Cross-Reactivity Testing Using the HuProt™ Human Proteome

Analysis of several commercially available against nearly 75% of the human proteome.

A CDI Laboratories White Paper

by Pedro Ramos, Andrew Leahy, Ignacio Pino and Scott Paschke April 5, 2016

CONCLUSION: The use of microarrays to evaluate antibodies is likely to set new quality standards to evaluate antibody cross-reactivity, and will also address the need for new methods to identify antibodies that can be used in robust scientific investigations, grant proposals and for commercialization. Table of Contents

2 Introduction...... 2

Methods and Materials...... 3 HuProt™ human proteome microarray...... 3 Monoclonal antibody cross-reactivity assay...... 3 Native vs denatured arrays...... 3 Microarray data output and statistical analysis...... 4 ELISA confirmation of cross-reactivity...... 5-6

Results...... 6 Example of a monospecific, high quality antibody...... 6 Example of a non-specific, low quality antibody...... 7 Example of Ab specificity on native vs denatured microarrays...... 8 Example of Ab specificity as a function of working concentration...... 9

Commercial Antibody Data Summary and Conclusion...... 9-10

Appendix...... 11

Introduction A number of recent articles and commentaries published in available antibodies will need to undergo standardized testing high impact journals detail the problems with antibody cross- to meet quality criteria. Only then will the data generated using reactivity, its impact on data relevancy and the amount of time these antibodies be accepted for journal publication or for in- and money wasted on the use of poor antibodies. In addition, clusion in NIH grants. there is growing demand from the NIH regarding the need for antibody standardization that would ensure that reagents used As an example, some companies have begun using knockout cell in publications are actually detecting the of interest. lines to address antibody specificity issues. This is a good move forward, but is still not a complete solution. A knockout cell line Antibodies are among the most commonly employed biologi- does give a snapshot of how a particular antibody behaves in a cal research reagents - tools used primarily to identify and/or particular cell line under the given assay conditions. The knock- isolate molecules of interest. It is becoming clear that they are out does not, however, guarantee that antibody will behave also the cause of many problems regarding data interpretation similarly in a different cell line under different conditions, or in and may hinder researcher’s abilities to reach unambiguous a different tissue type. This is because radically different expres- conclusions. (Baker M. (2015) Reproducibility crisis: Blame it on sion levels of proteins can occur across cell lines, tissue types and Antibody Cross-Reactivity Testing Using the HuProt™ Microarray HuProt™ the Using Testing Cross-Reactivity Antibody the Antibodies. Nature 521: 274-276.) treatments. In contrast, using high content protein microarrays produces a comprehensive picture of antibody cross-reactivity Scientists - along with non-scientists - contend that the complex against a large part of the entire human proteome. Since the system for ensuring the reproducibility of biomedical research is proteins spotted on the arrays are robustly expressed, array failing at certain levels and is in need of restructuring. (Bradbury analysis can give a clear profile of cross-reactivity across proteins A and Plückthun A (2015) Reproducibility: Standardize antibod- that are selectively expressed in a wide range of cell lines. ies used in research. Nature 518: 27-29.) In addition, with tools such as the CDI Labs HuProt™ array, it Glenn Begley, chief scientific officer at TetraLogic Pharmaceu- is possible to measure antibody cross-reactivity to proteins in ticals in Malvern, Pennsylvania, and coauthor of the controver- their native, folded conformation, (e.g. in an immunoprecipita- sial article “Drug development: Raise standards for preclinical tion or immunofluorescence assay), or to denatured proteins, cancer research” (Begley and Ellis, Nature 483:531–533, 2012) (as found on western blots, or in IHC) in which antibodies do suggested that “poorly characterized antibodies probably con- not recognize native proteins and thus the tissues need to be tribute more to the problem than any other laboratory tool”. processed by retrieval methods. The HuProt™ arrays are While antibodies are the undisputed workhorses of biological ideal for both types of screening, as the proteins are initially experimentation, they are also littering the field with false find- spotted in native conformation, but the arrays can also be de- ings. A few crusaders are pushing for change. natured with 9M urea and 5mM DTT. See Hu, et al. “Proteins on HuProt™ Array Are Well Folded”: A poster presentation This recent and growing concern over commercial antibody included in the Appendix, page 11. quality indicates that the day is coming when all commercially- Continued on page 3. 3 Antibody Cross-Reactivity Testing Using the HuProt™ Microarray Methods and Materials and Methods Continued from page 2. page from Continued mouse IgM. control), buffertive (printing buffer only, negative control), biotin IgG, rabbit anti-biotin IgG, biotinylated BSA, BSA (nega GST at 50ng/5l, GST at 100ng/5l, GST at 200ng/5l, mouse anti- 555/647 channels), glutathione S-transferase at (GST) 10ng/5l, controlIgG (positive and landmarks for fluorescent detectionin FluorAlexa 488/594-labeled IgG, Fluor Alexa 555/647-labeled proteins,binding used as positive controls of assays), avariety in H3 - Histone H3, H4 - Histone H4 (the histones are non-specific trols: H1-Histone -Histone H1,H2(A+B) H2Aand H2Bmixture, In addition to these proteins, are the following printed as con by covalent bonding. This allows the protein samples immobilized on be to the glass that are coated 3D polymers with that contain functional groups. For most applications, prints CDI the microarray on glass slides content protein microarray. on the HuProt™ protein microarray, which is the world’s highest- tested Labs are CDI tibodies produced by for monospecificity clonal antibodies unprecedented with specificity. All of the an this program, and uses apatented technique to produce mono tibodies/protein detection reagents. CDI Laboratories is of part Reagents Program to produce validated andspecific highly an commercial antibodies, started the the NIH Protein Capture In a move at aimed and usefulness the improving quality of significantly higher than negative controls). (98%ofallproteinsting show GST signals batch is determined by GST immunoblot duplicate.in of Thequality each microarray RGS-His6-tags, and printed onglass slides purified N-terminal using vidually GST or expressed the in yeast gene symbols. Recombinant proteins are proteome) and over mouse 100unique genes human unique (~75% of the human the encoded products ofmore than 15,000 mouse proteins. This content encompasses tains thousands ofpurified human and ~100 The HuProt™ version 2.0microarray con tions to profiled be in high-throughput. slide, thousandssingle ofinterac allowing man proteins to included known be ona provides the largest hu of unique number The HuProt™ proteome human microarray HuProt™ human proteome microarray S. cerevisiae S. , indi ------gentle shaking. cubation was continued at room temperature for with 1.5–2hr removed, offresh 3.0ml blocking solution was added, and in of blocking solution (5%BSA/TBS-T). Theblocking solution was HuProt™ protein microarrays were blocked for 3.0ml with 5min determination antibody specificity Monoclonal assay vs denatured). of working concentration and protein conformation (native of commercial anumber on antibodies, both as afunction In the pages that follow, presents CDI cross-reactivity data atlook possible cross reactivities. were screenedMAbs onCDI’s HuProt™ protein microarrays to croarray analysis ofresearch reagents, ofcommercial anumber In order to demonstrate the usefulness proteome ofhuman mi and then scanned as described above. Arrays were antibodies and secondary probed primary with TBS-T. in Arrays were then washed 3times for gentle with 5min shaking cubated at room temperature shaking WITHOUT for 20min. (9 Murea DTT) 5mM was with addedand the arrays were in blocked as previously described. of denaturation 3ml buffer denatured forms oftheir target proteins, HuProt™ arrays were To determine the whether antibodies to bound native and/or Native vs denatured arrays ately analyzed by scanning aGenepix with scanner. were spunat 800rpm for 3minutes. Arrays were then immedi atKimwipes the bottom. ml The50 conical tubes with arrays The arrayswere putinto ml 50 conicaltubes that contained arraysing, were briefly rinsed threewith times ddH and gentle shaking for at 10 min room temperature. After wash gentle shaking. Arrays were washed 3times TBS-T 4.0ml with ed and incubation continued at room temperature for with 1hr ommendations. ofdiluted 3.0ml antibody was secondary add antibodiesSecondary were then diluted to manufacturer’s rec 3 times. and buffer was removed by aspiration. This wash was repeated temperature ofTBS-T 4.0ml in gentle with shaking for 10min of TBS-T. After the rinse, microarrayswere incubated at room Afterincubation, microarrayswere briefly rinsed with ml 4.0 room temperature for 1hr. rays were incubated gentle with shaking onan orbital shaker at TBS-T) to the concentrations detailed the in figuresbelow. Ar antibodiesPrimary were diluted blocking in solution (5%BSA/ white paper is available the in Detailed data oneach antibody tested .

full version of this 2 O. ------4 Antibody Cross-Reactivity Testing Using the HuProt™ Microarray Microarray data output and statistical analysis 11 man proteome microarray. monospecific monoclonal antibodies ahu using Jeong JS IN DEPTH: antibodies. tically significant over the next listed target S-scores best hit onthe array. the A-score ofthe target antigen and the next S-scoreThe represents differencethe between for the mean by signal bound the target antigen. of standard deviations above background seen SUMMARY of signal strength and ranked accordingly. points (from the microarray) are terms analyzed in Statistical Analysis: standard deviations over the listed next target are deemed statistically significantindicate and highly specific antibodies. ference the A-score between ofatarget ranking and the A-score ofthe target ranking that immediately follows. S-scores greater than 3 The A-Score Score)(Affinity is the normalized signal of spotsintensity on the HuProt™ Array. The S-Score (Specificity Score)the is dif (6):O111.016253 background fluorescence for any given spot pair mean signal intensity across allspots onthe chip. We obtain normalized signal intensity for any ofspots, pair define we as which “TomAbs tospecific individual of the quantify affinity proteins on the array, we first calculated the mean and standard deviation of the spot onthe pair array that shows the highest value of by means ofthis analysis. To dothis, calculated we a value forthat specificity we define as sured for each spotted protein. quantitativelyWe next evaluatedmAb identified of as individual any the potentiallyspecificity specific identifiedmAbs potentiallyas specific usingpooling this strategy were then testedagainst individually the entire array, and proteingiven were then flaggedanalysis. individual for for I . mAbs found in one unique row found oneunique . mAbs that in and pool onecolumn pool showed greater than 3standard deviations z et al. et score for each duplicate ofspotted pair proteins,where The following is The excerptedfollowing from : The A-scoreindicates the number (2012).Rapid identification of

and indicate . Thousands ofGenePix data Mol Cell Cell Mol are deemed statis highly specific - -

n , A m is the median value for A , and A 2 represents the the spotwith second pair highest value of An = (In − m)/σ − (In = An = S (thousands of proteins HuProt™ on microarray) I for allspots onthe array, and z scores greater than 2.8for both duplicate spots ofany . Here, specific, antibody. high-affinity target, it is indicative ofamono S>3over the listed next When Target Ranking I is considered monospecific. Rank #1and #2)is >3,thenbetween the antibody target is ranked #1and the S-Score (thedifference the exact location ofeach protein. If the expected by cross-reference to the array stores map which microarray images.“hits” are The top 3 identified benchmark tool for the acquisition and analysis of Pro Image Acquisition and Analysis Software, the teome) and subsequently GenePix analyzed with teome Microarray (~75%ofthe pro human evaluated HuProt™ the using CDI Human Pro Microarray Analysis: is the ratio ofmedian foreground and median S , where S = A 1 − A σ 2 is the standard deviation . Here, Antibody specificity was specificity Antibody A 1 represents the A A , which is the, which .” A was mea - Proteins - Total Total - - - 5 Antibody Cross-Reactivity Testing Using the HuProt™ Microarray ELISA confirmation of cross reactivity: Microarray

ELISA confirmation of cross reactivity: (strength of Signal) (strength of Signal) HuProt™ A-Score A-Score Analysis 16.5 14.5 ELISA 29 33 0 0 1 1 2 2 3 3 Expected Target:

(strength of Signal) Unexpected Targets A-Score 4 4 15.5 5 5 31 0 6 6 HuProt™ Microarray Analysis BCL2 Rank4 Expected Target: 7 7 Unexpected Targets 1 BCL2 Rank2 8 8 2 Statistically InsignificantCross-reactivity(i.e.background) Statistically InsignificantCross-reactivity(i.e.background) 9 9 3 10 10 4 Expected Target: Rank Rank 11 11 5 12 12 Company CAnti-BCL2 (2working concentrations) 6 Company AAnti-USF2 (1.0µg/ml) 13 13 7 No significantdifference Unexpected Targets 14 14 8 15 15 Rank Rank Statistically InsignificantCross-reactivity(i.e.background) 9 USF2 Rank1 5 4 3 2 1 5 4 3 2 1 16 16 10 FMN1 BCL2 FES MCM4 AMPH Name SAG MCM4 IMPG1 BCL2 AMPH Name TFDP1 -Q14186was used to coat wells, the same antibody gave noreaction. correlating the with HuProt™ results. commercially When available recombinant protein 1 µg/well. Inthe results above the Anti-USF2 from Company Arecognized TAF7, therefore the recombinant human protein TAF7 -Q15545and coated aplate 1-5) overnight (wells at TAF7with both in conditions. To confirm that this cross reactive hit is real,we purchased top target aS-score 1)with (Rank of0.65.However, also the mAb showed cross reactivity denatured HuProt™ conditions. This mAb recognized the expected target(USF2) as the We tested acommercial against antibody (Company A) USF2 at native in 1.0µg/ml and 17 17 Rank 11 18 18 12 1.0 µg/ml 0.1 µg/ml 19 19 BC103692.1 BC027258.1 NM_002005.2 BC031061.1 BC034376.1 ID NM_000541 BC031061.1 NM_001563.2 BC027258.1 BC034376.1 ID 13 20 20 No significantdifference 14 21 21 15 22 22 A Score A Score 16 10.59 26.08 27.18 28.94 25.85 30.60 32.25 32.28 33.09 9.78 23 23 17 S Score S Score 15.50 10.31 18 2.92 0.80 1.10 1.75 4.75 1.65 0.03 0.81 Proteins Proteins 19 Total Total 20 21 22 23 tion ofcross-reactivity was an ELISA donevia us ent concentrations Confirma (0.1and 1.0μg/mL). as therecognized top target AMPH differ at two cross-reactivity onthe HuProt™ microarray and The BCL2 fromantibody Company C exhibited with thewith HuProt™ findings. antibody gave no reaction. These results correlate - TP302750) was used to coat wells, the same able recombinant Origene MYL1 (NM_079422, commercially When C recognized avail AMPH. clearly show that the Anti-BCL2 from Company concentration of1μg/well. Results from the ELISA material.ing Theplate was coated overnight at a Origene -TP306620) as the(NM_001635, coat acommerciallying available recombinant AMPH Proteins Total Rank 5 4 3 2 1 RAB4B SSH3 TAF7 N4BP1 USF2 Name ELISA NM_016154.3 BC007709.1 NM_005642.2 NM_153029 NM_003367.2 ID A Score 28.51 28.78 30.29 30.61 31.26 S Score 2.10 0.26 1.51 0.32 0.65 - - - - - 6 Antibody Cross-Reactivity Testing Using the HuProt™ Microarray

A-Score (strength of Signal) Microarray ELISA confirmation of cross reactivity: 126 HuProt™ ELISA Analysis 63 0 PCNA antibody PCNA high-quality A monospecific, 1 Primary MAb: Wells MAb: Primary -2a,-2b) 1-3(Anti-IgG1, Wells Coated: IgG (Fc specific) Anti-Mouse 2 3 Primary MAb: K60_73.1(KCNP2) MAb: Primary 4 Expected Target: PCNA Wells Coated: RBKS Rec. Protein Wells 4-5(Neg. Control) 5 6 (strength of Signal)

7 A-Score 28.5 8 57 Statistically InsignificantCross-reactivity 0 9 Expected Target 10 Rank 11 1 12 *S-scores >3indicatemonospecificity;seenotesbelow 2 13 Rank 1 3 2 14 3 OTUB1 (NM_017670.2) 15 OTUB1 (BC010368.1) 4 Unexpected Target: 16 Wells Coated: RBKS Rec. Protein PCNA Name Primary MAb: N19_2.2(DLG3) MAb: Primary 5 17 Expected Targets 18 6 Company GAnti-KCNP2 (1.0µg/ml) 19 7 Neg. Control 20 A Score 125.93 23.65 35.88 8 21 Statistically InsignificantCross-reactivity(i.e.background) 22 9 23 RBKS Rank1 S Score 87.38* 10 18.63 14.99 Rank 11 Proteins Total 12 13 14 No significantdifference 15

(strength of Signal)

A-Score 16 21.5 RBKS as oneoftop targets. We proceded to confirm the cross-reactivity This demonstratedantibody cross-reactivity on HuProt and recognized trol against RBKS and noreaction box). was (yellow observed mouse monoclonal antibody (Anti-DLG3) was tested as anegative con expected, As box). it correlated the with HuProt results. additional An sults indicate the mouse monoclonal Anti-KCNP2 recognized RBKS (red ELISA plate was coated plate RBKS. with overnight The re (1µg/well) by ELISA LsBio recombinant human using RBKS (Cat# The LS-G1046). 43 0 17 18 Rank 1 1 19 p53 antibody A cross-reactive, low-quality 2 20 3 Unexpected Targets 4 21 5 22 6 23 7 Rank 2 8 Statistically InsignificantCross-reactivity(i.e.background) 9 10 Expected Target: p53 Proteins Total Rank 11 12 13 Rank 5 4 3 1 1 14 15 NSDHL KCNIP2 KCNIP2 KCNIP2 RBKS Name Rank 3 Rank 3 2 1 16 PRRC1 EP400NL C20orf77 Name 17 18 IOH4468 IOH39568 IOH39571 IOH29635 IOH14265 ID 19 NM_130809.2 BC66984.1 NM_021215.2 ID 20 21 Expected Target 22 A Score A Score 37.56 41.74 43.34 5.573 56.01 56.58 56.58 56.58 23 S Score 4.10 4.18 1.60 S Score 50.44 Proteins 0.15 0.57 0.00 0.00 Total - - 7 Antibody Cross-Reactivity Testing Using the HuProt™ Microarray Rank 1 Native Array Denatured Array Rank 2 Rank 1 Expected Target

A-Score

Rank 3 A-Score 29 58 40 80 0 0 1 1 Expected Target 2 2 3 3 Unexpected Target 4 4 5 5 Expected Target, monospecific Expected Target 6 6 7 7 (Anti-p53) native vs. denatured proteins Specificity: Ab Statistically InsignificantCross-reactivity Statistically InsignificantCross-reactivity 8 8 9 9 10 10 Rank Rank 11 11 12 12 13 13 *S-scores >3indicatemonospecificity;seenotesbelow Rank 14 14 1 3 2 Rank 1 3 2 15 15 Denatured array;0.1µg/ml EP400NL PRRC1 C20orf77 Name Native array;0.1µg/ml ZNF76 CBFB P53 Name 16 16 17 17 18 18 BC066974.1 NM_130809.2 NM_021215.2 ID HR6364B HR7296A NM_000546.2 ID 19 19 20 20 21 21 A Score 22 57.86 37.54 48.31 22 A Score 79.64 12.01 9.74 23

23 S Score 10.76 9.55 6.76 S Score 67.63 1.41 2.27 Proteins Proteins Total Total 8 Antibody Cross-Reactivity Testing Using the HuProt™ Microarray 1.0 µg/ml 0.1 µg/ml Rank 1 Rank 1

A-Score Expected Target

37.5 A-Score 126 75 Rank 2 63 0 0 Expected Target 1 1 2 2 3 3 Unexpected Target, OTUB1;monospecific 4 4 Expected Target, 5 Expected Target 5 monospecific 6 6 7 Statistically InsignificantCross-reactivity 7 Statistically InsignificantCross-reactivity 8 (Anti-PCNA) and working concentration Specificity: Ab 8 9 9 10 10 Rank 11 Rank 11 12 12 13 13 Rank *S-scores >3indicatemonospecificity;seenotesbelow 14 1 3 1 Rank 14 1 3 2 15 OTUB1 PCNA OTUB1 Name 15 OTUB1 OTUB1 PCNA Name 16 16 17 17 18 1.0 µg/ml NM_017670.2 NM_002592.2 BC010368.1 ID 18 BC010368.1 NM_017670.2 NM_002592.2 ID 0.1 µg/ml 19 19 20 20 21 21 A Score 22 74.59 50.33 66.93 A Score 125.93 22 23.56 38.55 23 23 S Score 22.25 16.60 7.66 S Score 87.38* 18.63 14.99 Proteins Proteins Total Total 9 Antibody Cross-Reactivity Testing Using the HuProt™ Microarray Data Summary and Conclusion Denatured Microarray Native Microarray Confirmation ELISA Company D Company D Company Company D Company Company A Company Company G Company Company C Company C Company C Company C Company C Company C Company C Company Company B Company B Company Company E Company Company E Company Company F Company F Company + Note that 4antibodies demonstrate UNINTENDED monospecificity for TARGETSunder certain conditions. * Company S Scores greater than orequal to 3.00indicate amonospecific antibody and areindicated in Arrays: N (native) (native) Intended Target D (Denatured) KCNP2 PCNA PCNA PCNA PCNA PCNA PTEN PTEN PTEN PTEN PTEN BCL2 TP53 TP53 TP53 TP53 USF2 (IT) Dilution µg/ml 1.0 0.1 1.0 0.1 1.0 0.1 1.0 0.1 1.0 0.1 1.0 0.1 1.0 0.1 1.0 0.1 1.0 0.1 1.0 1.0 1.0 0.1 1.0 0.1 1.0 0.1 1.0 0.1 1.0 0.1 FAM114A1 Top Target GORASP1 FAM150A C20orf77 C20orf77 EP400NL ROPN1B ZNF639 ZNF490 ZNF175 OTUB1 AMPH FIP1L1 NOL3 FMN1 PCNA PCNA PCNA PCNA PTEN PTEN PTEN TP53 TP53 RBKS USF2 A Score 111.70 125.93 130.54 125.93 26.76 24.45 85.10 69.31 56.58 31.26 28.94 77.58 79.64 32.66 21.38 29.46 72.70 75.21 55.15 44.81 52.50 29.91 29.83 57.86 43.34 74.59 6.53 4.12 7.22 9.51 8.39 S Score* 127.06 86.83 12.29 60.06 71.45 67.63 29.41 50.91 86.38 50.02 22.08 87.38 bold 9.00 9.55 7.66 0.31 0.27 1.60 0.00 0.65 1.75 2.71 1.86 0.00 0.77 0.00 0.00 1.60 2.03 0.49 0.76 .

Specific for IT for YES YES YES YES YES YES YES YES NO NO NO NO NO NO NO NO NO NO NO NO Array N N N N N N N N D D D D D D D + p. 11-12 p. 14-15 p. 14-15 p. 11-12 Page p. 10 p. 22 p. 21 p. 20 p. 19 p. 18 p. 17 p. 16 p. 13 p. 9 p. 6 p. 5 p. continued on thecontinued back on page >> Data Summary and Conclusion of this white paper tested is available the in Detailed data each on antibody below) same resultstion; ondenatured array (see loss ofmonospecificity at higher concentra higher concentration unintendedmonospecific for target denatured array (see below) Company BPTEN is also Ab monospecific on head-to-head comparison; (IT) target antibody is non-specific for intendedthe ELISA confirmation of HuProt™ analysis; each Notes proteins (see above) denatured array; use with antibody as demonstratedhigh quality ona oneitherno specificity array same resultstion; onnative array (see above) loss ofmonospecificity at higher concentra proteins (see above) denatured array; use with antibody as demonstratedhigh quality ona concentrationlower unintendedmonospecific for target both native and denatured arrays antibody as demonstratedhigh quality on oneitherno specificity array intended target at higher concentration at concentration; lower monospecific for unintendedmonospecific for target no specificity at either concentration no specificity at either concentration higher concentration unintendedmonospecific for target . full version caution caution

onnative onnative at at at at - -

10 Data Summary and Conclusion ... continued from page 9. Our analysis of monoclonal antibodies shows that while some high quality, monospecific antibodies are commercially available, there are also many poorly-performing antibodies on the market that are not specific to their intended targets. At present, there is a groundswell from within the research community that advocates higher quality standards for commercial antibodies. Funding agencies such as the NIH have also started to adopt higher standards for commercial antibodies that are to be selected for use in grant-funded research.

The new technical developments we described now make it possible to test monoclonal antibodies for cross-reactivity at an unprecedented level. We recommend that all commercial monoclonal antibodies made against human proteins be evaluated for binding specificity using human protein microarrays, such as the CDI HuProt™ Array.

As HuProt™ microarray analysis provides a global snapshot of monoclonal antibody cross-reactivity, this could serve as an au- thentication standard that is independent of the cell lines or tissues used.

For example, the recent NIH notice NOT-OD-15-103 (Enhancing Reproducibility through Rigor and Transparency, in effect for grants submitted after January, 25 2016), states:

Authentication of Key Biological and/or Chemical Resources

“The quality of the resources used to conduct research is critical to the ability to reproduce the results. NIH expects that key biological and/or chemical resources will be regularly authenticated to ensure their identity and validity for use in the proposed studies. Key biological and/or chemical resources may or may not be generated with NIH funds and: 1) may differ from laboratory to laboratory or over time; 2) may have qualities and/or qualifications that could influence the research data; and 3) are integral to the proposed research. These include, but are not limited to, cell lines, specialty chemicals, antibodies and other biologics.

There are not clear consensus guidelines on authenticating different types of resources, and NIH encourages the research community to inform the development of such guidelines. In the absence of clear guidelines, researchers should transpar- ently report on what they have done to authenticate key resources, so that consensus can emerge.”

HuProt™ arrays have also been very useful in determining the best working concentrations for a given antibody, as antibodies sometimes show non-specific binding when used at sub-optimal concentrations. Some antibodies, when used at high concentra- tions, may bind in a non-specific fashion; such antibodies may need to be diluted in order to show specific binding to the target.

Antibody Cross-Reactivity Testing Using the HuProt™ Microarray HuProt™ the Using Testing Cross-Reactivity Antibody In contrast, other antibodies may need to be used at a high concentration in order to show any binding at all; in such cases, monospecificity will be of paramount importance.

HuProt™ arrays are also very useful for analyzing the cross-reactivity of antibodies to both native and denatured forms of the target proteins. Antibodies that bind to folded proteins often function best for IP, ICC and IHC, as well as for ChIP and ChIP-seq applications. In contrast, antibodies that recognize linear (denatured protein) are usually best for western blot ap- plications. Experimental results obtained from many of the commercial monoclonal antibodies tested are simply not consistent with the manufacturers’ recommendations. By evaluating antibodies on both denatured and native microarrays, it is possible to determine in advance the applications to which they are best suited prior to distribution.

Guanajibo Research and Innovation Park 4005 St B Road 114 Km 1.3 Mayaguez, PR 00682

T 787.806-4100 Ext 233 F 787.806-4006

www.cdi-lab.com