multiple-analyte fluoroimmunoassay using an integrated optical waveguide sensor

Multiple-Analyte Fluoroimmunoassay Using anIntegrated Optical Waveguide Sensor

T. E. Plowman, J. D. Durstchi,† H. K. Wang,† D. A. Christensen,† J. N. Herron,† and W. M. Reichert*

Center for Emerging Cardiovascular Technologies, Department of Biomedical Engineering, Duke University,Durham, North Carolina 27710

A silicon oxynitride integrated optical waveguide was usedto evanescently excite fluorescence from a multianalytesensor surface in a rapid, sandwich immunoassay format.Multiple analyte immunoassay (MAIA) results for two setsof three different analytes, one employing polyclonal andthe other monoclonal capture antibodies, were comparedwith results for identical analytes performed in a single-analyte immunoassay (SAIA) format. The MAIA protocolwas applied in both phosphate-buffered saline and simu-lated serum solutions. Point-to-point correlation valuesbetween the MAIA and SAIA results varied widely for thepolyclonal antibodies (R2 ) 0.42-0.98) and were accept-able for the monoclonal antibodies (R2 ) 0.93-0.99).Differences in calculated receptor affinities were alsoevident with polyclonal antibodies, but not so with mono-clonal antibodies. Polyclonal antibody capture layerstended to demonstrate departure from ideal receptor-ligand binding while monoclonal antibodies generallydisplayed monovalent binding. A third set of three anti-bodies, specific for three cardiac proteins routinely usedto categorize myocardial infarction, were also evaluatedwith the two assay protocols. MAIA responses, overclinically significant ranges for creatin kinase MB, cardiactroponin I, and myoglobin agreed well with responsesgenerated with SAIA protocols (R2 ) 0.97-0.99).

Contemporary trends in biosensing focus on the detection ofmultiple analytes through immunoassay. In a typical solid-phasemultiple-analyte immunoassay (MAIA), an array of capture anti-bodies is first immobilized on a solid support by physicaladsorption, covalent linkages, indirect anchors, or laser printingand stamping methods.1 A mixture of analytes, derived from asingle sample, is then reacted with the multicomponent capturelayer and detected with labeled, tracer antibodies. Detection isroutinely achieved using radiometric, enzymometric, gravimetric,amperometric, or fluorometric labels.2 Optical methods for detect-ing biochemical events at surfaces are well characterized andgenerally possess equivalent, if not better, sensitivities thancompeting measurement techniques.2

The literature relevant to MAIA employing optical detectionis categorized by transducer type in Table 1. Kakabakos et al.and Ekins et al. were first to propose methods for MAIA basedon time-resolved fluorescence and confocal microscopy, respec-tively.3,4 More recent techniques make use of multiple internalreflection elements such as planar waveguides, 5-12 planarwaveguide interferometers,13-15 and bundled fiber optics,16,17 whichtend to achieve higher sensitivities due to their large optical pathlengths. Also employed in optical MAIA are the reflectometricmethod of surface plasmon resonance (SPR)18 and capillary flowsystems with fluorescent detection.19 Fluorescent chemometricmethods have also been proposed that, as an advantage, do notrequire spatial separation to detect multiple analytes.20

The optical transducers used most often in MAIA are theintegrated optical waveguide (IOW) or the internal reflectionelement (IRE). Although many have proposed different MAIAschemes, no one to our knowledge has presented completebinding curves (i.e., response of the sensor from background tosaturating levels) for IOW MAIA when all analytes are assayed

* Corresponding author: [email protected]; (phone) (919) 660-5151; (fax)(919) 684-8886.

† Current address: Center for Biopolymers at Interfaces, Department ofPharmaceutics and Bioengineering, University of Utah, Salt Lake City, UT 88504.(1) Gopel, W.; Heiduschka, P. Biosens. Bioelectron. 1995, 10, 853-883.(2) Brecht, A.; Gauglitz, G. Biosens. Bioelectron. 1995, 10, 923-936.

(3) Kakabakos, S.; Christpoulus, T.; Diamandis, E. Clin. Chem. 1992, 38, 338-342.

(4) Ekins, R.; Chu, F.; Biggart, E. Clin. Chim. Acta 1990, 194, 91-114.(5) Klainer, S.; Coulter, S.; Pollina, R.; Saini, D. Sens. Actuators B 1997, 38-

39, 176-182.(6) Ligler, F.; Conrad, D.; Golden, J.; Feldstein, M.; MacCraith, B.; Bladerson,

S.; Czarnaski, J.; Rowe, C. Proc. SPIE 1998, 3258, 50-55 (Proceedings ofMicro- and Nanofabricated Structures and Devices for Biomedical Environ-mental Applications).

(7) Wadkins, R.; Golden, J.; Ligler, F. J. Biomed. Opt. 1997, 2, 74-79.(8) Wadkins, R.; Golden, J.; Pritsiolas, L.; Ligler, F. Biosens. Bioelectron. 1998,

13, 407-415.(9) Zhou, Y.; Magill, J. V.; De La Rue, R. M.; Laybourn, P. J. R.; Cushley, W.

Sens. Actuators, B 1993, B11, 245-250.(10) Brecht, A.; Klotz, A.; Barzen, C.; Gauglitz, G.; Harris, R.; Quigley, G.;

Wilkinson, J.; Sztajnbok, P.; Abuknesha, R.; Gascon, J.; Oubina, A.; Barcelo,D. Anal. Chim. Acta 1998, 362, 69-79.

(11) Misiakos, K.; Kakabakos, S. Biosens. Bioelectron. 1998, 13, 825-830.(12) Silzel, J.; Cercek, B.; Dodson, C.; Tsay, T.; Obremski, R. Clin. Chem. 1998,

44, 2036-2043.(13) Schneider, B.; Edwards, J.; Hartman, N. Clin. Chem. 1997, 43, 1757-1763.(14) Lukosz, W.; Stamm, C.; Moser, H.; Ryf, R.; Dubendorfer, J. Sens. Actuators,

B 1997, B39, 316.(15) Klotz, A.; Brecht, A.; Gauglitz, G. Sens. Actuators, B 1997, B39, 310-315.(16) Healey, B.; Li, L.; Walt, D. Biosens. Bioelectron. 1997, 12, 521-529.(17) Michael, K.; Taylor, L.; Schultz, S.; Walt, D. Anal. Chem. 1998, 70, 1242-

1248.(18) Berger, C.; Beumer, T.; Kooyman, R.; Greve, J. Anal. Chem. 1998, 70, 703-

706.(19) Narang; Gauger, P.; Kusterbeck, A.; Ligler, F. Anal. Biochem. 1998, 255,

13-19.(20) Piehler, J.; Brecht, A.; Giersch, T.; Kramer, K.; Hock, B.; Gauglitz, G. Sens.

Actuators, B 1997, 38-39, 432-437.

Anal. Chem. 1999, 71, 4344-4352

4344 Analytical Chemistry, Vol. 71, No. 19, October 1, 1999 10.1021/ac990183b CCC: $18.00 © 1999 American Chemical SocietyPublished on Web 08/27/1999

simultaneously. This paper examines how accurately MAIA datacorrelate to those data obtained when each element of the MAIAis assayed separately in a single-analyte immunoassay (SAIA).Such comparisons can provide information on the assay format’stendencies to misidentify proper analyte levels due to cross-reactivity, receptor heterogeneity, and nonspecific binding, as falsepositives are a typical problem in MAIA sensing.21

Our approach to performing IOW MAIA, Figure 1, is animprovement on the MAIA method of Kakabakos et al.3 becauseit is performed in one simple step. The surface is an array of threemillimeter-sized protein “channels” adsorbed onto a previouslydescribed, grating coupled silicon oxynitride (SiON) IOW trans-ducer operating in an evanescently excited fluorescence detectionformat.22 The channels contained either model monoclonal orpolyclonal antibodies or cardiac-specific antibodies to cardiactroponin I (cTnI), myoglobin (Mb), and creatine kinase of themuscle-brain form (CK-MB). A series of assays, in both SAIAand MAIA formats, was conducted to assess binding specificityand interchannel cross reactivity in terms of the correlationbetween assay data and therefore the tendency to generate falsepositives. Responses between buffer and serum matrix solutionswere subsequently compared by correlation. Biophysical param-eters, obtained via Langmuir curve fitting, provided furthercomparison between formats for both the model monoclonal andpolyclonal antibody systems.

Assay responses for the model monoclonal antibodies cor-related well between MAIA and SAIA formats. The goodness offit (R2) values for a straight-line correlation between formats werein the range 0.93-0.99. None of the predicted binding constantswere statistically different between assay formats and none of thereactions displayed a departure from ideal binding. Two of thethree polyclonal capture antibodies, however, showed a significantdifference between the predicted binding constants and a depar-ture from ideal binding in the MAIA format. Comparison of the

polyclonal antibody results between formats by a point-to-pointcorrelation did not suggest a linear relationship for these sametwo capture areas. When assays were performed in 10% simulatedserum versus assays performed in a phosphate-buffered saline(PBS) solution, only two of the six capture antibodies studied weresensitive to the presence of the 10% serum component. Finally,MAIA conducted for the cardiac proteins correlated well with SAIA

(21) Kricka, L. Clin. Chem. 1992, 38, 327-328.(22) Plowman, T. E.; Saavedra, S. S.; Reichert, W. M. Biomaterials 1998, 19,

341-355.

Table 1. Groups That Have Demonstrated or Are Developing MAIA Technologya

transducer ref MAIA configuration reagents assayed detection method MDC

IOW/IRE 9 etched wells r-, m-, gIgGs fluorescence 100s of ng/mL14b na proposed interferometry na

5b IC technology proposed fluorescence na15 na proposed interferometry na13b spatial arrangement proposed interferometry pM7, 8 spatial arrangement three toxic agents fluorescence ng/mL6 spatial arrangement ovalbumin, SEB fluorescence ng/mL

10 spatial arrangement proposed fluorescence NA11 spatial arrangement Av, biotin-bSA fluorescence NA12 DeskJet printing IgG sublasses fluorescence ng/mL

fiber 17 encoded microspheres AP, Av, biotin fluorescence naSPR 18b spatial arrangement hCG only angle of reflection namicroscope 4 spatial arrangement TNF, TSH confocal microscopy pg/mm2

slide/other 3 spatial arrangement LH, FSH, hCG, PRL time-resolved fluorescence 100s of ng/mL20 chemometrics three s-triazine

derivativesrelflectometric interference

spectroscopypg/mm2

19 multiple capillary tubes TNT, RDX fluorescence ng/mL

a Abbreviations: AP, alkaline phosphatase; Av, avidin; bSA, bovine serum albumin; FSH, follicle stimulating hormone; g, goat; hCG, humanchorionic gonadotropin; IC, integrated circuit; IOW, integrated optical waveguide; IRE, internal reflection element; LH, leutinizing hormone; MDC,minimum detectable concentration; m, mouse; na, not available; PRL, prolactin; r, rabbit; SEB, staphyloccocal enterotoxin B; SPR, surface plasmonresonance. b Known industrial affiliation.

Figure 1. Schematic representation of a MAIA as performed on agrating coupled SiON IOW. An idealized view of the surface (not toscale) shows three capture antibodies adsorbed to the surface inchannels separated by areas of blocking protein. Premixed analyteand tracer antibodies, specific for one channel each, are introducedby the sample inlet port and react with accessible binding sites.Fluorescent labels attached to the tracer antibodies absorb energyfrom the evanescent wave and re-emit it as fluorescence.

Analytical Chemistry, Vol. 71, No. 19, October 1, 1999 4345

measurements (R2 ) 0.97-0.99), but only one of the threecalculated detection limits in the MAIA format suggested theability to reliably detect trace amounts of analyte.

MATERIALS AND METHODSReagents. Polyclonal Immunoassay System. Capture antibodies

were anti (R) goat-, human-, and rabbit-IgG (RgIgG, RhIgG, andRrIgG) from Pierce (piercenet.com). Antibodies were of variablesubclass and their affinities were not provided. Analytes consistedof gIgG, hIgG, and rIgG-fluorescein isothiocyanate (FITC) in 1%bovine serum albumin (bSA) from Sigma (sial.com/sigma). Thetracer antibody was the monoclonal RFITC, isomer 5 fromGenzyme Diagnostics, Inc. (genzyme.com).

Monoclonal Immunoassay System. Capture antibodies wereRbSA, RgIgG, and Ravidin (RAv) (Sigma Product B9655, antibodyto avidin, had attached biotin groups.) from Sigma or RhCGR fromCalbiochem (calbiochem.com). All antibodies were of the IgG1subclass, and their affinities were not provided. Analytes were anythree of the following: Av-FITC, bSA-FITC, gIgG-FITC (Sigma),or hCG (Calbiochem). The tracer was, again, the RFITC fromGenzyme Diagnostics, Inc., or RhCGâ for the hCG analyte(Calbiochem).

Cardiac Immunoassay System (Lumenal Technologies, Salt LakeCity, UT). Both capture and tracer antibodies were one of twomonoclonal clones to the analytes: CK-MB, Mb, and cTnI(typically IgG1, affinities not provided).

Miscellaneous. Cyanine 5 dye (Cy5) was obtained from Re-search Organics (resorg.com) while porcine serum albumin (pSA)and trehalose were from Sigma.

Protein Preparations. Antibodies. Polyclonal antibodies fromPierce, the monoclonal tracer antibody from Genzyme, and thecardiac analytes from Lumenal Technologies were used asreceived without further purification, having already been sub-jected to fractionation and ion-exchange chromatographic purifica-tion procedures. Antibodies from Sigma were shipped in ascitesfluid or 1% bSA. In such cases, IgGs were purified with protein Gcolumns (Pharmacia Biotech, biotech.pharmacia.se). The finalconcentration of capture antibody was generally in the micromolarrange, as measured by absorbance (A280). Aliquots (100 µL) of allpurified proteins were frozen and stored until needed.

Labeling Tracer Antibody. The RFITC tracer antibody waslabeled with a Cy5 dye23 following the manufacturer’s procedure(Amersham Life Science, www.apbiotech.com). Generally fourdyes were attached to each antibody tracer. Aliquots of 100 µL(µM) were stored frozen.

Analyte Cocktails. The assays presented here required severalanalyte components and multiple-analyte cocktail mixtures, asoutlined in Table 2. Channel addressability and generic SAIA testsinvolved analyzing the binding of one analyte only. Therefore,nanomolar starting solutions containing the analyte of interestwere prepared in either PBS or 10% pSA. Generic SAIA assaysrequired diluting the starting solution through the ranges listedin Table 2. All solutions contained nanomolar amounts of RFITC-Cy5 tracer for detection. MAIA test solutions, in both thepolyclonal and monoclonal cases, contained a mixture of all three

(23) Mujumdar, R. B.; Ernst, L. A.; Mujumdar, S. R.; Lewis, C. J.; Waggoner, A.S. Bioconjugate Chem. 1993, 4, 105-111.

Table 2. Analyte Cocktails Used in the Various Assay Formatsa

assay proteins analyte concentrations buffer

analyte capture tracer channel tests single- and multiple-analyte tests PBS 10% pSA

polyclonalCA

rIgG-FITC p RrIgG m RFITC-Cy5 10 nM 300 pM-100 nM SAIAMAIACA

gIgG-FITC p RgIgG m RFITC-Cy5 1 nM 30 pM-30 nM SAIAMAIACA

hIgG-FITC p RhIgG m RFITC-Cy5 10 nM 300 pM-30 nM SAIAMAIA

monoclonalSAIA

Av-FITC m RAv m RFITC-Cy5 30 pM-100 nM MAIA MAIACA

IgG-FITC from goat m RgIgG m RFITC-Cy5 1 nM 30 pM-30 nM SAIA MAIAMAIACA

bSA-FITC m RbSA m RFITC-Cy5 10 nM 30 pM-30 nM SAIA MAIAMAIA

hCG m RhCG-R m RhCG-â-Cy5 50 nM CAcardiac

SAIAcTnI m RcTnI m RcTnI-Cy5 3-900 ng/mL MAIA MAIA

SAIACK-MB m RCK-MB m RCK-MB-Cy5 1-100 ng/mL MAIA MAIA

SAIAMb m RMb m RMb-Cy5 5-500 ng/mL MAIA MAIA

a Abbreviations: R, anti; Av, avidin; bSA, bovine serum albumin; CK-MB, creatine kinase of the muscle brain form; CA, channel addressabilityexperiment; FITC, fluorescein isothiocyanate (isomer 5); g, goat; h, human; IgG, immunoglobin G; Mb, myoglobin; PBS, phosphate-buffered saline;pSA, porcine serum albumin; r, rabbit; cTnI, cardiac troponin I; p, polyclonal; m, monoclonal; hCG, human chorionic gonadotropin.

4346 Analytical Chemistry, Vol. 71, No. 19, October 1, 1999

analytes in nanomolar tracer backgrounds as indicated in Table2. To simulate the presence of a serum background, 10% pSA wasadded to some of the monoclonal MAIA test solutions, to someof the cardiac MAIA, and to all of the cardiac SAIA test solutions(see Table 2).

Surface Preparation. IOW Silanization. Grating etched SiONsurfaces were immersed in a 5% (v/v) solution of hydrogenperoxide in sulfuric acid for 10 min, rinsed in ultrapure water andethanol, and allowed to dry under room-temperature vacuum. AllSiON surfaces were then modified using dimethyldichlorosilane(DDS, Sigma) in toluene to render the surface hydrophobic. After30 min, the IOWs were removed and cleaned in ethanol, ultrapurewater, and ethanol once again. SiON surfaces were then vacuum-dried at room temperature for at least 30 min. A 1% DDS/toluenesolution was found to produce a water contact angle of about 85°.Other DDS/toluene solutions (10, 0.5, and 0%) showed decreasingcontact angles with decreasing DDS concentration (105, 80, and0°). Test immunoassays at each DDS level indicated that a 1%solution produced the best capture layer (data not shown).

Capture Layer Depositions. To pattern antibody capture layers,a three-hole rubber gasket was clamped to an IOW to producethree adjacent rectangular wells (0.5 × 2 cm each) in whichcapture protein was adsorbed; the clamped wells also preventedcross-contamination of the different antibody solutions. Eachcapture antibody solution (0.5 mL of 0.1 µM solution), pipettedinto its designated well, was allowed to adsorb to the surface for2 h. At that point, the rubber gasket was removed and the surfacerinsed with PBS to yield three distinct regions of hydratedantibody separated by two sections of antibody-free SiON(0.33 × 2 cm). Physical adsorption was chosen over otherdeposition techniques, e.g., covalent attachment, primarily for itssimplicity. While covalent attachment is desirable for sensorstability and regeneration aspects, it was not necessary for ourone-shot transducers.

Postcoating Technique. After adsorbing capture antibody, theentire sensor surface (except the grating region) was coated witha passivating and preserving solution of pSA and trehalose sugar(5 and 1%, respectively, in water) for 30 min. The pSA was usedto block nonspecific sites while the sugar was used to stabilizeand preserve the adsorbed antibodies. The surface was removedfrom solution, rinsed in PBS, and vacuum-dried. Prepared surfaceswere stored under refrigeration (4 °C). All treated IOWs wereused within a day of preparation.

Assay Method. Rehydrated sensor surfaces, capture layer sideup, were sealed in a custom sample cell (Lumenal Technologies,Salt Lake City, UT) and placed in the path of a 10-mW, 635-nmdiode laser. The m ) 0 guided mode of the IOW was coupled viagrating diffraction, establishing a continuous streak of light at thewaveguide interface (although not explicitly shown in Figure 1).24

The evanescent field of the guided mode excites fluorescence verynear the waveguide/solution interface. After the sample cell wassituated and the guided mode coupled, the control fluorescencesignal from a plasma/PBS background solution (injected througha 1-cm3 syringe fitted with a plastic pipet tip), was measured overa 5-s exposure, every 30 s, for 5 min. Fluorescence measurementswere made using a spatially binned CCD camera (ST-6, Santa

Barbara Instrument Group) fitted with a band-pass filter totransmit only the fluorescence from the Cy5 dye. Time coursefluorescence readings were stored via LabView (National Instru-ments, natinst.com) in a KestrelSpec data file (KestrelSpec,rheacorp.com). This process was repeated until all samples in theexperiment had been assayed. Acquired data contained arrays offluorescence intensity as a function of both time and binned IOWposition.

Since the IOW sensor is ideally designed to be a one-shotdevice, regeneration of the surface was not considered, and eachentire assay represents a fresh surface on a different waveguide.Calibration of the assay did not involve correction for drift (which,unlike mass sensors, is not a major problem for fluorescencetransducers); however, a background was collected, as mentionedabove, and subtracted from all future intensity profiles acquiredin an assay. This background corrected for two effects: (1)fluorescence measured from bulk excitation and nonspecificallybound tracers and (2) attenuation of the guided mode as a functionof propagation distance. Thus, there will not be a noticeabledepreciation in signal intensity with propagation distance. Twononspecific channels between the specific binding areas were alsopresent and acted as on-board reference channels that gaugedthe level of tracer antibody nonspecific binding.

Data Analysis. Fluorescence intensities were collected as afunction of position over 5-s exposure times every 30 s during a5-min reaction. Since the exposure time was intermittent, effectsdue to photobleaching were insignificant. Integrated fluorescencevalues for each binding channel were plotted on a curve as afunction of assay time. A series of sample concentrations produceda step isotherm. Fraction bound, Fb, with respect to bulk analyteconcentration, c, was calculated by normalizing the data to themaximum intensity obtained in each experiment. Individuallymeasured intensities were compared between SAIA and MAIAmethods by a direct point-to-point correlation.

As in our previous work,25 the reactions were also treated asif in a quasi-equilibrium state and modeled analytically with aLangmuir equation that included a term for receptor nonideality,R (this symbol should not be confused with the same symbol thatdenotes an antibody)

where K is the binding constant for any particular antibody-antigen pair and R represents the standard deviation of an antibodyaffinity probability density function, centered about K, for any givenpopulation of receptors. This equation is an immunologicalanalogue to one originally intended to model a catalyst surface.26

It predicts well the sigmoid response typical of antibody-antigenreactions and has been applied to immunoassays previously.27 Thismodel is also especially useful when the response is not completelyhomogeneous due to diffusion limitations, surface activity, averageaffinity, and avidity with polyclonal antibodies.

RESULTSChannel Addressability. Experiments were devised to ex-

amine the response of individually addressed sensing areas (i.e.,

(24) Lee, D. L. Electromagnetic Principles of Integrated Optics; John Wiley andSons: New York, 1986.

(25) Plowman, T. E.; Reichert, W. M.; Wang, H. K.; Christensen, D. A.; Herron,J. N. Biosens. Bioelectron. 1996, 11, 149-160.

(26) Sips, R. J. Chem. Phys. 1948, 16, 490-496.(27) Kaufman, E.; Jain, R. Cancer Res. 1992, 52, 4157-4167.

Fb ) (Kc)R/(1 + (Kc)R) (1)


channels) arranged on the surface of an IOW. Two separate testswere run, one for the polyclonal capture antibodies and one forthe monoclonal capture antibodies. In each test, three analytesolutions, each specific to one of the three channels, weresequentially exposed to the sensor surface. Analyte concentrationswere selected to ensure that each channel bound sufficient analyteto elicit a signal. Results for the polyclonal and monoclonalantibody surfaces are displayed in Figures 2 and 3, respectively.

Figure 2a consists of three graphs, one for each analyteinjection, that display the channel-specific changes in the mea-sured fluorescence. Figure 2b plots the changes in integratedintensity of Figure 2a for each channel as a function of assay time.Similar representations are made in Figure 3a and b for mono-clonal antibodies. The final integrated intensities reached in eachsample channel, and in one of the two nonspecific areas betweenchannels, are summarized in Table 3. The jagged nature of thetraces in Figure 3a is most likely a result of inhomogeneities inthe surface chemistry and possible aggregation of tracer antibod-ies. It is not believed to be due to gaps in the evanescent field(the field is essentially continuous for an IOW) or attenuation ofthe guided mode (since the traces have already been backgroundsubtracted). A portion of the rightmost channel in both assayswas obscured from the camera’s field of view; however, this didnot affect the results.

Polyclonal and Monoclonal Assays. Dose-response curveswere constructed for the polyclonal and model monoclonal

antibody systems, in both SAIA and MAIA formats, by monitoringthe integrated fluorescence of spatially arranged antibody channelswith increasing analyte concentration. Figure 4 shows the poly-clonal binding curves for the SAIA (top panel) and MAIA (bottompanel) measurements. Figure 5 shows the monoclonal bindingcurves for the SAIA (top panel) and MAIA (bottom panel)

Figure 2. (a) Plots of fluorescence intensity versus relative waveguide position for polyclonal capture antibody areas specific to (left to right)hIgG, rIgG, and gIgG. (b) Three plots of integrated intensity versus time for the separate polyclonal capture channels.

Table 3. Comparison of Integrated Intensities forPolyclonal and Monoclonal Antibody Capture Areasa

integrated intensities,polyclonal capture antibodies

injected analytes RhIgG RrIgG RgIgG nsb

injection1 rIgG-FITC (10 nM) 71925 564950 9248 51962 gIgG-FITC (1 nM) 31654 159259 194038 63703 hIgG-FITC (10 nM) 372863 6624 9544 3271

integrated intensities,monoclonal capture antibodiesinjected analytes

RbSA RhCG RgIgG nsb

injection1 hCG (50 nM) 40492 815290 149637 227402 gIgG-FITC (1 nM) 270763 155830 1848772 472883 bSA-b (10 nM) 1194710 294640 362391 249178

a Abbreviations: R, anti; bSA-b, bovine serum albumin, biotinlabeled; g, goat; h, human; IgG, immunoglobin G; nsb, nonspecificbinding; r, rabbit.


measurements. Three replicates of each entire titration wereperformed. The solid curves in Figures 4 and 5 are best fits ofthe data to eq 1. The binding and nonideality constants derivedfrom these fits are listed in Table 4.

According to the R values, polyclonal antibodies acted in anideal manner in SAIAs; i.e., the antibodies bound antigen in alargely monovalent fashion. However, when the polyclonal anti-bodies were tested together in a MAIA, changes in the index wereapparent; most noticeably, the values for the RrIgG and RhIgGs

dropped from an ideal value of 1 to 0.51 ( 0.02 and 0.72 ( 0.06,respectively. Similar differences were noticed in the bindingconstants, i.e., 0.12 ( 0.01 to 0.05 ( 0.00 nM-1 (RrIgG) and 0.11( 0.01 to 1.59 ( 0.21 nM-1 (RhIgG). The only antibody unaffectedby the MAIA cocktail was the RgIgG antibody (Table 4). Mono-clonal antibodies displayed similar values between assay formatsfor each analyte tested. Nonideality constants were not signifi-cantly different from unity (p < 0.05). The binding constants forall monoclonal antibodies showed only slight variations betweenformats and were indistinguishable at or above the 90% confidencelevel.

Cardiac Assays. SAIA and MAIA were performed for threecardiac proteins: CK-MB, Mb, and cTnI.28-30 Table 2 gives theclinically relevant concentration ranges over which the measure-ments were conducted (units of ng/mL are used in agreementwith clinical convention). The immobilized capture proteins usedin all cases were monoclonal antibodies. All of the SAIAs employedsimulated serum solutions containing 10% pSA, while only one ofthe MAIAs included the 10% pSA matrix. Parts a and b of Figure6 show the average (n ) 3) dose-response binding relationships

(28) Antman, E.; Tanasijevic, M.; Thompson, B.; Schactman, M.; McCabe, C.;Canno, C.; Fischer, G.; Fung, A.; Thompson, C.; Wybenga, D.; Braunwald,E. N. Engl. J. Med. 1996, 335, 1342-1349.

(29) Ruzich, R. Postgrad. Med. 1992, 92, 85-89.(30) Adams, J.; Abendschein, D.; Jaffe, A. Circulation 1993, 88, 750-763.

Figure 3. (a) Plots of fluorescence intensity versus relative waveguide position for monoclonal capture antibody areas specific to (left to right)bSA, hCG, and gIgG. (b) Three plots of integrated intensity versus time for the separate monoclonal capture channels.

Table 4. Comparison of SAIA and MAIA BindingConstants and Nonideality Indexes for the Polyclonaland Monoclonal Antibody Systemsa

K (nM-1) R

analyte SAIA MAIA SAIA MAIA

polyclonalgIgG-FITC 1.40 ( 0.10 1.60 ( 0.10 1.10 ( 0.07 1.11 ( 0.10rIgG-FITC 0.12 ( 0.01 0.05 ( 0.00b 1.02 ( 0.08 0.51 ( 0.02c

hIgG-FITC 0.11 ( 0.01 1.59 ( 0.21b 1.06 ( 0.07 0.72 ( 0.06c

monoclonalbSA-FITC 0.76 ( 0.10 0.54 ( 0.07 0.87 ( 0.06 0.98 ( 0.10gIgG-FITC 0.28 ( 0.04 0.24 ( 0.03 1.00 ( 0.06 1.01 ( 0.04Av-FITC 0.13 ( 0.01 0.16 ( 0.02 0.79 ( 0.09 0.97 ( 0.05

a Abbreviations: R, nonideality index; K, binding constant. b Statis-tically different from corresponding SAIA value. c Statistically differentfrom 1.


for the cardiac-specific SAIA and MAIAs, respectively. Theresponses between assay formats follow similar trends, except forthe SAIA measurement of cTnI, which was assayed over a muchwider range and thus displayed inflection in the transition fromlower to higher analyte concentrations. Analytical sensitivities werecalculated (Table 5) according to the clinical chemistry definition,i.e., average background value plus two standard deviationsdivided by assay sensitivity. Analytical sensitivities were between3 and 15 ng/mL for the SAIA format, while operating in the MAIAformat caused the analytical sensitivity to rise significantly in allcases. Since these data do not represent complete isotherms but

only clinically significant ranges, binding and nonideality constantswere not calculated.

DISCUSSIONIn general, the individual channels for MAIA were addressable

when either polyclonal and monoclonal antibodies were used.However, the polyclonal rIgG capture antibody was cross reactivewith the single-analyte gIgG solution and the monoclonal bSAantibody was cross reactive with the single-analyte gIgG solution.The results further suggested that MAIAs with monoclonalantibodies were more selective than polyclonal antibodies asjudged by comparison with SAIA results. Monoclonal antibodieswere also more well behaved, according to the nonidealityconstant. Two of the three polyclonal antibodies displayedsignificant nonideality, indicative of cross-reactivity, nonspecificbinding, and/or, polyvalent binding. The results from the cardiacanalyte MAIAs also agreed well with SAIA results, although theanalytical sensitivity for two of the three analytes rose significantly.

Figure 4. (a) Plots of fraction bound (Fb) versus bulk analyteconcentration in SAIA format using polyclonal capture antibodies togIgG, rIgG, and hIgG. (b) Fb versus bulk analyte concentration inMAIA format using polyclonal capture to gIgG, rIgG, and hIgG.

Table 5. Analytical Sensitivities for the Cardiac Assaysin Both Single-Analyte and Multianalyte ImmunoassayFormats

analyte SAIA (ng/mL) MAIA (ng/mL)

CK-MB 2.8 4.5cTnI 15.9 26.9Mb 7.0 33.8

Figure 5. (a) Plots of fraction bound (Fb) versus bulk analyteconcentration in SAIA format using monoclonal capture antibodiesto bSA, gIgG, and avidin (Av). (b) Fb versus bulk analyte concentrationin MAIA format using monoclonal capture antibodies to bSA, gIgG,and Av.


Using the information contained in Table 3, it is possible toexpress the channel addressability data (Figures 2 and 3) into asignal-to-background ratio (SBR) format by dividing the channel-specific integrated intensities in each row by the tabulatednonspecific integrated intensity. The SBR measures how muchgreater an analyte signal is compared to the nonspecific back-ground level. Summing the corresponding SBR values down eachcolumn provides a total SBR level for each capture area. Thepercentage of an analyte’s SBR to the total capture area SBR is ameasure of the influence a particular analyte has on a givencapture area.

Figure 7 graphs the SBR calculations for both polyclonal andmonoclonal arrays. Considering both experiments, there does notseem to be a dependency on capture area position (advancingfrom the incoupling site toward the injection site is from left toright) or analyte injection order (number above each column) withpercent SBR. It is apparent that analyte binds primarily to thetarget channel; i.e., the tallest column of each set corresponds tothe specific analyte of the capture area (except for the RbSAchannel, which is discussed below). The SBR values for theaddition of gIgG to the polyclonal capture array suggest that thecross-reactive component of gIgG with the rIgG antibodies wasnot as pronounced as originally portrayed by the raw data inFigure 2.

Figure 7 also highlights two interesting features that appearedin the RbSA channel with monoclonal capture antibodies. First,the SBR produced by the bSA analyte with its capture layer wasabout 3 times less than that of hCG and IgG analytes with theirspecific capture areas. Although the maximum fluorescenceattained by the RbSA capture area (Table 3) was similar to theprevious two analyte responses, nonspecific binding across theentire sensor surface decreased the final SBR. This result wasnot completely unexpected, as bSA is known to be a surface-activeanalyte, especially at high concentrations.31 Improvements insurface chemistry to reduce nonspecific binding could potentiallyminimize or prevent this effect. (See for instance, http://www.biotul.com.) Second, the RbSA antibodies appeared to actively bindanalyte from the IgG sample (striped column). A trivial, butpossible, explanation for the false positive displayed by the RbSAantibodies to IgG can be attributed to residual bSA in the IgGstock solutions. Even though the purification process was assessedby SDS-PAGE (data not shown), not every stock solution’s puritywas verified.

From the comparison of MAIA results with SAIA results, it isclear that two of the three polyclonal capture areas were notsuitable when used together in MAIA. Specifically, both the affinityand nonideality constants (Table 4) for rIgG and hIgG captureantibodies point to some form of cross-reactivity between analytesand capture antibodies. One possible explanation for this behaviorcould be that antibody complexes, which form in the mixing andbinding stages of the assay, may attach to the capture areas withenhanced affinity.

Since the assays were performed over identical concentrationsbetween MAIA and SAIA, point-to-point correlations were calcu-lated, as a function of MAIA fraction bound versus SAIA fractionbound, to further compare the reponses for polyclonal andmonoclonal antibody capture layers (Table 6). Correlations forthe hIgG and rIgG antibodies are not quite significant (R2 < 0.90),suggesting that the relationship between SAIA and MAIA is notnecessarily linear. All other correlations for the polyclonal andmonoclonal antibody test assays performed in buffer weresignificantly linear and demonstrated one-to-one agreement (i.e.,slope, 1) between SAIA and MAIA formats.

(31) Ahluwalia, A.; Giusto, G.; De Rossi, D. Mater. Sci. Eng. C 1995, 3, 267-271.

Figure 6. Dose-response curves for the cardiac analytes CK-MB,cTnI, and Mb in both (a) SAIA and (b) MAIA formats.

Figure 7. Comparing the SBRs achieved with polyclonal antibodiesand monoclonal antibodies. Each bar represents one analyte and thepercentage of total intrachannel SBR it achieves during the experi-ment. The number above each set of bars indicates the order ofanalyte injection while the spatial arrangement of the capture areasis identical to their position on the IOW.


Serum albumin is the most prevalent blood plasma protein andhas the greatest potential to interfere with assay results.32 In anattempt to simulate the interfering effect of dilute serum, MAIAsusing monoclonal antibodies were conducted with 10% pSA addedto the injected analyte/tracer solution (Table 2). The correlationvalues for a MAIA run with monoclonal antibodies in buffer versusan identical MAIA run in 10% pSA are also listed in Table 6. Onlythe avidin channel displayed departure from one-to-one correlation.In this case, the added serum component impeded response; i.e.,the signal derived from buffer was 1.17 times that from the serummatrix over the range of concentrations tested (30 pM to 0.1 mM).Herron et al. performed a SAIA for CK-MB over the 0-100 ng/mL range in whole serum and observed a 60% decrease in assaysensitivity.33 A likely cause for this change in sensitivity is adecrease in analyte transport due to the viscosity of the serumsolution, possibly leading to the 15% decrease in the response foravidin. A similar decrease was observed for the cTnI cardiacanalyte.

The assayed cardiac samples were within the clinically sig-nificant ranges for assessing myocardial infarction, i.e., 0.5-100ng/mL for cTnI, 1.5-100 ng/mL for CK-MB, and 5-500 ng/mLfor Mb. However, detection limits were near to, or less than, 10ng/mL for all analytes in SAIA format (Table 5). When performedin MAIA format, the detection limit for CK-MB and cTnI rose byabout 60% while that for Mb increased by a factor of 4. As forcomparisons, to our knowledge, there are no published results

for a CK-MB, cTnI, or Mb assay on an integrated opticalwaveguide. The CK-MB and cTnI detection limits can be com-pared to those obtained by Herron et al.33 using a thick-filminternal reflection element. Our device performs comparably forCK-MB (2.8 ng/mL SAIA and 4.5 ng/mL MAIA vs 2.3 ng/mL)and less well for cTnI (15.9 ng/mL SAIA and 26.9 ng/mL MAIAvs 0.2 ng/mL). The 2 orders of magnitude difference for the cTnIdetection limit is difficult to explain, even in light of the fact thatno accepted convention exists for the conversion of cTnI inter-national units to true mass units.

CONCLUSIONSThe goal of this work was to demonstrate that MAIA was

possible on a planar SiON IOW using the methods of spatialarrangement, physical adsorption, and fluorescent tracer antibod-ies. Channel addressability experiments showed that it was,indeed, possible to separately activate each capture area intendedfor use in a MAIA. Comparison of SAIA and MAIA affinityconstants revealed that two of the six model capture antibodies,both of which were polyclonal, bound analyte differently betweenformats. Further, the nonideality constants of these two antibodieswere significantly different from the theoretical value and a point-to-point correlation between formats for each antibody did not infera linear relationship. Two of the analytes showed a decrease inassay sensitivity with the addition of a serum component. Of thethree cardiac analytes tested, only assays for CK-MB showed acomparable detection limit between formats while those for cTnIand Mb were adversely affected by the MAIA format. Theseresults suggest that polyclonal antibodies are prone to cross-reactivity in MAIA and that nonspecific binding can lead todegradation in the quality of MAIA, particularly in the presenceof serum. The solution to the latter lies in carefully controlledsurface chemistry.

ACKNOWLEDGMENTWe gratefully acknowledge support from NIH Grant HL 32132,

NSF grant BES-9402355, the NSF-sponsored Duke/North CarolinaCenter for Emerging Cardiovascular Technologies, the Center forBiopolymers at Interfaces at the University of Utah, and LumenalTechnologies of Salt Lake City, UT. The authors also thankanonymous reviewers for identifying critical omissions in theliterature review.

Received for review February 17, 1999. Accepted June 23,1999.

AC990183B

(32) Werner, M.; Dietz, Ed. Handbook of Clinical Chemistry; CRC Press: BocaRaton, 1989.

(33) Herron, J.; Wang, H.-K.; Terry, A.; Durtschi, J.; Tan, L.; Astill, M.; Smith,R.; Christensen, D. Proc. SPIE 1998, 3259, 54-64 (Systems and Technolo-gies for Clinical Diagnostics and Drug Delivery).

Table 6. Correlation Values between Various AssayFormats

fraction bound MAIA vs fraction bound (R2)

analytepAb SAIA

(n ) 3)mAb SAIA

(n ) 3)

10% pSA,mAb MAIA

(n ) 3)

hIgG 1.39 ( 0.19 (0.89)a

rIgG 0.67 ( 0.10 (0.42)a

gIgG 1.00 ( 0.09 (0.98) 1.00 ( 0.11 (0.99) 0.95 ( 0.08 (0.92)bSA 1.03 ( 0.09 (0.93) 0.97 ( 0.01 (0.99)av 0.86 ( 0.14 (0.95) 1.17 ( 0.04 (0.98)b

CK-MB 0.72 ( 0.21 (0.97)cTnI 1.16 ( 0.03 (0.99)b

Mb 0.96 ( 0.36 (0.99)

a Linear fit not necessarily valid. b Statistically different from 1.


multiple-analyte fluoroimmunoassay using an integrated optical waveguide sensor

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