Biosensors & Bioelectronics 14 (1999) 587–595

The use of regenerable, affinity ligand-based surfaces forimmunosensor applications

John Quinn a, Pritesh Patel b, Brian Fitzpatrick a, Bernadette Manning a, Paul Dillon a,Stephen Daly a, Richard O’Kennedy a,*, Marcos Alcocer c, Heather Lee c,

Michael Morgan c, Kenny Lang d

a School of Biotechnology, Dublin City Uni6ersity, Glasne6in, Dublin 9, Irelandb Department of Chemical Engineering/Biotechnology, Lund Uni6ersity, Lund, Sweden

c Institute of Food Research, Norwich, UKd Biotrin, Mount Merrion, Dublin, Ireland

Received 28 May 1998; accepted 19 May 1999

Abstract

The regeneration of antibody-binding surfaces is of major importance for re-usable sensor formats such as required for direct‘real-time’ biosensing technologies and is often difficult to achieve. Antibodies commonly bind the antigen with high avidity andmay themselves be sensitive to regeneration conditions. The interaction of polyclonal anti-chlorpyriphos antibody with animmobilised chlorpyriphos-ovalbumin (chlor-oval) conjugate and the interaction of soluble recombinant CD4 with covalentlyimmobilised anti-CD4 IgG are presented in order to highlight these difficulties. Affinity-capture is suggested as an alternativeformat as it facilitates surface regeneration, directed immobilisation and the attainment of interaction progress curves thatconform to the ideal pseudo-first-order kinetic interaction model. Protein A, protein G and polyclonal anti-mouse Fc-coatedsurfaces were used to observe the interaction of captured anti-GST monoclonal antibody with glutathione-s-transferase (GST). Itwas shown that a protein A affinity-capture surface produced ideal interaction progress curves while both protein G andpolyclonal anti-mouse Fc resulted in systemic deviations. © 1999 Published by Elsevier Science S.A. All rights reserved.

Keywords: Affinity capture; Antibody; CD4; Chlorpyriphos; Glutathione-s-transferase; Immobilisation; Protein A; Protein G; Pseudo-first-order;‘Real-time’ biospecific interaction analysis

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1. Introduction

Direct biosensing technologies combine mass sensi-tive transducers, biointerfacial chemistry and the spe-cificity of a wide range of affinity ligands, to createvaluable analytical and research tools (Lofas et al.,1991; Fortune, 1993). The BIACORE 1000™ employsa refractive index sensitive optical transducer based onsurface plasmon resonance (SPR) for mass detectionand an integrated microfluidic cartridge for delivery ofsample to the biospecific surface. The biospecific sur-face is composed of a carboxymethylated dextran (CM-dextran) hydrogel onto which biomolecules can be

covalently immobilised using well establishedchemistry.

Typically, ‘real-time’ biospecific interaction analysis(BIA) involves immobilising a partner of an affinitypair (e.g. antibody) onto the sensor surface and contin-uous monitoring of surface mass changes on exposureto a solution containing the other partner (e.g. antigen)(Stenberg et al., 1991). Bound antigen can be removedusing chaotrophic solutions, allowing the functionalisedsurface to be used repeatedly. This regeneration processis essential for the attainment of high quality data andis commonly difficult to achieve. Furthermore, im-munological reactions characterised by low dissociationrate constants and multivalent interactions of highavidity are often virtually irreversible (Quinn et al.,1997). Such interactions require extremely harsh regen-eration conditions that lower the surface binding capac-

* Corresponding author. Tel.: +353-01-7045105; fax: +353-01-7045997.

E-mail address: okennedr@ccmail.dcu.ie (R. O’Kennedy)

0956-5663/99/$ - see front matter © 1999 Published by Elsevier Science S.A. All rights reserved.

PII: S 0 9 5 6 -5663 (99 )00032 -9

J. Quinn et al. / Biosensors & Bioelectronics 14 (1999) 587–595588

ity. In practice, a compromise between complete regen-eration and maintenance of analyte binding activitymust be achieved. In addition, direct immobilisation ofantibodies using random amine coupling can lead tosteric hindrance of biomolecular interactions (Lu et al.,1996; Oddie et al., 1997), as evidenced by heteroge-neous binding progress curves that cannot be used toextract kinetic constants (O’Shannessy and Winzor,1996; Edwards and Leatherbarrow, 1997).

Affinity-based immobilisation (affinity-capture) is analternative format that facilitates surface regeneration,directed orientation of the antibody and the attainmentof ideal pseudo-first-order (Hall et al., 1997) bindingprogress curves. Essentially, affinity-based immobilisa-tion involves the non-reversible immobilisation of ahigh-affinity ligand to reversibly anchor the antibody ofinterest to the hydrogel. Replacement of the antibodyafter each interaction-regeneration cycle avoids losses inantigen binding activity, assuming that antibody cap-ture is reproducible. Crude dilute sources of antibodysuch as hybridoma supernatant and diluted serum maybe used. Protein A, protein G and polyclonal anti-mouse Fc antibody are widely used as affinity-captureligands allowing reversible immobilisation of antibodiesto biosensing surfaces (commonly glass or gold) (Mura-matsu et al., 1987). Protein A from Staphylococcusaureus possesses three homologous Fc binding sites thatbind the Fc region of IgG from a variety of mammalianspecies with high affinity (Kd�10−9 M) (Goding, 1978;Langone, 1982; Jendeberg et al., 1997).

Protein G from group G Streptococci represents amore general and versatile IgG binding reagent (Bjorckand Kronvall, 1984; A, kerstrom et al., 1985; A, kerstromand Bjorck, 1986), as it binds a greater range of IgGisotypes from a wider variety of mammalian specieswith higher affinity. Recombinant protein G that hasbeen genetically engineered to possess IgG-binding ac-tivity alone is commercially available and is a superioralternative to native protein G that possesses a serumalbumin binding site. Polyclonal anti-mouse Fc fromvarious mammalian species (e.g. goat, rabbit) are rou-tinely used for the affinity-capture of murine mono-clonal antibodies (mAb) for the kinetic evaluation ofantibody-antigen interactions (Malmborg et al., 1992).These antibodies can be inexpensively raised againstany target, thus avoiding the species isotype limitationof protein G and particularly protein A. Polyclonalantibody affinity-capture generates high avidity due tomultivalent binding. As a result, regeneration of thesesurfaces is not as efficient as that observed for proteinA-coated and protein G-coated sensor surfaces (unpub-lished results).

Immobilisation and regeneration difficulties werehighlighted for two unrelated interactions studied byreal-time BIA: (a) binding of polyclonal anti-chlorpy-riphos antibody (anti-chlorpyriphos Ab) to covalently

immobilised chlor-oval conjugate; (b) binding of solu-ble recombinant CD4 to covalently immobilised mono-clonal anti-CD4 IgG. The advantages ofaffinity-capture over direct covalent immobilisationwere investigated. In particular, protein G, protein Aand polyclonal anti-mouse Fc-coated sensor surfaceswere employed to anchor anti-GST IgG for kineticevaluation of the interaction with soluble glutathione-s-transferase (GST). Kinetic analysis is a suitable ‘testcase’ as it is a highly demanding application of ‘real-time’ interaction analysis and is very sensitive to sub-optimal ligand immobilisation and regenerationschemes.

2. Experimental

All reagents and chemicals were supplied by Sigma(Poole, Dorset, England), unless otherwise stated. BIA-CORE 1000™ and CM5 sensor chip were both sup-plied by BIACORE AB (Uppsala, Sweden). HEPESbuffered saline (HBS) buffer, pH 7.4, containing 10mM HEPES, 1 mM EDTA, 0.001% Tween-20 and 0.15M NaCl, was used as the constant flow buffer and as adiluent throughout all analysis unless otherwise stated.BIAevaluation 3.1™ was used for kinetic analysis ofdata (BIACORE AB, 5-75450, Uppsala, Sweden). Allbuffers and solutions used during BIACORE analysiswere made up using ultrapure water, degassed andsterile-filtered. Recombinant protein G was purchasedfrom Sigma (UK). Mouse monoclonal anti-chlorpy-riphos IgG was produced at the Institute for FoodResearch, Norwich, UK. Anti-GST mAb and GSTwere both donated by Biotrin, (Mount Merrion,Dublin, Ireland). Rabbit polyclonal IgG antibodieswere raised in New Zealand white rabbits.

2.1. Interaction of free chlorpyriphos withanti-chlorpyriphos antibody

2.1.1. Biotinylation of conjugateThe chlor-oval conjugate was dissolved (2 mg ml−1)

in coupling buffer (50 mM sodium bicarbonate buffer,pH 8.5, containing 0.15 M NaCl, 0.05% Tween-20).Biotin-NHS was dissolved to 2.5 mM in DMF. A 1:10molar excess of biotin reagent was added to the proteinsolution. The reaction was incubated at room tempera-ture for 1.5 h. The biotin-NHS was separated from thebiotinylated-protein by dialysis.

2.1.2. Coupling reactionActivation of the CM-dextran matrix was carried out

by mixing equal volumes of 400 mM ethyl-N-(dimethy-laminopropyl)carbodiimide (EDC) and 100 mM N-hy-droxysuccimide (NHS) (freshly prepared in ultrapurewater) and injecting the mixture over the sensor surface

J. Quinn et al. / Biosensors & Bioelectronics 14 (1999) 587–595 589

for 7 min at a flow rate of 5 m1 min−1. Streptavidin (50mg ml−1) was dissolved in 10 mM acetate buffer, pH5.0, and injected over the surface for 15 min at a flowrate of 2 ml min−1. Capping of the unreacted sites wasachieved by injection of 1 M ethanolamine, pH 8.5, for7 min. The biotinylated chlor-oval conjugate waspassed over the surface for 50 min at a flow rate of 2 mlmin−1.

2.1.3. Sample preparation for BIACORE™Serial dilutions of antibody stock solution (3 mg

ml−1) were prepared using HBS buffer, containing 2mg ml−1 ovalbumin, as diluent. The antibody dilutionswere pre-incubated for 2 h at room temperature tocompetitively inhibit potential non-specific binding tothe ovalbumin carrier protein.

2.1.4. Sample analysisSamples were injected sequentially in order of in-

creasing concentration at a flow rate of 5 ml min−1. TheBIACORE 1000™ system continuously monitors thechange in mass at the sensor surface and dedicatedsoftware displays this in response units as a function oftime. The entire analyses was performed on a singlechlor-oval conjugate-coated surface.

2.1.5. RegenerationRegeneration of the surface was performed by inject-

ing a single 30 s pulse of 20% (v/v) acetonitrile in 1 Methanolamine, pH 12.0.

2.2. Direct immobilisation of antibody

Anti-CD4 mAb was produced by culturing theOKT4 hybridoma cell line (European Animal Cell Cul-ture Collection) and harvesting the supernatant. Solu-ble recombinant CD4 (CD4) was purchased fromIntracel (London W1Y 3WD, UK). Anti-CD4 mAbwas immobilised in accordance with the procedure out-lined for streptavidin above.

2.3. Affinity-capture

Coupling of protein G, protein A and anti-mouse FcIgG was performed in accordance with the procedureoutlined above for streptavidin. However, the couplingbuffer for protein G, protein A and anti-mouse Fc IgGwas adjusted to pH 4.3, 4.4 and 5.0, respectively.

2.3.1. In6estigation of protein G and protein A stabilityA total of 25 ml of rabbit polyclonal IgG (0.7 mg

ml−1) was passed over the protein G-coated surface at5 m1 min−1. Regeneration was achieved using alternatepulses of 20 mM HCl and 20 mM NaOH for the first90 cycles while alternate pulses of glycine–HC1 (pH1.7) and glycine–NaOH (pH 12.5) were employed for

the final 90 cycles. A total of 180 binding–regenerationcycles were completed. Murine mAb IgG3b (15 ml) waspassed over the protein A-coated surface at 5 m1 min−1

and the surface was regenerated using a single 60 spulse of 10 mM HCl. A total of 124 binding–regenera-tion cycles were completed.

2.3.2. Titration cur6eMurine monoclonal IgG samples were prepared from

0.040 to 40 mg ml−1 and passed over the proteinG-coated surface for 5 min at a flow rate of 2 ml min−1

(5 replicates). Random order sampling was employed.The surface was regenerated using a 30 s pulse of 20mM HCl followed by a 30 s pulse of 20 mM NaOH.Non-specific binding of IgG to the CM-dextran wasdetermined for the highest concentration of IgG andwas found to be 0.290.2 RU.

2.3.3. Affinity-capture for kinetic e6aluationProtein A was immobilised (�494 RU) onto a sen-

sor chip using conventional EDC/NHS coupling. Anti-GST mAb (25 mg ml−1 in HBS buffer) was injectedover the protein A-coated surface for 2 min at 10 mlmin−1 giving a binding response of �500 RU. A 5min waiting period allowed the baseline to stabilise andGST (100 nM in HBS buffer) was injected giving abinding response of 100 RU. The surface was regener-ated using two 30 s pulses of 25 mM HCl. An identicalbinding–regeneration cycle was repeated where HBSbuffer was substituted for the GST sample giving asuitable reference curve. The reference curve was sub-tracted from the active curve to eliminate baseline driftdue to dissociation giving corrected curves (e.g. Fig.7a).

3. Results and discussion

3.1. Interaction of free chlorpyriphos withanti-chlorpyriphos antibody

3.1.1. BIAcore analysisCoupling of proteins onto the CM-dextran hydrogel

is normally achieved using EDC/NHS amine coupling.However, this technique could not be employed as theconjugate was poorly soluble in low ionic strengthbuffers. BIACORE pre-concentration studies revealedthat the pI was very low (B4.0). Consequently, immo-bilisation of the conjugate had to be accomplishedwithout pre-concentration, in high ionic strength buffer.Affinity-capture using streptavidin-biotin was selectedas it is commonly used for the immobilisation of nucleicacids, polysaccharides and highly acidic proteins (Re-hak et al., 1994; Hoshi et al., 1995). Streptavidin is atetrameric protein containing four identical subunitseach possessing a high affinity (Kd�1015 M−1) binding

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site for biotin (Ebersole et al., 1990). The streptavidin-biotin association is practically irreversible and is rela-tively insensitive to chaotrophic agents and extremes ofpH and ionic strength.

Streptavidin was covalently immobilised (�8000RU) by standard amine coupling (Experimental, Sec-tion 2.1.2.) and the biotinylated conjugate was subse-quently captured (�3000 RU). The interaction ofanti-chlorpyriphos antibody with the immobilisedchlor-oval conjugate was characterised by very highavidity as evidenced by the low dissociation rate. Theinteraction was resistant to a wide variety ofchaotrophic agents (20% acetonitrile, 50% DMSO, 10%DMF, 70% methanol, 70% ethanol), pH extremes (0.1M NaOH, 0.1 M HCl, 0.1 M phosphoric acid, 20%formic acid, 1 M ethanolamine, pH 12, 0.1 M glycineHCl, pH 2.0, 0.1 M glycine, pH 12.0), detergents (0.1%(v/v) triton-x-100, 0.1% Tween-20) and high ionicstrength solutions (1 M NaCl, 1 M ethanolamine.Many combinations of the above solutions yielded poorresults with the exception of 20% acetonitrile in 1 Methanolamine, pH 12.0. This solution removed �90%of the bound antibody with �4% loss in surfacebinding capacity for each interaction-regeneration cy-cle. In addition, this solution combines high ionicstrength, extreme pH and chaotrophic properties toremove bound protein. Such harsh conditions may belimited to applications where the immobilised moleculeis extremely robust and/or devoid of endogenous bio-logical binding activity (e.g. hapten-carrier protein con-jugates). Careful analysis of regeneration profiles wasimportant as appreciable leaching of the covalentlybound streptavidin and the ovalbumin-bound chlorpy-riphos can occur under these conditions. In addition,the stability of the streptavidin-biotin linkage and thebiotin-conjugate linkage during regeneration is critical.

Fig. 1(a) is a titration curve for the interaction de-scribed above. Moderate non-specific binding (88.797.0 RU) of the anti-chlorpyriphos antibody preparationto a streptavidin-coated sensor surface occurred. Thetitration curve appears satisfactory with relatively lowstandard error values. However, there was significantaccumulation of anti-chlorpyriphos antibody (�2000RU) on the surface over the course of the experiment(Fig. 1b) and further investigations revealed that quan-titative removal of bound antibody resulted in excessiveleaching of the conjugate from the surface. Moreover,the standard errors for the analysis (Fig. 1a) wouldhave been much greater if the samples had beenanalysed in random order, as the undesirable accumula-tion of antibody at the surface would not have followedthe stepped increase apparent in Fig. 1(b). It is clearfrom these results that surface regeneration is not atrivial matter and that optimal regeneration cannot beguaranteed despite rigorous experimental optimisation.This experiment constituted a preliminary investigation

of the interaction and further optimisation was notpursued. However, affinity-capture of the conjugateusing a polyclonal anti-ovalbumin IgG would avoidregeneration difficulties imposed by the high avidity ofthe antibody-chlorpyriphos interaction thus facilitatingthe development of a competitive immunoassay forquantification of free chlorpyriphos.

3.1.2. Direct immobilisation of antibodyDirect immobilisation of the antibody of interest via

covalent amine coupling results in random site attach-ment to the hydrogel. Heterogeneous immobilisation ofthe antibody has been implicated as a source of errorwhen fitting kinetic interaction models to interactioncurves (Edwards et al., 1995). However, direct immobil-isation is suitable for the majority of applications suchas epitope mapping (Johne et al., 1993) and concentra-

Fig. 1. (a) Titration curve for the interaction of anti-chlorpyriphosantibody with covalently immobilised chlor-oval-conjugate. Serialdilutions of the anti-chlorpyriphos polyclonal antibody were preparedusing HBS buffer as diluent. Sampling was conducted by running fivereplicates sequentially, in low to high order of concentration. Aftereach binding interaction, the surface was regenerated using 10%acetonitrile in 1.0 M ethanolamine, pH 12.0. (b) Plot of residualantibody accumulation as a function of the sample concentration forthe titration curve (above). The undesirable accumulation of residualantibody clearly reflects the sequential order of sample analysis. Atlow concentrations of antibody a slight decrease in surface mass,resulting from the undesirable removal of chlorpyriphos-ovalbuminconjugate and/or streptavidin, is apparent. As expected, the graphdemonstrates a stepped increase in residual antibody mass withincreasing sample concentration.

J. Quinn et al. / Biosensors & Bioelectronics 14 (1999) 587–595 591

Fig. 2. Sensorgram showing the preliminary optimisation of regenera-tion conditions, for the interaction between directly immobilisedanti-CD4 IgG (580 RU) and CD4. The flow rate was kept constant at10 m1 min−1. (1) Stable baseline with anti-CD4 IgG-coated surface(580 RU). (2) Baseline after 1 min injection of 33.3 nM CD4, givinga binding response of 131 RU. (3) Baseline after 30 s pulse of 10 mMHCl resulting in negligible regeneration (B14% CD4 removed). (4)Baseline after 30 s pulse of 25 mM HCl, giving 98% regeneration ofthe surface. (5) Re-injection of CD4 under identical conditions out-lined in (2), giving a binding response of 100 RU. (6) A 30 s pulse of25 mM HCl, giving 98% regeneration of the surface. (7) A 3 mininjection of 150 nM CD4 giving a binding response of 127 RU. (8)Regeneration of the surface as outline for (3) and (6).

tion conditions are denaturing the extremely sensitiveanti-CD4 IgG thus necessitating the adoption of analternative format such as affinity-capture.

3.1.3. Protein G, protein A and polyclonal antibody asaffinity-capture ligands

Affinity-capture allows controlled and reproduciblecapture of the antibody and avoids repeated exposureof the antibody to harsh regeneration conditions thatare likely to result in denaturation. In addition, theantibody is anchored to the surface in a homogenousmanner in contrast to the conventional random aminecoupling method. These properties facilitate the reduc-tion of undesirable surface heterogeneity (Schuck,1996).

3.1.4. Stability of protein G and protein APolyclonal rabbit IgG samples were adsorbed onto a

protein G-coated sensor surface and subsequently des-orbed over 180 consecutive binding–regeneration cy-cles. Fig. 3 demonstrates the stability of immobilisedprotein G through 180 binding–regeneration cycleswithout loss of surface binding capacity. The antibodybinding response is consistent throughout the analysis.The apparent increase in the antibody binding fromcycle 100–150 is due to the sub-optimal regenerationresulting in the accumulation of residual antibody, ascan be seen from the corresponding increase in thebaseline response. A similar experiment was conductedon a protein A-coated surface for 124 binding–regener-ation cycles (Fig. 4). In contrast to the protein G-coated surface, regeneration was optimal giving a stablebaseline throughout the analysis. A relatively consistentIgG-binding level is maintained through 124 bindingregeneration cycles. These results show that protein Aand protein G-coated surfaces can be used repeatedlywith little deterioration in Ab-binding capacity.

tion determination. The success of these applications isdependent on the ability to regenerate the surface undernon-denaturing conditions. Clearly, surface heterogene-ity could be avoided if directed covalent immobilisationto the hydrogel could be achieved through a uniquefunctional group distant from the antibody’s bindingsite. Such a functional group is not commonly found innature, but recombinant protein engineering technologymay allow the design and production of these deriva-tives. Unfortunately, antibodies vary significantly withrespect to stability and antigen affinity, two propertiesthat dictate the performance of any given antibody.Antibodies of high affinity and low stability are com-monly found and are not suitable for direct covalentcoupling strategies.

Fig. 2 is a sensorgram illustrating preliminary optimi-sation of regeneration conditions for the interaction ofCD4 with covalently immobilised monoclonal anti-CD4IgG. 25 mM HCl proved effective at removing boundCD4, but re-injection of the CD4 (33.3 nM) sampleunder identical conditions resulted in a �24% reduc-tion in the CD4 binding level. Initially, 580 RU ofanti-CD4 IgG was covalently immobilised giving anexpected CD4 saturation capacity of 425 RU. Theactual CD4 (150 nM) saturation response was 127 RU,which represents B30% of the ideal. Clearly, regenera-

Fig. 3. Binding response and regeneration profiles of 180 binding–re-generation cycles for the interaction between immobilised protein Gand rabbit polyclonal antibody. The initial 90 cycles employed two 30s regeneration pulses of 20 mM HCl and 20 mM NaOH, respectively.The remaining cycles were regenerated using consecutive 30 sec pulsesof 0.1 M glycine–HCl, pH 1.7 and 0.1 M glycine–NaOH, pH 12.5.

J. Quinn et al. / Biosensors & Bioelectronics 14 (1999) 587–595592

Fig. 4. Binding response and regeneration profiles of 124 binding–re-generation cycles for the interaction between immobilised protein Aand murine monoclonal IgG2b. The surface was regenerated aftereach binding cycle using a 1 min pulse of 10 mM HCl.

Fig. 6. Overlay of both working and reference sensorgrams obtainedfor the GST-anti-GST interaction. Protein A was immobilised (494RU) onto a planar sensor chip using conventional EDC/NHS cou-pling. (A) Baseline response on protein A coated surface. (B) A 2 mininjection of anti-GST mAb at 10 ml min−1 giving a binding responseof �500 RU. (C) A 5 min waiting period to allow the dissociation ofanti-GST mAb from the immobilised protein A to stabilise. (D) A 2min injection of GST (100 nM) giving a response of �100 RU. Ablank sensorgrarn involved substituting the GST sample with HBSbuffer giving a suitable reference curve. (E) Dissociation of bothanti-GST and GST followed by regeneration using two injections of25 mM HCl. (F) Baseline response re-established (i.e. all non-cova-lently attached material has been removed). The reference curve wasthen subtracted from the active curve to eliminate baseline drift dueto dissociation giving curves similar to Fig. 7.

3.1.5. Titration cur6eOuantitative assays generally require maximising the

yield of immobilised antibody while kinetic analysisnecessitates the adsorption of very low concentrations(100 200 RU) of IgG in order to prevent mass transportlimitation. A typical titration curve for selecting theappropriate concentration of specific antibody is shownin Fig. 5. Complete regeneration of the surface aftereach run was achieved using a 30 s pulse of 20 mM HCland 20 mM NaOH.

3.1.6. Affinity-capture for kinetic analysisTypically, kinetic analysis involves the evaluation of

a set of interaction curves obtained for different con-centrations of analyse according to a chosen interactionmodel. It is essential that the dissociation rate of theantibody-capture ligand complex is low in order toreduce baseline drift. Fig. 6 is an overlay of bindingprogress curves using the protein A affinity-capture

format. The downward drifting baseline caused by dis-sociation of the captured antibody from the protein Acan be eliminated by subtracting a reference curve toproduce corrected interaction progress curves similar tothose in Fig. 7(a).

The suitability of protein A, protein G and poly-clonal anti-mouse Fc antibody as affinity ligands forkinetic analysis was investigated. Fig. 7(a) is a sensor-gram showing overlaid binding progress curves for theinteraction of GST (100 nM) with affinity-capturedmonoclonal anti-GST IgG conducted on protein A,protein G and goat polyclonal anti-mouse Fc IgG-coated surfaces. All three curves have been correctedfor baseline drift and bulk refractive index variations.Visual inspection reveals that all three curves comparerelatively well with respect to the association phase butdeviate during the dissociation phase. Pseudo-first-or-der kinetic analysis requires analysis of the dissociationphase in order to determine the dissociation rate con-stant (kd). Having determined the kd it is possible toevaluate the association phase allowing calculation ofthe association rate constant (ka).

BIAevaluation software was used to assess the good-ness of fit of these curves with simulated curves. Thesimulated curves were generated by fitting pseudo-first-order integrated rate equations to the actual data set.

Fig. 5. Typical titration curve for the interaction of mouse mono-clonal IgG with immobilised protein G. Serial dilutions of mAb wereprepared from 40 ng ml−1 to 40 mg ml−1 in HBS buffer, pH 7.4.Sampling was conducted randomly with five replicates. The mean9S.E. is plotted above.

J. Quinn et al. / Biosensors & Bioelectronics 14 (1999) 587–595 593

Both the protein G-coated and polyclonal anti-mouseFc-coated surfaces lead to substantial deviations fromideal dissociation. In particular, the positive dissocia-tion phase slope on the protein G-coated surface sug-gests that non-specific protein-protein interactionswere occurring. The entire association phase data setwas fitted to the simple 1:1 Langmuir associationisotherm. The residual plot (Fig. 7b) displays the dif-ference between the ideal and actual curves at eachdata point. Clearly the protein A-coated surface leadsto very little deviation (Fig. 7b) from ideal behaviour(91 RU) and is therefore the most suitable affinity-capture ligand for kinetic analysis. As expected fromthe dissociation phase curves, both protein G-coatedand polyclonal anti-mouse Fc-coated surfaces lead tohigher residuals. Therefore, protein G and polyclonalanti-mouse Fc appear unsuitable for kinetic analysisdespite the many desirable properties outlined earlier.

These results are not altogether unexpected asprotein A binds antibodies via the Fc region, a regionthat is the natural binding site for Fc receptors pos-sessed by macrophage cells and complement factors.In contrast, the protein G antibody-binding site islocated between the Fab and Fc region and may

cause steric hindrance of the antigen-antibody interac-tion. In addition, the positive dissociation phaseslope, obtained for the GST-anti-GST interactionconducted on the protein G-coated surface, suggeststhat matrix cross-linking is occurring (Karlsson andFalt, 1997). Matrix cross-linking increases the proba-bility of interference due to steric hindrance (‘over-crowding’) and mass transport limitations within thehydrogel itself (Schuck, 1996) and may account forthe deviations observed.

4. Conclusions

The anti-chlorpyriphos antibody was shown to spe-cifically bind to the chlor-oval conjugate with highavidity. This property is advantageous for solid statenon-reversible immunoassays (e.g. ELISA) but im-poses serious limitations for biosensor analysis whereregeneration of the surface is necessary. Interferencefrom incomplete regeneration resulted in considerableerror, which increased with each binding–regenerationcycle. The utility of direct covalent attachment of an-tibodies to the sensor surface is dependent on the

Fig. 7. (a) Overlaid binding progress curves for the interaction of GST with affinity-captured anti-GST mAb, using three different affinity-captureligands. Anti-GST mAb was injected over the functionalised sensor surface at 10 ml min−1 for 2 min (B500 RU immobilised). After 5 min, 100nM GST was injected over the surface at 30 ml min−1 for 3 min and allowed dissociate for a further 1.5 min. This analysis was repeated on proteinG-coated, protein A-coated and anti-mouse Fc-coated sensor surfaces. The curves obtained were then subtracted from reference curves in the samemanner as described in Fig. 6. The curves were normalised with respect to each other to allow visual comparisons. (b) Simulated integrated rateequations based on ideal pseudo-first-order behaviour were superimposed on the above curves and the difference between the actual and idealcurves at each data point is displayed above for the association phase.

J. Quinn et al. / Biosensors & Bioelectronics 14 (1999) 587–595594

antigen binding avidity and antibody resistance toharsh regeneration conditions. The anti-CD4 IgG wasshown to be highly sensitive to regeneration condi-tions demonstrating the limitations of direct attach-ment. In addition, surface heterogeneity is assuredmaking kinetic analysis difficult. In contrast to theabove interactions, the affinity-capture format, usingprotein G and protein A, facilitated repeated regener-ation under mild conditions with negligible deteriora-tion of surface binding capacity. However, thesereagents are limited to the immobilisation of a spe-cific subset of antibody species. Polyclonal antibodiesare more versatile as they can be tailored to allowdirected immobilisation of virtually any affinity ligandbut do not perform as well in terms of regenerability.In conclusion, direct immobilisation of biomoleculescan often impose serious limitations on applicationdevelopment. However, affinity capture provides asimple solution to this problem and was shown to besuitable for kinetic analysis which is a highly demand-ing application of ‘real-time’ interaction analysis.

Kinetic analysis of biomolecular interactions iscomplicated by mass transport limitation, steric hin-drance, matrix cross-linking and surface heterogeneityeffects. It is difficult to determine, with certainty,which of these effects is responsible for observed devi-ations from ideal pseudo-first-order behaviour. Never-theless, the protein A affinity-capture formatproduced ideal binding progress curves for the inter-action of GST with anti-GST mAb. Protein G-coatedand polyclonal anti-mouse Fc-coated surfaces led tonon-conformity to the pseudo-first-order interactionmodel. It is worth noting that without the benefit ofthe comparison one could easily mistake the observeddeviations as an inherent property of the interaction.Consequently, it is prudent to investigate alternativeformats when performing kinetic analysis.

Acknowledgements

This study was facilitated by financial support fromEnterprise Ireland, the Irish Science and TechnologyAgency and the EU FAIR research grant CT96 1181.

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