Sensors and Actuators B 99 (2004) 416–424

Quartz crystal biosensor for real-time kinetic analysis of interactionbetween human TNF-� and monoclonal antibodies

Yang Liua, Wei Zhangb, Xiao Yua, Hongwu Zhanga, Rui Zhaoa, DiHua Shangguana,Yan Li b, Beifen Shenb, Guoquan Liua,∗

a Center for Molecular Science, Institute of Chemistry, Chinese Academy of Sciences, Zhongguancun, Beijing 100080, PR Chinab Institute of Basic Medical Sciences, Beijing 100850, PR China

Received 7 June 2003; received in revised form 8 December 2003; accepted 11 December 2003

Abstract

Quartz crystal microbalance (QCM) biosensor integrated in a flow injection analysis (FIA) system was used for the real-time investigationof the interaction between human TNF-� (hTNF-�) and its monoclonal antibodies (mAbs). Biologically active hTNF-� trimer wasimmobilized on the gold electrodes of the piezoelectric crystals and the binding interactions of the two immobilized antigen with twoanti-hTNF-� monoclonal antibodies (Z8 and Z12) were followed as changes in the resonant frequency of the modified crystals. Theresultant sensorgrams were rapidly analyzed by using an in-house kinetic analysis software based on genetic algorithm (GA) to derive boththe kinetic rate constants (kassandkdiss) and equilibrium dissociation constant (KD) for antigen–antibody interactions. For the interactions,KD were 2.31× 10−7 and 5.42× 10−7 M for Z8 and Z12, respectively. The generally good agreement found between biosensor data andthe affinities known from conventional equilibrium enzyme-linked immunosorbent assay (ELISA) validates this analytical method. Thestudy shows the potential usefulness of QCM biosensor technology for biomolecular interaction analysis.© 2004 Elsevier B.V. All rights reserved.

Keywords:Quartz crystal biosensor; Kinetic analysis; Human TNF-�; Genetic algorithm; ELISA

1. Introduction

Tumor necrosis factor-� (TNF-�) is a pleiotropic cy-tokine mainly produced by activated macrophage[1]. Thebiologically active TNF-� molecule is a trimer with amolecular weight of 51 kDa, while monomeric TNF-� isconsidered to be biologically inactive. Although TNF-� isoriginally defined for its ability to kill tumour cell in vitroand plays a key role in a variety of physiological responses,it has also been shown to be implicated in septic shock, in-flammation, cachexia and other pathophysiologic responsesas an endogenous mediator, and therefore, be a useful targetfor specific immunotherapy[2]. A number of reports haveprovided strong evidence that the blockade of TNF-� resultsin the amelioration of some diseases symptoms. For exam-ple, anti-TNF antibodies lessen pain, joint swelling, anemiaand elevated erythrocyte sedimentation rates in patientswith rheumatoid arthritis[3]. Encouraging results have alsoemerged from the use of anti-TNF antibodies in the treatment

∗ Corresponding author. Tel.:+86-10-62557910;fax: +86-10-62559373.E-mail address:liugq@iccas.ac.cn (G. Liu).

of Crohn’s disease[4]. Apparently, anti-hTNF-� mono-clonal antibody (mAb) characterized as neutraling hTNF-�biological activity is, therefore, of great therapeutic interest.

Real-time kinetic analysis of biospecific interaction isimportant implement for studying the interaction betweenbiomolecules and understanding the relationship betweenthe structure of a molecule and its biological function[5,6].Previously, kinetic analyses were restricted to only selectsystems with unique spectral properties. Fortunately, withthe development of biosensors, the kinetics of most molec-ular interactions is now routinely accessible.

Quartz crystal microbalance (QCM) is a highly sensitivedevice among the biosensing methods. Many advantagesexist for QCM biosensor, i.e., simplicity, convenience, eco-nomical cost and real-time response. Further, QCM biosen-sor integrated in a flow injection analysis (FIA) system hasthe advantage to work continuously and to monitor on-linethe binding of the analyte[7–9]. No complicated and ir-reproducible drying procedures are needed and a highlyautomated test performance can be achieved. In addition,the binding results in a change of resonance frequency ofthe crystal and thus immunological marker, such as en-zyme or fluorescence labeled tracers, are not necessary.

0925-4005/$ – see front matter © 2004 Elsevier B.V. All rights reserved.doi:10.1016/j.snb.2003.12.010

Y. Liu et al. / Sensors and Actuators B 99 (2004) 416–424 417

Such a QCM–FIA system is suitable for rapid analysis ofbiospecific interactions and the reported successful applica-tions for kinetic studies include immunochemical reactions,hybridization of DNA, interaction of nucleic acids withproteins and drugs, interaction of protein with amino acidand drugs[10–13].

In this paper, QCM–FIA system with a new kinetic anal-ysis software based on genetic algorithm (GA) was set upand used for real-time kinetic analysis of the interaction be-tween hTNF-� trimer and its two neutralizing anti-hTNF-�mAbs (Z8 and Z12, whose structure only differs in CDR3region of heavy chain). The results obtained by QCMbiosensor were in good agreement with those determinedby conventional ELISA. The obtained quantitative informa-tion on the kinetics of binding was vital to understand thestructural/functional relationships of different mAbs on amolecular level.

2. Materials and methods

2.1. Materials

hTNF-� trimer and anti-hTNF-� mAbs (Z8 and Z12)were a gift of the Molecular Immunity Laboratory, BeijingInstitute of Basic Medical Sciences. The mAb stock solu-tions (5.0 mg/ml protein concentration) were prepared in10 mmol/l phosphate buffer (pH 7.4), stored at 4◦C in the re-frigerator and diluted to the desired concentration for exper-imental use.dl-Dithiothreitol (DTT), human serum albumin(HSA) and bovine serum albumin (BSA) were all purchasedfrom Sigma (St. Louis, MO, USA). Ethanedioldiglycidylether and sodium borohydride were from Aldrich (St. Louis,MO, USA). 2,2-Azinobis-3-ethylbenzthiazoline–sulfonicacid (ABTS) used as the substrate of ELISA was purchasedfrom Sigma. About 100 mmol/l of glycine·HCl solution (pH2.0) was used as regeneration reagent. Unless otherwisenoted, all the solutions of the reagents were in 10 mmol/lphosphate buffered solution (PBS), pH 7.4. All other chem-icals were analytical reagent grade and doubly distilled wa-ter was used throughout. Prior to use, all solutions preparedwere filtered (0.22�m).

The QCM sensors employed were commercially available10 MHz AT–cut quartz crystals (14 mm diameter) with goldelectrodes (8 mm diameter and 50.2 mm×2 mm of geomet-ric area) on both sides purchased from Beijing 707 Factory(Beijing, China). The crystals were polished to a surfaceroughness of less than 5�m, which produced a mirror-likefinish on the gold electrodes.

The reading instrument of ELISA was purchased fromAnthos (Australia), whose model is Labtec 2001.

2.2. Immobilization of hTNF-α antigen on piezoelectriccrystals[12,13]

The immobilization procedure is described in the follow-ing:

• The gold electrodes were cleaned with a boiling solutionconsisting of H2O2 (33%), NH3 (33%) and doubly dis-tilled water in a 1:1:5 ratio to remove adsorbed compo-nents disturbing the self-deposition of thiols. The crystalswere immersed in the boiling solution for 10 min and thenthoroughly washed with doubly distilled water and usedimmediately afterwards.

• The freshly cleaned gold surfaces were immersed in a20 mmol/l ethanol solution of DTT. The unstirred solutionwas kept at room temperature, in the dark, for 24 h. Thecrystal was then washed with ethanol and doubly distilledwater and sonicated for 10 min in ethanol to remove theexcess of thiols[14]. A stable self-assembled monolayerof DTT was formed on the gold surfaces.

• The monolayer was then modified by activation of thesulfhydryl groups with bisoxiranes under weak basic con-dition. The gold surfaces were immersed in the mixturesolution consisting of 1 ml of ethanediol diglycidyl etherand 4 ml of 0.1 mol/l Na2CO3 buffered solution (pH 8.5)containing 8 mg of sodium borohydride. The reactionswere performed at 30◦C for 4 h and stopped by washingthe surfaces with a large volume of water. In this way, thesurfaces were modified forming epoxide groups[15].

• The prepared crystal was immersed for 2 h in the basicsolution containing 1.5 mg of hTNF-� trimer in 5 ml of0.1 mol/l Na2CO3 buffered solution (pH 11.0) and thereaction temperature was kept at 40◦C.

• Finally, ethanolamine hydrochloride (pH 8.6, 1 M watersolution), a blocking agent for the prevention of nonspe-cific binding, was then applied to block the residual re-acting sites for 30 min.

2.3. Measurement setup—QCM–FIA system

The measurement setup mainly consisted of two parts:QCM system and FIA system. QCM system was the corepart, which comprised the QCM sensors, oscillator circuit,the frequency counter and personal computer. FIA systemconsisted of a fluid circuit with a flow-through cell, an HPLCinjection valve and a pump (seeFig. 1).

The modified crystal was fixed in a home-madeflow-through cell (40�l) with two organosilicon rubbero-rings. Only one side of the crystal was in contact withthe flowing solution. The crystal was powered by an oscil-lator circuit constructed from a transistor–transistor logicintegrated circuit (TTL–IC). The frequency output wasmonitored with an EE3386 Universal Counter (NanjingTelecommunication Instrumental Factory, Nanjing, China)controlled by a personal computer via RS 232 interface.An 1 s interval between two experimental points was set inorder to achieve 1 Hz resolution from the counter. The SB-2micropiston dispenser pump (The Beijing Oriental Science& Technology Company, Beijing, China) was positionedin line before the cell and provided a continuous flow ofbuffer liquid (60�l/min) through the flow-through cell.A Rheodyne 7125 injection valve allowed for injecting a

418 Y. Liu et al. / Sensors and Actuators B 99 (2004) 416–424

Fig. 1. Setup of the QCM–FIA system.

certain volume of the sample into the liquid circuit and,thus, onto the sensor. A water bath was used to keep thesensor at 25± 0.5◦C over the experimental period.

2.4. Measurement and regeneration procedure

The fresh-coated quartz crystal was mounted in theflow-through system and rinsed with PBS (pH 7.4,10 mmol/l) continuously until the frequency had stabilizedunder flow conditions (60�l/min). By means of an injec-tion valve, 200�l of various protein solutions were injectedinto the fluid system. The permanent frequency shifts ver-sus time curves were recorded and the binding process wasmonitored in real time. After one binding measurement,600�l of glycine·HCl solution (100 mmol/l, pH 2.0) wasinjected into the system with the injection valve to disso-ciate the bound biomolecules and free-binding sites in thereceptor layer for the next binding.

2.5. Kinetic data analyses

2.5.1. Basic principle of kinetic analysesSince the QCM is a continuous, real-time detector, the

possibility for assessing the kinetics of interaction exists.The reaction between the immobilized compound (B) andthe molecule in solution (A) often assumed to follow pseudo-first-order kinetics[16]. For the reversible interaction,

A + Bkass�kdiss

AB

The rate of formation of the product, AB, at timet may bewritten as

d[AB]

dt= kass[A][B] − kdiss[AB] (1)

wherekass is the association rate constant andkdiss is thedissociation rate constant. After some reaction time,t,[B] = [B]0 − [AB]. Substituting into (1) gives

d[AB]

dt= kass[A] ([B]0 − [AB] ) − kdiss[AB] (2)

where [B]0 is the concentration of B att = 0.The rate of dissociation of the formed complexes, AB, is

described by

d[AB]

dt= −kdiss[AB] (3)

Considering the Sauerbray equation (the decrease of fre-quencyF is directly proportional to the attached mass) andusingFmax as the frequency change after a complete satura-tion of the surface of the crystal with A, the concentrationof the free B is proportional to (Fmax− F ), and the concen-tration of the complex AB toF. Eqs. (2) and (3)can thus beexpressed respectively as

dF

dt= kassC(Fmax − F) − kdissF (4)

dF

dt= −kdissF (5)

whereC is the concentration of the free A, held constant ina continuously flowing solution.

Eqs. (4) and (5)are integrated to giveFor association:

F = − kassCFmax

kassC + kdiss(e−(kassC+kdiss) − 1) (6)

For dissociation:

F = −F0e−kdiss(t−t0) (7)

wheret is time,t0 is time at start of reaction andF0 frequencyat t0 [17].

Y. Liu et al. / Sensors and Actuators B 99 (2004) 416–424 419

The integrated rate equations [Eqs. (6) and (7)] are usedfor fitting the sensorgram data to derive the rate constant(kass and kdiss). The equilibrium constantKD for the ABcomplex can be obtained as a ratio

KD = kdiss

kass.

2.5.2. Kinetic analysis software based on GAFor SPR, kinetic analysis software such as BIAE valuation

software (Pharmacia, Sweden) and Fastfit software (AffinitySensors, UK) are commercially available. While for QCM,there is no special kinetic analysis software used for evalu-ation of results and extraction of kinetic data. Some QCMresearchers have used the non-linear fitting procedure fromOrigin (Microcal Software, USA) to calculate the kineticconstants, but such data handling process is discommodious.Here, we developed new kinetic analyses software based ongenetic algorithm (GA)[13]. The genetic algorithm, whichwas first proposed in Holland[18], is one of the most impor-tant stochastic optimization methods[19–21]. Our kineticanalyses software based on GA was written in C++. Theprograms involve the following procedure: creation of pop-ulation, evaluation of cost, mate selection, reproduction andmutation. Using the procedures of mate selection, reproduc-tion and mutation, a new population is created and the wholecircle is repeated until a proper stop criterion is fulfilled.The cost function was given by

χ2 = 1

n

n∑

i=1

{yif(yi, ai)}2

where theyi are the experimental data,f(yi, ai) is the functionchosen as model and theai are the model parameters. Thequality of the fitted data is evaluated by comparison betweencalculated and experimental curves and by the magnitudeof χ2. After repeated tests, the parameters related to GAare set as follows: the population size is 200, the maximumnumber of generation is 200, crossover probability is 0.3and mutation probability is 0.3. This software is carried outon a PC with a Pentium III processor and fast and preciseinformation on the kinetic rate constants could be obtained.

2.6. The measurement of affinity constant of monoclonalantibodies by ELISA

Based on ELISA method described by Loomans et al.[22], we first determined the optimal concentration ofhTNF-� through a pilot experiment. The optimal concen-tration of hTNF-� was 5�g/ml recognized by Z8 or Z12 ofwhich the concentration was 10�g/ml. Then, the 96 wellmicrotitre plate was coated with 50�l (5 �g/ml) of purifiedhTNF-� antigen diluted in 0.05 mol/l bicarbonate coatingbuffer (pH 9.6) overnight. To establish specificity, controlswere performed with only coating buffer. Washed threetimes after each incubation with PBS-Tween (PBST). Toprevent non-specific reaction, blocked the plate with 200�l

of 10% fetal calf serum (FCS) for 2 h at room temperature.At the same time, the antigen/antibody reaction was carriedout in tubes with constant concentration 10�g/ml of anti-body Z8 or Z12, and with different concentration of antigenhTNF-� about 0 (adding no antigen), 100 and 250�g/ml,respectively. The mixtures in tubes (200�l) were incubatedat 37◦C for 1 h, to which the antigen–antibody reactionhad reached a state of equilibrium. The plate, after washingwith PBST, was incubated for 1.5 h at room temperaturewith 50�l antigen/antibody reactants. Subsequently, afterwashing, the wells were incubated with 50�l rabbit anti-mouse IgG conjugated to horseradish peroxidase for 45 minat room temperature. The final washing procedure was fol-lowed by adding 50�l of the substrate solution 2,2-azinobis-3-ethylbenzthiazoline–sulfonic acid. The reaction wasterminated, after 30 min incubation in the dark, by adding50�l of 2N sulphuric acid solution, and then measuredthe absorbance values at 405 nm immediately. The affinityconstant was accounted by the following equation[22]:

KD = a0A

A0 − A

whereKD is the equilibrium dissociation constant (mol/l);a0 is the concentration of hTNF-� in tube reacting withantibody (mol/l);A0 is the OD405 nm value measured withthe tube without antigen;A is the OD405 nm value when theantigen concentration isa0.

3. Results and discussion

3.1. Immobilization of hTNF-α antigen

The functionalization of gold surfaces could be done us-ing self–assembled monolayer of thio-compounds. Dithio-threiol was chosen as the functional reagent to derivatizegold electrodes of piezoelectric crystals. The sulfhydrylgroups were easily activated by ethanediol diglycidyl etherunder weak base condition[23] and the crystal surfaceswere modified by epoxide groups. hTNF-� trimer, was thenbound directly to the epoxide groups on the modified sur-faces through amino groups. The structure of biorecognitionlayer was depicted inFig. 2.

To evaluate the progress of immobilization, the reso-nance frequencies of the dried crystals were determinedafter all reaction steps and the amount of bound materialwas determined according to Sauerbray’s equation[24],1�g ≈ 457 Hz over the linear range of 0.01–25.0�g. Theamount of the immobilized antigen ligand was calculated.About 0.036 nmol was immobilized on each surface ofcrystal (0.036 nmol antigen/mm2 gold electrode area).

This immobilization procedure has two significant ad-vantages. First, the gold electrode surfaces were coveredwith self-assembled layer of dithiothreitol, which provideda hydrophilic environment suitable for most biomolecularinteractions and a chemical handle for the stable coupling

420 Y. Liu et al. / Sensors and Actuators B 99 (2004) 416–424

Fig. 2. The structures of biorecognition layers of hTNF-� trimer modified sensor.

immobilization. Free and rich hydroxyl groups of dithio-threitol could form an effective hydrophilic layer to reduceconsiderably the possibility of the nonspecific adsorption ofprotein on the gold surface. Second, the bisoxirane, ethane-diol diglycidyl ether, could provide not only active epoxidegroups for the coupling of ligands, but also an appropriatespacer. This long spacer or arm could help the antigen lig-and to turn around freely and locate correctly in the bindingsite of mAb.

3.2. Specific Interaction of hTNF-α immobilized sensorswith mAbs

Prior to experiments, the hTNF-� modified crystal placedin the flow-through cell was rinsed with PBS until a steady

Fig. 3. Interaction of hTNF-� antigen immobilized sensor with various proteins (protein concentration: 0.5�g/�l).

baseline was obtained. About 200�l of various protein so-lutions such as BSA, HAS, Z8 and Z12 (0.5�g/�l, preparedin 10 mM PBS, pH 7.4) were injected respectively into thefluid system to monitor the interactions that could occur onantigen modified surface of the crystal. The results wereshown inFig. 3.

It was clearly shown inFig. 3 that there was no nonspe-cific adsorption of BSA or HSA to the antigen modifiedgold electrodes. As the residual epoxide groups reactingsites have been blocked by ethanolamine and the goldsurfaces were hydrophilic, the nonspecific adsorption ofthese hydrophobic proteins to the gold surfaces was almostimpossible.

In contrast, when 200�l of Z8 or Z12 was injected intothe cell, the result was quite different (shown inFig. 3). The

Y. Liu et al. / Sensors and Actuators B 99 (2004) 416–424 421

frequency decreased about 120 Hz. With the rinsing by thebuffer solution, the frequency did not return back to the ini-tial baseline. This result confirmed that strong interaction be-tween hTNF-� and mAb (Z8 or Z12) could occur. Since thebinding interactions of two mAbs to immobilized hTNF-�are carried out in similar conditions (flow rate, temperature,concentration and mAb molecular weight) and on the samesensor chip, their kinetic binding curves can be compared di-rectly. The association course was quite closely, while therewas significant difference in the dissociation course. Z12showed faster dissociation rate than Z8. Based on the equa-tion,

KD = kdiss

kass

it could be deduced straightway that Z12 would haveweaker affinity to immobilized antigen than Z8. Appar-ently, the subtle difference in mAb’s structure resulted inthe significantly different affinity to hTNF-�. This compar-ison also suggested that QCM–FIA system is quite suitablefor rapid affinity ranking of closely related substancescombination.

For repeated uses of the sensor, it is important to re-generate the sensor. Hence, the efficiencies in removingadsorbed IgG and maintaining sensor sensitivity of variousregeneration reagents: glycine·HCl buffer (100 mM, pH2.0), 0.5 M NaCl solution (pH 7.4, PBS buffer), 10 mMHCl solution and 10 mM NaOH solution, were com-pared. The mild glycine·HCl buffer (pH 2.0) was chosenas the best one and a typical binding and regenerationsensorgram was obtained as shown inFig. 4. In orderto verify reproducibility, 0.5�g/�l of Z8 solutions arechosen and determined for five times, respectively (fig-ures were not shown). The relative standard deviation

Fig. 4. The typical binding and regeneration sensorgram. 0.5�g/�l of Z8 or Z12 was prepared in 10 mmol/l phosphate buffer (pH 7.4). 100 mmol/l ofglycine·HCl solution (pH 2.0) was used as regeneration reagent.

(R.S.D.) of frequency shift (F) is calculated and obtainedas 2.8%.

3.3. Calculation of the apparent binding constants

For obtaining affinity constants, the binding and dissoci-ation of various concentrations of Z8 with the immobilizedhTNF-� were measured and recorded as sensorgrams, re-spectively. The individual curves obtained were shifted verti-cally for graphic display and the results were shown inFig. 5.The detection limit for Z8 was estimated as 6.5×10−8 mol/l(S/N = 3). The Mw= 150, 000 for the molecule of mAbwas used[25].

The sensorgram data were analyzed using our in-housekinetic analysis software based on GA to obtain the bind-ing constants. Data were fitted toEq. (6) for the on-rateconstant and toEq. (7) for the off-rate constant. Then,equilibrium constant (KD) was determined as a ratio ofkdiss/kass. Magnitude of theχ2 and comparison betweencalculated and experimental curves were used to evaluatethe quality of the fitted data. Two examples of curves fitto frequency change data for the determination ofkass andkdiss were shown inFig. 6. Results of the kinetic analy-ses of the interaction between immobilized antigen andZ8 in various concentrations were summarized inTable 1.The same kinetic analysis method was applied to deter-mine the affinity binding constants of Z12 (data handlingprocess was not shown). The results of kinetic and equi-librium analysis for the two mAbs were summarized inTable 2.

From Table 2, it could be seen that Z8 showed higheraffinity to immobilized antigen than Z12, which meansthat Z8 will be a more suitable mAb for the blockade ofhTNF-�.

422 Y. Liu et al. / Sensors and Actuators B 99 (2004) 416–424

Fig. 5. The individual binding curves for several concentrations of Z8 with immobilized hTNF-� antigen.

Fig. 6. Example of curve fits (solid lines) to frequency change data (dotted line) for the determination ofkass andkdiss. Frequency modulation in Hz dueto: (A) specific adsorption of Z8 (1�g/�l) to the quartz crystal coated with hTNF-� antigen; (B) specific desorption of Z8 (1�g/�l) from the quartzcrystal coated with hTNF-� antigen.

Table 1Kinetic and affinity data from the QCM analysis of interaction between Z8 mAb and immobilized hTNF-� antigen illustrated inFig. 5

Concentration (�g/�l) kass {l/(mol s)} χ2 kdiss (s−1) χ2 KD (M)

0.1 466 0.998 1.04× 10−4 2.19 2.23× 10−7

0.25 461 1.03 1.01× 10−4 2.14 2.19× 10−7

0.5 471 0.992 1.12× 10−4 2.01 2.38× 10−7

1 484 0.974 1.18× 10−4 1.97 2.44× 10−7

Table 2The kinetic and equilibrium constants of Z8 and Z12 mAbs interacting with immobilized hTNF-� antigen by QCM analysis

mAbs kass {l/(mol s)} kdiss (s−1) KD (M)

Z8 470± 9.98 (1.08± 0.08) × 10−4 (2.31 ± 0.119)× 10−7

Z12 462± 12.5 (2.49± 0.221)× 10−4 (5.42 ± 0.488)× 10−7

Y. Liu et al. / Sensors and Actuators B 99 (2004) 416–424 423

Table 3The measurement of affinity constant of monoclonal antibodies by ELISA

mAb 100�g/ml of hTNF-� in tube reaction 250�g/ml of hTNF-� in tube reaction KD

Z8 KD = 3.12 × 10−7 M KD = 2.63 × 10−7 M 2.88 × 10−7 MZ12 KD = 3.47 × 10−7 M KD = 4.21 × 10−7 M 3.84 × 10−7 M

3.4. Comparison of conventional immunoassay (ELISA)with QCM biosensor for affinity constant determination

Conventional ELISA was used for the determination ofequilibrium affinity constant and the result was shown inTable 3.

The generally good agreement found between biosensordata and the affinities known from ELISA validates ourabove kinetic analysis method. However, the QCM biosen-sor offers several advantages over conventional ELISA:QCM biosensor requires low amounts of reagents withoutany special preparation; does not require antibody label-ing or a second reagent for immunocomplexes detection;allows measurements in real time with high precision andreproducibility; and sharpening yes/no results.

4. Conclusion

The present results suggest that QCM biosensor integratedwith a FIA system is a convenient and valuable tool forreal-time kinetic analysis of antigen–antibody interaction. Anew home-made kinetic analysis software based on geneticalgorithm is used for evaluation of results and extractionof kinetic data. Fast and precise information on the kineticconstants could be obtained expediently. This study providesvaluable insight into the structural and functional basis ofhTNF-�–mAbs interactions by quantitating the strength andspecificity of their binding.

Acknowledgements

We are grateful to the National Natural Science Founda-tion of China (No. 20035010 and No. 20075033), National863 Hitech Project and Chinese Academy of Sciences forfinancial support of this work.

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