA scores corresponding to the cell lysates demonstrating a strong correlation.association, and thus we are not so bold asto call this a “recommendation.” Instead,we provide what we feel is a compromisebetween rigor and lab economics thatresults in a level of evidence sufficient fordata dissemination. Our algorithm forantibody validation (Figure 3) is especiallyfocused for the end u

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 (E