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The Bead Array StrategyTraditional biochemical methods often detect orquantitate proteins individually. Unfortunately,individual analysis prevents researchers fromelucidating complex biochemical pathways andprocesses. Today, protein arrays enable researchers tosimultaneously measure multiple analytes. LuminexxMAP technology was the first — and continues tobe the most successful — bead array strategydesigned for medium-density, medium-throughputarray applications. The technology uses spectrallyaddressable microspheres as array elements to enablethe simultaneous analysis of up to 100 analytes inonly a few microliters of sample (see Figure 1). Thelarge surface area of suspension arrays and the highconcentration of reactants on the surface of thebeads improve binding kinetics, reducing assay timeand increasing assay sensitivity (de Jager et al. 2003).The Bio-Plex™ suspension array system integratesthis powerful bead array technology with instrumentcontrol and data analysis software, system validationand calibration tools, multiplex immunoassays, andall the supplies needed to perform xMAP-basedassays in a 96-well microplate format.

This article highlights some of the validation andcorrelation issues involved when applying xMAPtechnology to specific applications. We will look attwo popular cell-signaling applications: cytokinetesting and phosphoprotein testing. Theseapplications leverage the strengths of xMAPsuspension array technology — analysis of multipleproteins in complex biomolecular processessimultaneously. This technology also has potentialutility in biomarker quantitation. The bead arraystrategy for testing allows the end user to mix andmatch targets rather than committing to a specificset of analytes (Figure 2). Furthermore, analysis takesplace in a liquid medium, where proteins can beanalyzed in their native or near-native conformation,rather than on the solid medium of a chip (seesidebar on next page).

xMAP Suspension Bead Array Technology

Challenges in Comparing Results to Those of Traditional Methods for Selected Applications

Assay Data CategoriesBefore we discuss applications of a given method, itmay be useful to categorize assays or applications bywhether they are quantitative or nonquantitative(see Table). By breaking assay applications into thesecategories, we can gain insight into expectations andthus their performance. One way of looking at this isto sort assays into four distinct categories: definitivequantitative, relative quantitative, quasi-quantitative,and qualitative (Bowsher and Smith 2002).

Illustration by Audra G

eras

by Jim Torrence, Bio-Rad Laboratories, Inc., Hercules, CA USA

Akt

JNK

p38 MAPK

Fig. 1. Multiplexing usingxMAP technology. xMAPtechnology utilizes 100 beadcolors. The dyed 5.6 µmbeads are automaticallycategorized into beadregions. By coupling thecapture antibody to thedifferent bead regions, xMAP technology allowssimultaneous detection ofeach capture antibody.

Table. Comparison of the statistics and studies that can be performed on relative quantitationassays vs. quasi-quantitation assays.

Relative Quantitation Quasi-Quantitation(Example: Cytokines) (Example: Phosphoproteins)

Accuracy Spike recovery Not applicable

Precision Replicate testing Replicate testing

Sensitivity LOD or LLOQ LOD

Specificity Cross-reactivity studies Cross-reactivity studies

Linearity Ratio of observed to expected Not applicable; however, the values for diluted sample dose response of a diluted

sample may suggest linearity

Matrix effects Dilution studies Dilution studies

Recombinant standard Test vs. native protein Not applicable

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Protein Profiling — CytokinesCytokines are important extracellular signalingproteins, mediating a wide range of physiologicalresponses, including immunity, inflammation, andhematopoiesis (Jenmalm et al. 2003). They are alsoassociated with a spectrum of diseases ranging fromtumor growth to infections to Parkinson’s disease.While cytokines do exist intracellularly, mostresearch involves measuring extracellularconcentrations — generally in cell culturesupernatants or in serum.

Cytokines are typically measured byimmunoassay, most commonly using a sandwichassay format. The cytokines of interest are capturedby an antibody that is specific for the target anddetected by a secondary antibody directed against adifferent epitope. Presence of the target cytokine isread optically, and the data are displayed digitally (as OD when obtained by ELISA or MFI whenobtained by bead-based assays using xMAPtechnology). The data obtained for several knownconcentrations generate a curve. Unknown sampleconcentrations are then determined by interpolationbased on the standard curve.

Issues That Cloud Relative Quantitation of CytokinesLack of Endogenous Standard MaterialNo endogenous standard exists for cytokines today.The only alternative to a true reference standard incytokine testing is the National Institute forBiological Standards and Control (NIBSC)standards. However, the NIBSC standards do notqualify as true reference materials because many arerecombinant proteins that may differ from nativelyexpressed proteins found in a biological sample.Because they are recombinant, the possibility ofposttranslational modification — such asglycosylation, which may alter an assay’s response —cannot be ignored (Wadhwa et al. 1999). Makingmatters even cloudier, the NIBSC does not assay itsstandards for concentration (for example, pg/mlvalues reported by immunoassay methods), but rather reports in international units (IU) basedon bioassay activity. Thus, many assay manufacturersprovide IU conversion factors that link solubleconcentration to biological activity.

When manufacturers and researchers createcytokine assays, they generally use recombinantmaterial readily available to them — meaning eachmay be slightly different (Ledur et al. 1995, deKossodo et al. 1995). This is not to say that theresults of these assays are meaningless. On thecontrary, there is generally a reasonable correlationbetween different assays. But to make sure there is areasonable correlation, the assay curve must betested against native material (for example,lipopolysaccharide-stimulated peripheral bloodmononuclear cell supernatant).

Antibody VariationAnother key component of an immunoassay is thereagent used for analyte capture and detection.Antibodies are perhaps the most widely usedreagents in protein microarrays, because they can bemade highly specific for their targets and are oftenable to differentiate subtle protein modificationssuch as phosphorylation. The sandwich immunoassayformat is often used, making use of two antibodiesthat simultaneously bind the same target (Figure 3).Sandwich assays offer the advantage of very highspecificity, given that both antibodies must recognizea common target for successful measurement.Nevertheless, the use of antibodies can complicatethe direct correlation of results between platforms, aseach platform may use antibodies whose epitopesmay differ between sets of capture and detectionreagents. While recognition by one antibody for anepitope may be masked or obscured throughbiomolecular interactions involving soluble targets,another antibody’s target epitope may be free fromsuch interference, thereby recognizing bothcomplexed and uncomplexed forms of the targetmolecule. This may or may not be desirable,depending on the context of the experiment and thespecific molecular target. Furthermore, recognitionmay differ between native and recombinant proteins.

To reduce the possibility of variation, monoclonalantibodies are the first choice for an assay becausevariability in the affinity for the epitope is minimal.Unfortunately, specific monoclonal antibodies arenot always available for novel targets, and the highdegree of antibody characterization that is desirableis difficult and often complex (Wadhwa et al.1999). This complexity is further compoundedwhen developing a multiplex kit — each antibody,either detection or capture, must be checked forcross-reactivity against all other antibody pairs thatare to be combined. Generally speaking, high-multiplex assays require increasingly cumbersomeand difficult validation compared to their low-multiplex counterparts.

The ideal situation for the user is a definitivequantitative assay — meaning that there is adefined, endogenous reference standard available.While ideal, this usually is not the case for mostnovel markers, particularly in life science research. A good example is cytokines. While cytokines havebeen quantitated for years by ELISA, fewendogenous, purified reference materials exist.Therefore, cytokine assays today fall into the relativequantitative category.

The quasi-quantitative result is defined by havinga measurement that is characteristic, but thatcharacteristic cannot be readily compared. Activityassays fall into this category. For example, assayingkinases (measuring phosphorylation) is possible bywestern blot, ELISA, and bead-based assays. Westernblots will show lighter and darker bands, ELISAs willread a higher or lower optical density (OD), andbead-based assays will read a higher or lower medianfluorescence intensity (MFI). But what does it meanif we want to compare results across methods?Unfortunately, the ability to generate a number on ascanner or plate reader can mislead us into graspingfor quantitation-based data.

The last category, qualitative data, is generally apositive or negative type of result. For the purposes ofthis article, we will not be discussing qualitative results.

Once we’ve defined the parameters for evaluatingmethods, we can look at the challenges thatdifferent categories of assay data present.

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p38 MAPKJNKAkt

Mix and match selected targets(target specificity identified by bead color)

Perform assay (mix sample and beads)

TNF-α

IL-2

IFN-γ

Fig. 3. Multiplex sandwichimmunoassay. Twoantibodies simultaneouslybind different epitopes on thesame target protein, allowinga high degree of specificity.

Fig. 2. Bead array strategy. Based on a mix-and-match approach, 5.6 µm coupled beads insuspension allow mixing of appropriately coupled regions in a single well. This enables multiplexing ofup to 100 analytes simultaneously.

Protein Bead Arrays — A Compelling ApproachOligonucleotide and cDNA arrays helped revolutionize genomicresearch, in part due to the tremendous amount of informationavailable from a single experiment. Similarly, protein arrays aremaking large-scale proteomics more feasible, and coupled withpowerful computational methods are facilitating a more system-wide approach to biological research. Protein microarrays (chips)are a logical extension of the DNA array, but while microarraysmonitor gene expression levels in a particular organism, only proteinmicroarrays can provide similarly comprehensive information aboutthe activity and function of those gene products.

While traditional biochemical methods have been invaluable inelucidating protein function on an individual basis, such methodscannot reasonably be used to evaluate an entire cellular proteomeor tease apart a complex network of biomolecular processes.Deciphering even a single protein’s function in a complexbiochemical pathway typically requires years of intense study usingtraditional methods. It is here that protein arrays may have thegreatest impact, for they provide both the high-density format andthe multiplexing capability that are required for such global analysis.

Current assay tools, such as 2-D PAGE, mass spectrometry,enzyme-linked immunosorbent assay (ELISA), and similartechniques are proven methods for expression proteomics and drugdevelopment. While effective, these methods can be cumbersome,suffering from low throughput, low data density, or both. Protein

arrays using microbeads combine the simplicity of ELISAs with themultiplexing ability of microarrays. Protein bead arrays, like theirnucleic acid counterparts, are poised to drive the study of proteinsystems by offering both higher sample throughput and a higherdensity of results for a given sample (Willis et al. 2003).

Protein arrays utilizing microbeads enable parallel analysis ofmultiple proteins, often requiring much smaller sample volume for asingle experiment compared to an ELISA or western blot. Inaddition, bead arrays are typically amenable to a range of samplematrices, enabling analysis of many types of biological samples,including blood filtrates (Hutchinson et al. 2001), culturesupernatants (Rutella et al. 2002), tissue homogenates (Hulse et al.2004), and cell lysates. Because of the variety of samples that canbe analyzed using protein arrays, a correspondingly large number ofapplications can benefit from the use of protein bead arrays.

Quantitation of proteins in biological samples is one applicationthat is particularly amenable to multiplex analysis, leading to proteinexpression profiling or a molecular signature approach to sampleanalysis. However, protein bead arrays are not limited to expressionanalysis: Preferential enzyme target recognition, protein regulationand signaling activation (e.g., phosphorylation), binding siterecognition and competition, and other functional assays have beendeveloped, and several other assay formats are possible using thesame core technology. Thus, like DNA microarrays, protein beadarrays provide a “high content” solution to protein analysis.

Extracellular SamplesPotential effects of the matrix (for example, serumor plasma) can be addressed by making a dilutionseries of spiked material in the matrix of interest(de Kossodo et al. 1995). It is always possible that insome samples, high levels of interfering factors maycause unusual results. How do we rule this out? It isour experience that running dilutions of the(unexpectedly) high-concentration sample will leadto the correct answer. Upon dilution, the interferingsubstance(s) become dilute enough that nonspecificbinding drastically drops, leading to a nonlineardilution series for the sample of interest. If it is infact a nonlinear dilution series, it can be inferredthat an interfering factor is likely to have beenpresent in the sample.

For clinical study support, characterizing normaland test populations of patients is important(Findlay et al. 2000). We tested the sera of 50apparently normal human samples using the Bio-Plex human cytokine 17-plex panel andobserved a range of values (Figure 4).

Monitoring Signal Transduction —PhosphoproteinsBead array phosphoprotein assays allow thesimultaneous analysis of the phosphorylation state of several target proteins within a single cell lysatesample. The principle of phosphoprotein assays usingxMAP internally labeled beads is similar to that of acapture sandwich immunoassay. An antibody directedagainst the desired phosphoprotein target iscovalently coupled to 5.6 µm fluorescently dyedbeads. The coupled beads are allowed to react with asample containing an unknown amount ofphosphoprotein target. After a series of washes toremove unbound protein, a biotinylated detectionantibody specific to a different epitope on the targetprotein is added to the reaction. The result is theformation of a sandwich of antibodies around onlyphosphorylated target protein. The reaction mixture isdetected by the addition of streptavidin-phycoerythrin,

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in which the streptavidin binds to the biotinylateddetection antibodies.

Western blot analysis is the most commonmethod of testing for phosphoproteins today. Inshort, the western blot method consists ofdenaturation of proteins (heated in buffer containingSDS and 2-mercaptoethanol), gel separation, andtransfer onto nitrocellulose membrane, followed byspecific antibody probing. While western blotanalysis has proven to be a commonly used methodof testing phosphorylated proteins, it tends to betime-consuming and is subject to variability betweenruns. Phosphoprotein testing by western blottinggenerally involves testing a control sample that isunstimulated or normal against a test sample that isstimulated or treated with an inhibitor. A significantdifference in signal (as measured by a scanner) isoften used to compare the control against thestimulated sample (for example, signal is 3-foldhigher in the stimulated sample vs. the control).

Issues That Cloud Quasi-Quantitation of PhosphoproteinsLack of Phosphorylated Standard MaterialIt is important to make the distinction inquantitation categories at this point. As opposed tothe relative quantitation of cytokines, phosphoproteintesting is quasi-quantitative. No phosphoproteinstandard is used in western blot analysis, which iswhy a control sample is run against a test sample. Sohow do we correlate results from a western blot andan immunoassay method? One common method ofanalysis for western blots is a ratiometric approach,resulting in a “fold-change” of a test sample over acontrol. However, there are potential dangersinvolved in applying this same analyticalmethodology to other platforms, in part due to thelack of standard material for calibration purposes.Fold-change, as determined by most assay platforms,does not necessarily correlate directly with theabsolute magnitude of protein expression ormodification. By necessity, a fold-change calculationrequires a linear response over the test sample range.Furthermore, fold-change will often vary betweenplatforms because the linear region of the dynamicrange is highly platform dependent, even showingsignificant variation between different methods ofstaining or visualization. Results from western blotexperiments are typically obtained by image analysisand densitometry, and that digitized, “quantitative”information then forms the basis for further numericmanipulation and analysis.

Finally, numerical analysis must be comparable.For example, to minimize variation between westernblotting and Bio-Plex assay, it is recommended touse the same lysate (Han et al. 2003) and labeleddetection antibodies. Ratiometric results (fold-change) should likewise be calculated in asimilar manner, including background subtraction,signal averaging, etc.

Antibody VariationAs mentioned before, any immunoassay is subject to differences between antibodies used. A majordifference between a western blot and a bead assay(or any sandwich assay) is the number of antibodiesneeded; a western blot requires only a singledetection antibody, whereas bead arrays require anantibody pair to complete the “sandwich”. On onehand, this may lend better specificity and reducedbackground to the bead-and-sandwich approachbecause it means that two highly specific antibodies,directed at different epitopes, are needed to run abead assay. On the other hand, the sandwichmethod requires two highly specialized reagents,potentially complicating development.

A major issue that plagues phosphoprotein assaydevelopment in any platform is the lack of phospho-specific antibodies. The limited availability of high-specificity, high-affinity antibodies is compounded bythe fact that the antibodies must be further modifiedby immobilization onto beads or labeled withfluorophores without damaging their functionality.

Intracellular SamplesIn contrast to cytokines, which are generally testedextracellularly (that is, in serum, plasma, etc.),phosphoproteins are intracellular proteins and thusrequire cell lysis for accurate measurement. Assuch, the separation of phosphoproteins of interestfrom their intracellular complexes becomes asignificant consideration in an assay system. Thistask is further complicated by the many cell andtissue types, each of which may respond differentlyto the wide variety of available cell lysis methods.Commercial phosphoprotein blotting kits generallyinclude cell lysates that serve as control samples todemonstrate antibody specificity against the targetof interest. Lysates provided with Bio-Plexphosphoprotein and total target assays serve thesame purpose.

As more assays are introduced, the threat of cross-reactivity becomes an issue. There will likely come a point where it will not be possible to introducenew assays into a high-multiplex assay withoutencountering antibody cross-reactivity with othertarget proteins. Therefore, it may be necessary todivide samples over two or more assays in order toaccomplish a high-multiplex analysis involvingmany targets.

ConclusionAll methodologies have strengths and weaknessesthat are often judged based on a correlation ofresults to traditional methods. Multiplex bead-basedassays are no different — they are compared toexisting immunoassay methods (e.g., ELISA,western blotting, etc.). Many of the differencesamong immunoassay methods arise from the factthat immunoassays depend on the antibodies andproteins used (Findlay et al. 2000). With increasing

acceptance of the bead-based xMAP technology,issues regarding sensitivity, specificity, andcalibration will be further clarified by each newvalidation study. The growing number of studies inthe literature that employ bead-based xMAPtechnology suggests that this strategy for proteintesting is here to stay.

ReferencesBowsher RR and Smith WC, A practical approach for clinicaldrug development — analytical validation of assays for novelbiomarkers, AAPS Newsmagazine, 18–25 (2002)

de Jager W et al., Simultaneous detection of 15 humancytokines in a single sample of stimulated peripheral bloodmononuclear cells, Clin Diagn Lab Immunol 10, 133–139 (2003)

de Kossodo S et al., Assaying tumor necrosis factorconcentrations in human serum, A WHO InternationalCollaborative study, J Immunol Methods 182, 107–114 (1995)

Findlay JW et al., Validation of immunoassays for bioanalysis: apharmaceutical industry perspective, J Pharm Biomed Anal 21,1249–1273 (2000)

Han H et al., Application of the Bio-Plex cell lysis kit in westernblot analysis of phosphoproteins, Bio-Rad bulletin 3033 (2003)

Hulse RE et al., Optimization of multiplexed bead-based cytokineimmunoassays for rat serum and brain tissue, J NeurosciMethods 136, 87–98 (2004)

Hutchinson KL et al., Multiplex analysis of cytokines in the bloodof cynomolgus macaques naturally infected with Ebola virus(Reston serotype), J Med Virol 65, 561–566 (2001)

Jenmalm M et al., Bio-Plex cytokine immunoassays and ELISA:comparison of two methodologies in testing samples fromasthmatic and healthy children, BioRadiations 111, 38–39 (2003)

Ledur A et al., Variable estimates of cytokine levels produced bycommercial ELISA kits: results using international cytokinestandards, J Immunol Methods 186, 171–179 (1995)

Rutella S et al., Role for granulocyte colony-stimulating factor inthe generation of human T regulatory type 1 cells, Blood 100,2562–2571 (2002)

Wadhwa M et al., Standardization and calibration of cytokineimmunoassays, Revue de l’ACOMEN 5 (1999)

Willis E et al., The emergence of suspension bead arrays,BioRadiations 111, 30–35 (2003)

Proteomic Applications: Chip vs. Bead ArrayAs applied in proteomics research, suspension bead arrays rival the advantagesof the most promising protein chips. Synthesis of suspension arrays takesadvantage of established and readily available protein capture molecules, such asspecific antibodies and binding proteins. Multiplex protein assay protocols arestraightforward, often similar to classic immunoassays. The relative ease of assaygeneration and simplicity of protocols make suspension bead arrays a valuableresearch tool for protein expression profiling, detection of protein posttranslationalmodifications, and screening for protein functions and interactions.

While chips may have an advantage in content per sample, bead arraysclearly have an advantage in flexibility. Protein chips consist of a solid medium(e.g., glass slide) that is “spotted” to capture the analytes of interest. Thepredetermined number of analytes on the medium cannot be modified. Incontrast, bead arrays consist of different populations of beads directed againstdifferent targets. This allows the user to mix and match beads directed againstspecific targets (Figure 3).

Fig. 4. Cytokine expression profile. Sera from 50 normal human samples were tested using theBio-Plex human cytokine 17-plex panel. Results for each analyte are shown. For analytes that displayfewer than 50 points, the remaining values were below the 2 pg/ml limit of detection.

10,000

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IL-5

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