selective binding of polychlorinated biphenyl congeners by a monoclonal antibody: analysis by...

Selective Binding of Polychlorinated BiphenylCongeners by a Monoclonal Antibody: Analysis byKinetic Exclusion Fluorescence ImmunoassayYa-Wen Chiu,†,§ Qing X. Li,*,‡ and Alexander E. Karu†

Department of Nutritional Sciences, University of California, Berkeley, California 94720, and Department of MolecularBiosciences and Biosystems Engineering, University of Hawaii at Manoa, Honolulu, Hawaii 96822

A previously described monoclonal antibody, S2B1, washighly selective for coplanar (non-ortho-chlorinated) PCBcongeners in enzyme immunoassays that measured bind-ing at equilibrium. In the present study, kinetic exclusionfluoroimmunoassay (KinExA) was used to determine thedissociation constants (Kd) and on and off rates (kon, koff

) for binding of various PCB congeners to affinity-purifiedS2B1 IgG and Fab fragments in solution. This methodrevealed that mono- and di-ortho-chlorinated PCBs werebound by S2B1, but the on rates were slower, and the offrates faster by 6-60-fold, than with congeners that hadno ortho chlorines. Although the sensitivity of immunoas-says may be improved by using competing haptens thatS2B1 binds more weakly than the parent PCB, theKinExA results demonstrate that congener specificity isan intrinsic property of S2B1 and does not require weakerbinding haptens. KinExA also provided new informationon the percentage of active binding sites, valence, andeffects of buffer, solvent, and biotinylation on S2B1. Theadvantages and drawbacks of KinExA for measuringantibody-ligand binding are described.

Polychlorinated biphenyls (PCBs), originally valuable for manyindustrial applications, are now known to be among the mosthazardous and widely distributed synthetic substances. Com-mercial PCB formulations, synthesized by controlled chlorinationof biphenyl, may be thought of as combinatorial libraries, withvarious mole fractions of congeners that differ in number andposition of the chlorine atoms. Congeners with and without orthochlorines differ in their most energetically favored conformationsbecause ortho chlorines exert a repulsive force that causes rotationof the phenyl rings about the bond that connects them. Of the209 congeners possible with 1-10 chlorines, 20 have no orthochlorines, and their phenyl rings can lie in the same plane.1-3

The coplanar congeners 3,4,3’,4’-tetrachlorobiphenyl (PCB 77)and 3,4,3’,4’,5’-pentachlorobiphenyl (PCB 126) mimic the size and

shape of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), one of themost toxic organic compounds known. PCBs 77, 126, and 169,and, to a lesser extent, other coplanar congeners, bind to the arylhydrocarbon receptor (AhR), estrogen receptors, and otherproteins. Most of their toxicological effects appear to originatefrom these binding events.4-7 Noncoplanar PCBs are much moreabundant and diverse than the coplanar congeners in commercialPCB formulations, and their toxicity mechanisms are not yet aswell defined.8-11 Thus, the ability of congeners to assume thecoplanar conformation provides a structural basis for differenttoxicological modes of action.

Congener-specific PCB analytical methods are the most neededtools for contamination surveys, risk assessment, remediationmonitoring, and toxicological and epidemiological research.12-15

Congener-specific instrumental analysis is expensive, time-consuming, and requires skilled personnel, specialized recoveryand cleanup methods, and sophisticated data interpretation.2,15,16

Immunoassays and immunoaffinity methods are an attractivealternative. PCB immunoassay kits, validated by the U.S. Envi-ronmental Protection Agency (EPA) for particular uses, arecommercially available and increasingly used for regulatorypurposes.17-20 However, the kits are not congener-specific.

* Corresponding author: (phone) 808-956-2011; (fax) 808-956-5037; (e-mail)[email protected].

† University of California, Berkeley.‡ University of Hawaii at Manoa.§ Current address: Environmental Protection Administration, Heng-Yang

Road, Taipei, Taiwan, R.O.C.(1) McKinney, J. D.; Singh, P. Chem.-Biol. Interact. 1981, 33, 271-283.(2) Creaser, C. S.; Krokos, F.; Startin, J. R. Chemosphere 1992, 25, 1981-2008.(3) Egolf, D.; Jurs, P. Anal. Chem. 1990, 62, 1746-1754.

(4) Kafafi, S. A.; Afeefy, H. Y.; Ali, A. H.; Said, H. K.; Ad-Elazem, I. S.; Kafafi, A.G. Carcinogenesis (Oxford) 1993, 14, 2063-2071.

(5) Kafafi, S. A.; Afeefy, H. Y.; Ali, A. H.; Said, H. K.; Kafafi, A. G. Environ.Health Perspect. 1993, 101, 422-425.

(6) Safe, S. CRC Crit. Rev. Toxicol. 1984, 13, 319-396.(7) Safe, S. CRC Crit. Rev. Toxicol. 1994, 24, 87-149.(8) Tilson, H. A.; Kodavanti, P. R. Neurotoxicology 1997, 18, 727-743.(9) Shain, W.; Bush, B.; Seegal, R. Toxicol. Appl. Pharmacol. 1991, 111, 33-

42.(10) Hansen, L. G. Environ. Health Perspect. 1998, 106, 171-189.(11) Research Triangle Institute. Toxicological profile for polychlorinated biphenyls;

U. S. Department of Health and Human Services, Public HealthService, Agency for Toxic Substances and Disease Registry, Atlanta, GA,1997.

(12) McFarland, V. A.; Clarke, J. U. Environ. Health Perspect. 1989, 81, 225-239.

(13) Wolff, M. S.; Camann, D.; Gammon, M.; Stellman, S. D. Environ. HealthPerspect. 1997, 105, 13-14.

(14) Kodavanti, P. R. S.; Tilson, H. A. Neurotoxicology (Little Rock) 1997, 18,425-441.

(15) Mes, J.; Conacher, H. B. S.; Malcolm, S. Int. J. Environ. Anal. Chem. 1993,50, 285-297.

(16) Erickson, M. D. Analytical chemistry of PCBs, 2nd ed.; CRC/Lewis Publish-ers: Boca Raton, FL, 1997.

(17) U.S. EPA Office of solid waste and emergency response (OSWER). Screeningfor polychlorinated biphenyls by immunoassay (Method 4020); 1996; http://www.epa.gov: 80/epaoswer/hazwaste/test/4020.pdf.

Anal. Chem. 2001, 73, 5477-5484

10.1021/ac0102462 CCC: $20.00 © 2001 American Chemical Society Analytical Chemistry, Vol. 73, No. 22, November 15, 2001 5477Published on Web 10/06/2001

Immunoassay of individual PCB congeners or small groupsof congeners poses a very challenging problem in biomolecularrecognition. The hydrophobicity and similar shapes and electro-static charge distributions of PCBs leave very few options fordeveloping congener-specific antibodies and immunoassays byconventional methods.21-23 Antibody engineering is a possiblealternative, but more must be learned about PCB-antibodyinteractions. In 1995, we reported the derivation and propertiesof a monoclonal hybridoma antibody (MAb), S2B1, that was highlyspecific for coplanar PCBs 77 and 126 in competition ELISAs(cELISAs).24 More recently, we described the molecular cloning,DNA sequence, and performance in cELISAs of a recombinantFab fragment (rFab) from S2B1, which was functionally identicalto the MAb.25 Sequence comparisons and computational modelingprovided a scientifically plausible three-dimensional structure andsuggested bonding interactions in the PCB binding site. In thepresent paper, we describe additional data on the kinetics of PCBcongener binding to S2B1 IgG and Fab in solution. These resultsare proving helpful for testing predictions from the models, suchas orientation of PCB 126 in the binding pocket and involvementof framework amino acid side chains in binding (Pellequer, J-L;Roberts, V. A.; et al., in preparation), and genetically engineeringvariant rFabs with new useful binding properties.

Although we might have used any of several classical affinitydetermination methods, a new type of flow injection fluorescenceimmunoassay instrument became available at the time we beganthese experiments. This method and the instrument used for itare known as kinetic exclusion immunoassay (KinExA). Its majoradvantage is that it measures true liquid-phase equilibriumdissociation constants (Kd) and association rate constants (kon)using very small amounts of antibody. These experiments weredone to learn more about the congener selectivity of MAb S2B1,rather than to compare KinExA with other methods. However,KinExA reproducibly gave us the data we sought, and someaspects of its use are presented.

MATERIALS AND METHODSSafety Precautions. All PCB-containing solutions, solid wastes,

and containers were handled and disposed of as previouslydescribed.25 Protective glasses, gowns, and double nitrile gloveswere worn.

Reagents and PCB Reference Standards. Spectrogradeorganic solvents and deionized glass-distilled water were usedthroughout. All reagents were obtained from Aldrich ChemicalCo. (Milwaukee, WI), Fisher Scientific (Pittsburgh, PA), or SigmaChemical Co. (St. Louis, MO) unless otherwise indicated. PCBcongeners, > 99% purity, from AccuStandard, Inc. (New Haven,CT) were used to prepare reference solutions of 200 ppm (PCBs77 and 126) in 2-propanol and 100 ppm (PCBs 35, 52, and 70) inmethanol. These were stored at 4 °C in glass vials with Teflon-lined screw caps. The detecting antibody used for KinExAmeasurements was fluorescein isothiocyanate (FITC)-conjugatedgoat antimouse IgG H+L chain antibody (Catalog No. 115-095-003, Jackson Immunoresearch Labs, West Grove, PA). Aliquotswere diluted to 0.5 mg/mL in 50% glycerol and stored at -20 °C.

Buffers for KinExA and Enzyme Immunoassay (ELISA).Beads for KinExA were adsorptively coated with hapten-BSA orhapten-cytochrome c conjugates as described below. The coatingbuffer was 0.015 M Na2CO3-0.035 M NaHCO3-0.003 M NaN3,pH 9.6, Blocking buffer and diluent were 0.1% bovine serumalbumin (BSA)-0.1% NaN3n-0.005% Tween 20-10% methanol inphosphate-buffered saline (PBS).25 Washing buffer was PBScontaining 0.1% BSA-0.1% NaN3-0.005% Tween 20, pH 7.4. DirectcELISAs were done in the streptavidin-biotin format as previouslydescribed,24 with PBS-0.01% gelatin-0.005% Tween 20 as blockingbuffer, and this buffer with 5% (v/v) methanol as diluent. ThisTween concentration was one-tenth of that used in most immu-noassays. As previously reported, higher concentrations of Tween20 adversely affect assays with MAb S2B1.24

Haptens and Conjugates. The previously described haptenderivatives, 6-[(3,3′,4-trichlorobiphenyl-4-yl)oxy]hexanoic acid (hap-ten I) and 4-(3,4-dichlorobenzoyl)butyric acid (3,4-keto),24 werenewly synthesized and purified, and their structures were veri-fied.25 Portions were converted to active esters and conjugated toBSA and horseradish peroxidase (HRP) as before.24 Figure 1shows the general structure of PCBs, the hapten derivatives, andthe chlorination patterns of the congeners used in this study.

Purification and Biotinylation of S2B1 IgG and FabFragments. S2B1 IgG was affinity-purified on protein A-Sepharose,IgG concentration was determined from the absorbance at 280nm, Fab fragments were prepared by papain digestion of affinity-purified S2B1 IgG using an Immunopure Fab preparation kit(Pierce Chemical Co., Rockford, IL), and portions of MAb and

(18) U.S. EPA Region I: New England Quality Assurance Unit Staff, Immunoassayguidelines for planning environmental projects (QA Unit Fact Sheet 10/96;New Immunoassay Guidance), 1996; http://www.epa.gov/region01/measure/ia/iaguide.html.

(19) U.S. EPA. Immunoassay: Principles, Procedures, Advantages, and Limita-tions. In U.S. EPA Field Analytical Technology Evaluation Encyclopedia(FATE). U.S. EPA Technology Innovation Office, 1999; http://fate.clu-in.org/immunoassay_index.asp?techtypeid)45.

(20) Hurt, A. Field Analytical Measurement Technologies, Applications, andSelection; California Military Environmental Coordination Committee, Cali-fornia State Water Quality Control Board, Sacramento, CA, 1996.

(21) Carlson, R. E. In Immunoanalysis of Agrochemicals: Emerging Technologies;Nelson, J. O., Karu, A. E., Wong, R. B., Eds.; ACS Symposium No. 586;American Chemical Society: Washington, DC, 1995; pp 140-152.

(22) Franek, M.; Hruska, K.; Sisak, M.; Diblıkova, I. J. Agric. Food Chem. 1992,40, 1559-1565.

(23) Goon, D. J. W.; Nagasawa, H. T.; Keyler, D. E.; Ross, C. A.; Pentel, P. R.Bioconjugate Chem. 1994, 5, 418-422.

(24) Chiu, Y.-W.; Carlson, R. E.; Marcus, K. L.; Karu, A. E. Anal. Chem. 1995,67, 3829-3839.

(25) Chiu, Y.-W.; Chen, R.; Li, Q. X.; Karu, A. E. J. Agric. Food. Chem. 2000, 48,2614-2624.

Figure 1. Structure and nomenclature of PCB congeners andhapten derivatives used in this study: Generic PCB structure (topleft); IUPAC number and chlorination positions (bottom left); haptenI (top right); 3,4-keto (bottom right).

5478 Analytical Chemistry, Vol. 73, No. 22, November 15, 2001

Fab were biotinylated, all as previously described.24,26 Aliquotswere stored at -70 °C.

Adsorption of Hapten-BSA Conjugates to Beads. Poly-(methyl methacrylate) (PMMA) beads and polystyrene-12%divinylbenzene (PS-DVB) beads of 98-µm diameter were obtainedfrom Sapidyne Instruments, Inc. (Boise, ID). Dry beads (200 mg)in 1.5-mL Eppendorf tubes were suspended and settled twice in 1mL of 6 N NaOH and then three times successively in glassdistilled water, PBS, and coating buffer. They were then suspendedin 1 mL of coating buffer containing 0.2 mg of hapten I-BSA or3,4-keto-BSA and rolled at room temperature for 3 h. After theconjugate solution was discarded, the beads were washed threetimes with PBS, then resuspended in blocking buffer, and rolledagain at room temperature for 1 h. Coated bead preparations inblocking buffer were stable for 7-10 days at 4 °C.

All experiments were done with 3,4-keto-BSA-coated PS-DVBbeads, unless otherwise indicated. On the day of use, 200-mgaliquots of coated beads were uniformly suspended in 29 mL ofPBS to provide the concentration required for KinExA. For eachsample analyzed, a new bed of hapten conjugate-coated beadsexactly 4 mm high (to match the width of the excitation beam)was deposited over the mesh trap in the capillary flow cell. Thebed height proved very important for reproducibility and, thus,was checked with a small metal template before each run.

KinExA Principles and Operation. KinExA is a flow injectionsystem designed to measure the unoccupied binding sites afteran antibody and analyte are allowed to react in solution. Aschematic of the device and principles of operation were describedon the manufacturer’s Web site (www.sapidyne.com) and by Blakeet al.27,28 The antibody-analyte mixture is pumped through acapillary flow cell of 1.6-mm inside diameter, at the bottom ofwhich is a bed of beads, 4 mm deep, coated with hapten-proteinconjugate, retained on a very fine mesh. The beads lie in the pathof a fluorescence excitation beam and an emission detector. Asthe sample, in about 50 µL, is passed through the beads, antibodieswith unoccupied binding sites are bound. This occurs so quickly(on the order of 240 ms when the flow rate is 0.5 mL/min) thatthere is negligible time for an antibody molecule to release fromthe hapten and reassociate with soluble ligand. The test antibodyattached to the beads is exposed to excess fluorescent-labeledgoat anti-mouse secondary antibody and then unbound secondaryantibody is washed away, and the fluorescence that remains onthe beads (directly proportional to the amount of primaryantibody) is measured. When these steps are completed, the beadsare automatically pumped out and replaced with a new aliquotfor the next measurement.

All experiments were done on a first-generation instrumentgenerously loaned by Dr. Steve Lackie (Sapidyne Instruments,Inc., Boise, ID). The KinExA was controlled, and data wereacquired through an IBM-compatible personal computer with Intel486 or later microprocessor, running Microsoft Windows. Theoperating parameters (volume, flow rate, sequence, time interval)

were configured and run by Sapidyne’s control software. Experi-mental data were imported into a Microsoft Excel spreadsheet.

Experimental Procedures. Total protein was determined byUV spectrophotometry or by micro-BCA assay (Pierce Chemicals,Rockford, IL), using crystalline BSA as a standard. The percentageof active binding sites was estimated from a Scatchard plot andalso from an iterative nonlinear fit of the percent of unoccupiedsites versus site concentration. To determine Kd, various amountsof antibody and PCB analyte were mixed and allowed to reachequilibrium for 30 min at room temperature, and then the solutionswere passed through the KinExA’s beads to capture and quantifythe unoccupied binding sites. An estimate for Kd was calculatedby iterative least-squares fit of the parameters [A0], [B0], Sig0%,and Sig100%, corrected for the percentage of functional binding sites.To measure kon, a fixed amount of antibody and various concentra-tions of analyte were mixed by simultaneous injection, and themixture was passed through a tube of known length, and throughthe haptenated beads, at a predetermined rate. Each Kd determi-nation was made with five different PCB concentrations intriplicate, i.e., 15 separate runs. The kon determinations were madewith 8-12 concentrations of PCB congeners in triplicate, rangingfrom 0 to 100% binding site occupancy. The koff values werecalculated; koff ) konKd.29

Data Analysis. Analyses and curve fitting were done inMathCad Plus 6.0 (MathSoft, Inc., Cambridge, MA), on a Macin-tosh PowerPC computer, using worksheets developed by Sapi-dyne. The calculations and theoretical basis have been extensivelydescribed in the literature.29-31 Three types of determinations weremade: estimated percentage of functional binding sites, equilib-rium dissociation constant (Kd), and kon. The instrument’s fluo-rescence signal was related to antibody and ligand concentrations

by the classical binding equation, [A] + [B] y\zkon

koff[AB], where the

concentrations of analyte, active antibody binding sites, andantibody-analyte complex are [A], [B], and [AB], respectively.Kd, is defined as koff/kon. Therefore, [AB] ) [B][A]/Kd. Atequilibrium, the total analyte concentration [A0] ) [A] + [AB],and the total concentration of functional binding sites [B0] ) [B]+ [AB]. The experimental data are the fluorescence signals, inmillivolts, measured at various values of [A]. The equation thatrelates the instrument readings to molecular species and kineticparameters is

where signal is the instrument signal for [B] and Sig0% is the signalwhen [B] ) 0, i.e., the baseline nonspecific background when noprimary antibody is bound to the haptenated beads. Sig100% is themaximum signal when no soluble analyte-antibody complex was(26) Harlow, E.; Lane, D. Antibodies: A Laboratory Manual; Cold Spring Harbor

Laboratory; Cold Spring Harbor, NY, 1988.(27) Blake, D. A.; Khosraviani, M.; Pavlov, A. R.; Blake, R. C. In Immunochemical

Technology for Environmental Applications; Aga, D. S., Thurmann, E. M.,Eds.; ACS Symposium Series 657; American Chemical Society: Washington,DC, 1997; pp 49-60.

(28) Blake, R. C., 2nd; Pavlov, A. R.; Blake, D. A. Anal. Biochem. 1999, 272,123-134.

(29) Day, E. D. Advanced Immunochemistry, 2nd ed.; Wiley-Liss: New York, 1990;pp 298-299.

(30) Steward, M. W.; Steensgard, J. Antibody affinity: Thermodynamic aspectsand biological significance; CRC Press: Boca Raton, FL, 1983; pp 5-11.

(31) Glass, T. R.; Lackie, S. Kinetic Exclusion Fluoroimmunoassay (KinExA); 1997;http://www.sapidyne.com/.

signal )Sig100% - Sig0%

2[Bo](([Bo] - Kd - [Ao]) +

x[Bo]2 + 2[Bo]Kd - 2[Bo][Ao] + Kd2 + 2[Ao]Kd + [Ao]2) +

Sig0%

Analytical Chemistry, Vol. 73, No. 22, November 15, 2001 5479

formed, and all of the active antibody ([B0]) is captured on thebeads.29-31

For calculation of kon, the Kd, molar concentration of activebinding sites, and the time allowed for the analyte and antibodyto react were all known. The data were fitted using an iterativenonlinear least-squares algorithm, as in the Kd determination. The95% confidence limits for [B0] (concentration of active bindingsites), Kd, and kon, were computed by varying the best-fit valueand reoptimizing the remaining variables at each point, using analgorithm developed by Sapidyne.32

RESULTS AND DISCUSSIONThe PCB-MAb S2B1 interaction is particularly interesting

from the standpoint of structural biology, because binding mayinvolve a conformational change of the ligand, part of the antibody,or both. In this study, the primary target analytes were thecoplanar PCBs 77 and 126, as defined by the specificity of MAbS2B1 and its proteolytic Fab fragments in cELISAs.24,25 The haptenderivatives used as competitors were the structures chemicallydefined in Materials and Methods, depicted in Figure 1, andabbreviated as hapten I and 3,4-keto for consistency with ourearlier papers. The experiments described here were done (a) toobtain quantitative data for the differences in binding of PCBcongeners and hapten derivatives that resulted in the highselectivity for coplanar congeners in ELISAs and (b) to comparethe binding properties of whole IgG and Fab fragments and choosean optimal hapten derivative for panning display phage to selectrFabs.

Most present-generation immunoassay methods required apolyclonal antiserum or MAb that was empirically found to havestrong binding and high selectivity. By contrast, kinetic analysishas become essential to support the recent advances in antibodyengineering, selection from combinatorial libraries, multianalyteimmunoassays, discrimination within large groups of chemicalanalogues such as PCBs, and sensor applications that involvetemporal as well as end point binding. The KinExA method wasattractive for two reasons. First, only nanogram-to-microgramamounts of antibody, analyte, and hapten conjugate were neededfor each measurement; sample volumes could be as small as 50µL. Second, KinExA directly measured bimolecular, solution-phasebinding kinetics, without the expense and artifacts possible whenantibody or hapten conjugate is immobilized in surface plasmonresonance (SPR) and resonant mirror instruments such asBIAcore and IASYS.33-39

Technical Aspects of KinExA Use. Some standard KinExAprotocols had to be modified for experiments with the PCB-hapten conjugates and hydrophobic PCBs. PMMA beads re-mained monodisperse when coated with hapten I-BSA, but theyclumped if coated with 3,4-keto-BSA. PS-DVB beads did not

clump when coated with 3,4-keto-BSA. Clumping causes unevenbead distribution, inconsistent exposure of ligand for antibodybinding, and unacceptable variance in baseline and maximumfluorescence (Sig100%) values. The amount of beads automaticallydelivered to the flow cell from the stirred reservoir can be adjustedby varying the rate and duration of withdrawal, but only if thesuspension is monodisperse.

Several pre-and postexperimental procedures proved essentialto maintain precision and reproducibility. These included adegassing cycle to clear air bubbles from the tubes, a procedureto prime the rotary valves, a washing cycle between replicatesand new samples, a “night wash” program to automatically washthe entire system 30 times, and a “morning rinse” to conditionthe system with PBS 3-10 times before starting a series ofmeasurements. To prevent carry-over of antibody and ligand, allruns were begun with the most dilute samples and progressed tothose with higher concentrations. An additional wash was includedbetween measurements of the most concentrated sample fromone datum set and the most dilute sample of the next set. In someexperiments, nonspecific binding in the flow cell caused thebaseline fluorescence to drift upward in each consecutive mea-surement. When it exceeded 0.1 V, experiments were interrupted,and the system was washed three times each with neat methanol,0.1 M NaOH, and PBST.

In the prototype instrument we used, the range of measurablekon values was limited by the available sample injection flow rates(0.25-0.75 mL/min) and the flow path (length of tubing) betweenthe mixing chamber and the flow cell. The kon determinations inthis paper were done using a tube with a minimum transit timeof 12 s. Fortunately, the kon measurements for S2B1 MAb andFab could be obtained by adjusting the antibody and ligandconcentrations so that equilibrium was not reached in 12 s. Otherantibody-ligand combinations that we tested had kon values thatapproached or exceeded 108 M-1 s-1. At the dilutions necessaryto bring those complexes into a measurable time frame, thefluorescence signal was too weak to use (Bell, C. W.; Karu, A. E.,unpublished). The present version of the instrument (KinExA3000) has much broader measurement ranges: 10-2-10-13 M forKd (the reciprocal of affinity), and 103-109 M-1 s-1 for kon. Withinthese bounds, koff values from 10-6 to 1.0 s-1 can be calculated.31

Analysis of PCB Binding. For all KinExA measurements,fluorescence in the bead bed was continuously recorded as afunction of time for each sample. Plots of the raw data resulted in“KinExAgrams” essentially identical to the “sensorgrams” pro-duced by BIAcore (SPR) and IASYS (resonant mirror) instru-ments.37,38 These plots provided real-time information on baselinedrift, monodispersity of the beads, and other factors that affectdata quality. Figure 2 shows the superimposed KinExAgrams forequilibrated mixtures of MAb S2B1 and PCB 126. The signal usedfor analysis was the difference between the baseline (t ) 100-180 s) and the steady-state fluorescence after excess probe waswashed away (t ) 420-500 s). Figure 2 also shows that the netsignal was linearly proportional to the amount of S2B1 IgG (i.e.,active binding site) over the entire concentration range used inthese experiments.

The kinetic analyses were done in three steps: estimation ofthe percentage of active binding sites, determination of Kd, andmeasurement of kon. All methods for determining affinity and rate

(32) Lackie, S.; Glass, T. Kd mathematics; 2000; www.sapidyne.com/kdmath.html.(33) Nieba, L.; Krebber, A.; Pluckthun, A. Anal. Biochem. 1996, 234, 155-165.(34) Schuck, P. Biophys. J. 1996, 70 (A212), 1230-1249.(35) Schuck, P. Annu. Rev. Biophys. Biomol. Struct. 1997, 26, 541-566.(36) Edwards, P. R.; Maule, C. H.; Leatherbarrow, R. J.; Winzor, D. J. Anal.

Biochem. 1998, 263, 1-12.(37) Malmborg, A.-C.; Borrebaeck, C. A. K. J. Immunol. Methods 1995, 183,

7-13.(38) Lowe, P. A.; Clark, T. J.; Davies, R. J.; Edwards, P. R.; Kinning, T.; Yeung,

D. J. Mol. Recognit. 1998, 11, 194-199.(39) Adamczyk, M.; Moore, J. A.; Yu, Z. Methods (Orlando, Fla.) 2000, 20, 319-

328.


constants are affected by the percentage of binding sites thatremain functional after purification, storage, or chemical modifica-tion.30,40,41 This percentage was estimated by comparing resultswhen either the PCB concentration or the protein concentrationwas used as the reference. The theoretical maximum concentra-tion of binding sites was based on total protein concentration.KinExA data were collected on equilibrium solutions containinga fixed amount of S2B1 IgG or Fab (13.3 nM, equal to 2 µg ofIgG/mL), with no PCB, a saturating amount of PCB 77 or 126,and five to eight intermediate concentrations in triplicate. AScatchard plot (the ratio of unoccupied to occupied antibodyversus the amount of occupied antibody) was linear, as expectedfor a MAb binding a single small-molecule analyte (Figure 3). Thex-axis intercept gave the moles of active binding sites per mole ofMAb or Fab, where each molecule has two binding sites. Theactive binding site concentration was also estimated from anonlinear fit of the percent of unoccupied sites versus site

concentration, using Sapidyne’s standard Kd analysis program.Various preparations of S2B1 IgG and proteolytic Fab had from60 to 90% active binding sites. All subsequent experiments weredone with one batch of affinity-purified S2B1 IgG that hadapproximately 75% active sites and one batch of proteolytic FabS2B1 with approximately 67% active sites. These correction factorsreduced the Kd and kon estimates by 10-17%.

Effects of Blocking Protein and Buffer Composition onKinExA. For direct cELISAs with biotinylated S2B1 IgG capturedon streptavidin-coated microwells, the optimal diluent and blockingbuffer was PBS with 0.005% Tween 20 and 0.01% gelatin.24 Whenthis buffer was used in the KinExA, the baseline increased byapproximately 0.035 V per run. This drift was partially eliminatedby using 0.1% BSA instead of gelatin. In many immunoassayformats, surfactants such as Tween 20 are commonly added toPBS at 0.05% (v/v) to reduce nonspecific binding. However,diluents containing 0.01% (v/v) or more Tween 20 significantlyreduced PCB binding by MAb S2B1 in cELISAs.24 KinExA wasaffected even more; the highest usable concentration of Tween20 was 0.005%. The optimum diluent and wash buffer consistedof PBS with 10% methanol, 0.1% BSA, and no surfactant. This keptthe background stable within 0.05-0.1 V over many consecutiveruns.

Solvent Effects. Immunoassays for very hydrophobic analytessuch as PCBs generally require a minimal amount of water-miscible organic solvent, surfactant, or both to keep the analytessoluble. However, larger amounts of these solubilizers oftendegrade assay performance. We previously found that additionof 5-10% methanol (MeOH) or dimethyl sulfoxide (DMSO) tothe diluent changed the selectivity as well as sensitivity of directcELISAs with MAb S2B1.24 When the diluent contained 10%DMSO instead of 5% methanol, the half-maximal inhibition (I50)by PCB 35 was 80 nM, rather than 1240 nM. Similar effectsoccurred in the KinExA. In the standard diluent, PBS-10%methanol-0.1% BSA, 0.05 mL of a 10 nM solution of S2B1 IgG,captured on beads coated with 3,4-keto-BSA, gave a net signalof 0.8 V. This was reduced by approximately 80, 87.5, or 90% ifthe methanol was replaced by 10, 15, or 20% DMSO, respectively,in the antibody capture step. However, substitution of 10% DMSOfor the 10% methanol in the diluent improved the Kd for PCB 35roughly 6-fold, from 2.2 ( 0.01 to 0.37 ( 0.001 nM. Thus, in bothcELISA and KinExA, DMSO in the diluent weakened binding ofS2B1 to the hapten but increased the affinity for the coplanarcongener PCB 35.

Hapten Preference. In KinExA, the Kd of a particularantibody-analyte pair should be independent of the hapten usedto trap unoccupied antibody on the beads. To test this, the Kd forPCB 77 binding by S2B1 IgG was determined in separateexperiments using PMMA beads coated with hapten I-BSA andPS-DVB beads coated with 3,4-keto-BSA (PMMA beads coatedwith 3,4-keto-BSA clumped, as noted above). The difference inKd values, 2.8 ( 0.05 and 1.7 ( 0.03 nM, respectively, was probablynot significant with respect to between-assay variation withseparate preparations of antibody and beads coated with eitherhapten conjugate. Unfortunately, this comparison could not bemade using S2B1 Fab, because it bound 3,4-keto-BSA-coated PS-DVB beads too weakly to allow Kd measurements. This wasconsistent with our previous observation that S2B1 proteolytic and

(40) Karlsson, R.; Fagerstam, L.; Nilshans, H.; Persson, B. J. Immunol. Methods1993, 166, 75-84.

(41) Adamczyk, M.; Mattingly, P. G.; Shreder, K.; Yu, Z. Bioconjugate Chem.1999, 10, 1032-1037.

Figure 2. Example of KinExA output (KinExAgram). Superimposedfluorescence responses from five equilibrium mixtures of S2B1 IgGand various amounts of PCB 126, used for determination of Kd. Thebaseline was established from 0 to 200 s, as unoccupied S2B1 wascaptured by the immobilized haptens. Fluorescein-labeled secondaryantibodies were added over the next 200 s, followed by a wash toremove unbound secondary antibodies. The signal (delta) used forcalculations was the difference between the average values from 450to 550 s and the baseline.

Figure 3. Scatchard plot for estimation of active antibody. Thecrosses are datum points (average of triplicates), and the solid lineis the theoretical best fit. The ordinate is the ratio of bound/free ofS2B1 IgG, the slope is 1/Kd, and the X intercept indicates that nearly100% of the binding sites in this sample are active.


recombinant Fabs did not bind to microplate wells coated with3,4-keto conjugates of BSA and cytochrome c.25

Kinetics of PCB Congener Binding. Table 1 summarizesthe Kd and kon values determined by KinExA, and the koff valuescalculated from them, for MAb S2B1. Also included are Kd valuesestimated from I50 data in a previously reported direct cELISA,24

as described by Muller.42 These are for relative comparison only.The absolute values cannot be compared because direct cELISAsmeasure competition of soluble analyte and hapten for binding toantibody immobilized on a solid phase, and the results dependon the affinity for the competing hapten-enzyme conjugate (3,4-keto-HRP in this experiment). An experiment analogous to directELISA could have been done by KinExA. For example, the bindingof 3,4-keto-HRP to beads coated with MAb S2B1 IgG could havebeen measured with a fluorescent anti-HRP conjugate as thereporter. However, this was peripheral to our main objectives,which were (a) to determine whether KinExA would detectbinding of mono- or di-ortho-chlorinated PCB congeners to S2B1,when ELISA could not, and (b) to select a hapten conjugate forpanning S2B1 rFab displayed on M13 phagesa procedureanalogous to indirect ELISA.

PCBs 77, 126, and 35 have no ortho chlorines, PCB 70 hasone, and PCB 52 has two. The Kd and kon values determined by

KinExA for PCBs 77, 126, and 35 were nearly identical. Bycomparison, PCBs 70 and 52 had weaker affinities, 5-56-foldslower kon, and 5-23-fold faster koff. The data in Table 1 reflectthe two greatest advantages of KinExA, compared with cELISA;KinExA measures binding kinetics in solution, and it is consider-ably more sensitive. In cELISAs, PCB concentrations as high as5 µg/mL (5 parts per million) were required to demonstrate thatMAb S2B1 had roughly 1% cross-reactivity with PCB 70 andnegligible cross-reactivity with PCB 52.24 KinExA gave reproduc-ible Kd and kon estimates at much lower PCB concentrations. TheKd values for PCBs 70 and 52 corresponded to 19 and 672 ng/mL(parts per billion), respectively.

Error Estimates. Figure 4 shows the Kd analysis for PCB 77binding by MAb S2B1 and error estimates that indicate the qualityof the data. Panel A shows the data and best-fit curve. Panels Band C are plots of the residual errors in the fitted Kd and the activebinding site estimates, respectively. The nonlinearity and sharpminimums indicate how sensitive the affinity determination is tothe percentage of active binding sites and other experimentalconditions. Figure 5 is the summary for determination of kon andits estimated error. Panel A demonstrates how the kon estimatebecomes less accurate as the concentration of antibody used inthe experiment approaches the Kd. Panel B shows the upper andlower 95% confidence levels for kon, and steep increase of residual(42) Muller, R. J. Immunol. Methods 1980, 34, 345-352.

Table 1. PCB Binding Characteristics of MAb S2B1

Kd (nM)PCBno. chlorine positions KinExAa ELISAb

kon(M-1 s-1)a × 104

koff(s-1)

77 3,4,3′,4′-tetrachloro 1.7 ( 0.03 24 150 ( 4 0.003126 3,4,3′,4′,5′-pentachloro 2.5 ( 0.01 31 120 ( 6 0.00335 3,4,3′-trichloro 2.2 ( 0.01 1240 120 ( 6 0.002670 2,5,3′,4′-tetrachloro 66 ( 0.6 6900 22 ( 1.4 0.01452 2,5,2′,5′-tetrachloro 2300 ( 20 ncc 2.7 ( 0.05 0.06

a Data are corrected values and are best-fitted for 95% confidence limit. b Direct cELISA with biotinylated S2B1 immobilized on streptavidin-coated microwells, and 3,4-keto-HRP as competitor, as described in Chiu et al.24 c nc, no competition in cELISA by up to 5 ppm of a PCB congener.

Figure 4. Summary data for Kd of S2B1 IgG with PCB 77. A fixed amount of S2B1 IgG was mixed with 0, 2, 4, 6, 8, 10, 12, 14, 16, and 100ppb of PCB 77 in triplicate, as described in Materials and Methods (panel A, left). The plot of binding site occupancy versus antibody concentration(panel A, right) shows the actual data (diamonds; average of triplicate measurements) and the theoretical best-fit curve (solid line). The residualerror of the best-fit values for Kd and active antibody concentration are shown in panels B and C, respectively.


error on both sides of the best fitted value. Other data qualitystatistics included the ratio of antibody concentration to Kd ([Ab]/Kd) in Kd determinations and how far from equilibrium theconditions were in kon determinations. A ratio of [Ab]/Kd > 1indicates that the estimate of binding site concentration wasaccurate, but the Kd determination was less so. A ratio of [Ab]/Kd < 1 indicates that the active binding site concentration estimatewas less reliable, but the Kd value was more accurate, given theavailable data.

Antibody Valence. Theoretically, Kd determinations by Kin-ExA should be independent of antibody valence (the number ofbinding sites per molecule of antibody that can function underthe particular experimental conditions).28 A separate experimentwas conducted to compare the Kd values of S2B1 whole IgG andFab for PCB 77. Equilibrium assays were performed with hapten-1-BSA-coated PMMA beads. Whole S2B1 IgG (10 nM) andproteolytic Fab (30 nM) gave Kd values of 2.8 ( 0.05 and 1.6 (0.03 nM, respectively, corrected for the percentage of activebinding sites. These values, with small intra-assay differences,were close enough to each other, and to the result in Table 1, toexpect that Fab and intact IgG would perform similarly in cELISAsand other applications.

KinExA can be used to verify the quality of chemically modifiedantibodies. A portion of affinity-purified S2B1 IgG was biotinylatedand gave highly reproducible, sensitive results in direct cELISAsfor more than one year.24 The Kd values for binding of PCB 35 byunmodified and biotinylated IgG were 2.2 and 0.68 nM, respec-tively, using beads coated with 3,4-keto-BSA hapten conjugate.Thus, PCB 35 binding by the biotinylated IgG was not significantly

different, or may have been slightly better, within experimentalerror.

CONCLUSIONSThe initial strategy for developing MAbs specific for coplanar

PCB congeners required two components: an immunizing haptenthat most closely mimicked coplanar congeners and a competitorhapten that retained features essential for recognition, but wouldbe bound more weakly than the target PCBs in competitionassays.21,24 In cELISAs, which are based on binding at equilibrium,MAb S2B1 showed no detectable binding of noncoplanar (mono-and di-ortho-chlorinated) PCBs. The KinExA analyses showed thatrepresentative noncoplanar congeners were transiently bound byS2B1 in solution, but the off rates were faster and on rates wereslower than those of the coplanar congeners by 5-60-fold. Thisestablishes that S2B1 is intrinsically congener-selective, and itspotential applications are not limited to competitive binding assayformats. However, use of weaker binding competitor haptensincreased the sensitivity for coplanar congeners, i.e., resulted inlower I50 values in cELISAs.24

KinExA and cELISA revealed different aspects of PCB bindingby S2B1. Ortho-chlorinated PCBs 70 and 52, which gave noobservable binding in cELISAs, gave measurable, reproduciblevalues in KinExA. The coplanar 3,4,3’-trichlorobiphenyl (PCB 35)was only about 2-10% cross-reactive, compared to PCBs 77 and126, in a direct cELISA, yet all three of these congeners behavedsimilarly in KinExA. Comparison of Kd values for one batch ofbiotin-conjugated and unconjugated IgG demonstrated that kineticmeasurements are useful for evaluating the functionality of

Figure 5. Example of results for kon determinations. A fixed amount of MAb S2B1 was injected and mixed with solutions of PCB 77 to givefinal concentrations of 0, 5, 10, 15, 25, 35, 60, and 100 ppb, in triplicate. Panel A shows the input data and calculated results at left. The plotat upper right shows the actual data (diamonds), the theoretical best-fit curve (solid line), and the response predicted if the solutions had reachedequilibrium (dashed line). Panel B shows the residual error and 95% confidence limits of the best-fit kon value.


chemically modified antibodies. Our limited data were consistentwith results on other antibody-antigen combinations reported byAdamczyk et al., using a SPR instrument.41

At present, the most widely used instruments for measuringligand-binding kinetics include those based on surface plasmonresonance (BIAcore), resonant mirror technology (IASYS), andisothermal titration calorimetry (ITC). The BIAcore and IASYSrequire immobilization of antibody or ligand and are subject todiffusion and rebinding artifacts.33-36,38 ITC is gaining use as ameans of getting true solution-phase binding kinetics and ther-modynamic data.43-45 These instruments are all more expensiveto purchase, maintain, and operate than the current model of theKinExA. Despite these shortcomings, the instrumental methodsare more accurate and require less manual work than cELISAs.Several ways to get approximate Kd estimates from solid-phaseradioimmunoassays and ELISAs have been published over manyyears.42,46-53 By far the most accurate method to estimate Kd froman ELISA is to measure the amount of unoccupied antibodyremaining in the liquid phase after equilibration with variousamounts of analyte.52 The procedure is similar to what occurs inKinExA, but more complicated and less reproducible.

The primary advantages of KinExA are the small amounts ofantibody needed (e100 ng in 50 µL per sample), the ability tomeasure the percentage of active antibody, analysis of truesolution-phase Kd and kon with good error estimates over a verywide range on one instrument, and the well-documented, user-

friendly control and data analysis software. When reagents andreaction conditions are optimal, KinExA data are very reproduc-ible, with coefficients of variation less than 5%. Disadvantagesinclude the empirical choice of beads, haptens, and haptenpresentation conditions, tedious procedures to prime and cleanthe system before and after a series of runs, and inability tomeasure kon when the analyte and antibody concentrations neededfor measurable signals would reach equilibrium in less than 2 s.As with all currently available instruments for binding analysis,the KinExA can be run as a flow injection device, but it can onlymeasure one sample at a time. A full analysis cycle for onereplicate, including the steps before and after the actual measure-ment, takes about 15 min. To date, the KinExA has been used tomeasure binding kinetics of MAbs that bind metals,27,54 metalchelates,55-57 opiate metabolites in urine,58,59 chlorophenoxyherbicides,60 and peptide libraries.61 A detailed comparison of eightprocedural and data quality criteria with the nine most commonmethods and instruments for measuring ligand binding kineticsmay be found on the Sapidyne web site (http://www.sapi-dyne.com/systcomp.html).

In previous-generation hybridoma and immunoassay technolo-gies, hapten design was basically the only way to evoke antibodieswith ideal characteristics for recognizing small molecules that havemany analogues. MAb development was, and still is slow,expensive, and influenced by unpredictable biological factors. Inthose circumstances, it was more practical to focus on optimizinghapten structure and immunoassay formats for the availableantibodies, as we did to produce S2B1. With the rapid advancesin antibody engineering and combinatorial library productionguided by computational modeling, target ligands and mimics arestill needed for antibody selection but are no longer required toevoke antibody production. Higher-throughput methods for kineticanalysis are critical for development of recombinant antibodiesand other ligand binding molecules, and instrumentation similarto KinExA is likely to evolve rapidly soon.

ACKNOWLEDGMENTWe thank T. Glass and S. Lackie (Sapidyne Instruments, Boise,

ID) for generously supplying the PMMA and PS-DVB beads,instruments, related software, training, and valuable advicethroughout the experiments. The interpretations and conclusionsherein are not necessarily those of Sapidyne, Inc., and the authorsare solely responsible for any errors. C.W. Bell and T.E. Chinalso provided valuable discussion and helped to develop experi-mental protocols. Portions of this work were included in Y.-W.C.’sPh.D. dissertation, filed at U.C. Berkeley in April 2000. The projectwas supported by a grant from the Consumer and EnvironmentalProtection Division of the Alameda County, California, DistrictAttorney’s Office. A.E.K. was an investigator in the NIEHSEnvironmental Health Sciences Center at the University ofCalifornia, Berkeley (Grant ES01896, Bruce N. Ames, Director).

Received for review February 28, 2001. Accepted August13, 2001.

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selective binding of polychlorinated biphenyl congeners by a monoclonal antibody: analysis by...

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