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Biosensors and Bioelectronics 22 (2006) 409–414

Dioxin immunosensor using anti-2,3,7,8-TCDD antibody which wasproduced with mono 6-(2,3,6,7-tetrachloroxanthene-9-ylidene) hexyl

succinate as a hapten

Jong-Won Park a, Shigeru Kurosawa a,∗, Hidenobu Aizawa a, Hirokazu Hamano b,Yoshitsugu Harada b, Satoko Asano c, Yutaka Mizushima c, Megumu Higaki c,∗a Institute for Environmental Management Technology, National Institute of Advanced Industrial Science and Technology (AIST),

1-1 Higashi, Tsukuba 305-8565, Japanb Biochemical Research Laboratory, Morinaga Milk Industry Co., Ltd., 1-83-5, Higashihara, Zama, Kanagawa 228-8583, Japan

c DDS Institute, The Jikei University School of Medicine, 3-25-8 Nishi-shimbashi, Minato-ku, Tokyo 105-8461, Japan

Received 7 September 2005; received in revised form 13 February 2006; accepted 4 May 2006Available online 8 June 2006

bstract

To detect dioxin using a quartz crystal microbalance (QCM) immunosensor, anti-2,3,7,8-tetrachloro-p-dibenzodioxin (TCDD) monoclonalntibodies (MAbs) were produced as types of IgG1 and IgM, with mono 6-(2,3,6,7-tetrachloroxanthene-9-ylidene) hexyl succinate (as a hapten)onjugated with bovine serum albumin (dioxin-BSA). Furthermore, ScFv was generated from hybridoma-producing IgG1 MAb. Among thesentibodies, ScFv showed excellent capability for dioxin detection using QCM immunosensors.

2006 Elsevier B.V. All rights reserved.

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eywords: Dioxin-BGG; Anti-dioxin monoclonal antibody; ScFv; QCM

. Introduction

Polychlorinated dibenzo-p-dioxins (PCDDs) and polychlo-inated dibenzofurans (PCDFs) are well-known groups ofidespread, persistent and extremely toxic environmental pol-

utants. They are typically found in commercial chlorophenolsnd their derived products, emissions from incineration sourcesnd in by-products from pulp and paper mills (Safe et al.,990). Nevertheless, these ubiquitous PCDDs and PCDFs existn a myriad of environmental matrixes including air, soil, sed-ment, fish and human adipose tissues and milk (Sakurai etl., 1996). For those reasons, regulatory agencies have inves-igated their potential for adverse effects on human health andnvironmental damage (Poland and Knutson, 1982; Kimbrough

t al., 1984; Muto and Takizawa, 1992; Giesy et al., 1994).he PCDDs have 75 positional congeners; their toxicity differsidely (Rappe, 1984). Particularly, 2,3,7,8-tetrachlorodibenzo-

∗ Corresponding authors. Tel.: +81 29 861 4746; fax: +81 44 977 8111.E-mail addresses: shigeru-kurosawa@aist.go.jp (S. Kurosawa),

h0402@jikei.ac.jp (M. Higaki).

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956-5663/$ – see front matter © 2006 Elsevier B.V. All rights reserved.oi:10.1016/j.bios.2006.05.002

-dioxin (TCDD) is known as the most toxic congener. The LD50alue of this compound is 0.6–2.0 �g/kg in guinea pigs (Kocibat al., 1978). The toxicity varies according to the congeners.herefore, analyses for PCDDs require identification and quan-

ification of each isomer and congener.In spite of the great need to monitor PCDDs and PCDFs, the

nly analytical technique with sufficient sensitivity (parts perrillion) and selectivity for determination of PCDDs including,3,7,8-TCDD is a combination of high-resolution gas chro-atography and high-resolution mass spectrometry (USEPA;ethod 1613). That analytical technique is expensive; more-

ver, it requires specialized equipment, highly trained analystsnd a dedicated laboratory. Depending on the amount of sam-le preparation needed, the analysts can take several days toomplete an investigation. Consequently, screening of numer-us samples has been limited and supplemental methods are inemand (Crummett et al., 1986). Ideally, these methods woulde sensitive, rapid, cost-effective, field-portable and specific for

he most toxic dioxin congeners.

Immunoassays satisfy many of those criteria and have beensed widely in environmental analyses and medical diagnostictudies because of their extremely high selectivity and sensi-

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e(v(wfmaiww(aascsEpB1ammimmunochromatography (Wako Pure Chemical Industries Ltd.,Osaka, Japan). Titration and competition were carried out withELISA using hapten-BGG (Fig. 1) as an antigen. Microtiterplates (Nunc, Roskilde, Denmark) were coated with 100 �l of

10 J.-W. Park et al. / Biosensors an

ivity. Several government and non-government organizationsn many countries are currently involved in the evaluation ofmmunoassays and the proposal of guidelines for their use aspproved analytical methods (Schwartz, 1995). Enzyme-linkedmmunosorbent assay (ELISA) and quartz crystal microbalanceQCM) immunosensors are usually used in the immunoassayeld; they are indispensable techniques for determining verymall amounts of environmental pollutants (Hayashi et al., 2004;hons and Dorman, 1972; Sota et al., 2002). In addition to theLISA method, QCM immunosensors present a more beneficial

echnique because such sensors require no labeled molecules,uch as fluorescence reagents or enzyme for detection of desirednalytes.

Immunoassay analyses of halogenated biphenyls, chlorinatedydrocarbon insecticides, halogenated dibenzo-p-dioxins andibenzofurans have not been as frequent as those of more water-oluble species (Meulenberg et al., 1995). Immunoassays areypically aqueous-based systems. Therefore, the low water sol-bility of these compounds makes the use of immunoassaysore challenging. Attempts to detect PCDDs by immunoassays

ave been reported (Albro et al., 1979; Kennel et al., 1986).he reported radioimmunoassay (RIA) was time-consuming andtilized polyclonal antibodies (PAbs). Monoclonal antibodiesMAbs) developed by Kennel et al. lacked selectivity for freeioxins in solution. Stanker et al. generated MAbs to dioxinnd developed MAb-based ELISA (Stanker et al., 1978, 1987;anderlaan et al., 1988; Watkins et al., 1989). The selectivityf the ELISA closely resembled that of the RIA. The opti-ized assay detected 200 pg/well 2,3,7,8-TCDD as the IC50

the analyte concentration giving 50% inhibition). Langley etl. reported development of PAb-based ELISAs that detectedng/well 2,3,7,8-TCDD as the IC50. Recently, Harrison andarlson (1997) developed a tube test and a microplate test usingne of Stanker’s Mabs; the two formats displayed respectiveetection limits of 100 and 25 pg/well 2,3,7,8-TCDD. Althoughhese results have led to increased sensitivity, further improve-

ents are necessary to approach the detection limits of GC/MSechniques (1 pg of 2,3,7,8-TCDD or less in a 1 g sample).

In this study, both IgG1 and IgM types of new anti-2,3,7,8CDD monoclonal antibodies (MAbs) were obtained with mono-(2,3,6,7-tetrachloroxanthene-9-ylidene) hexyl succinate con-ugated bovine gamma-globulin (dioxin-BGG) as a non-toxicew hapten. In addition, ScFv was generated from hybridoma-roducing IgG1 MAb. Subsequently, we developed a QCMmmunosensor for 2,3,7,8-TCDD via the evaluation of the affin-ty test according to the competitive reaction between dioxin-GG and 2,3,7,8-TCDD with Dx-� IgG1, Dx-M-� IgM andx-� ScFv No. 12H.

. Experiments

.1. Synthesis of new hapten-carrier

A complex of mono 6-(2,3,6,7-tetrachloroxanthene-9-lidene) hexyl succinate (as a hapten) and bovine serum albuminBSA)/bovine gamma-globulin (BGG; as a carrier), which werebtained from Sigma Chemical Co. (St. Louis, MO, USA),

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electronics 22 (2006) 409–414

as synthesized as follows: 10 mg (0.02 mmol) of mono 6-2,3,6,7-tetrachloroxanthene-9-ylidene) hexyl succinate (kindlyrovided by Morinaga Milk Industry Co. Ltd., Kanagawa, Japan)as dissolved in 5 ml of dimethylformamide under argon atmo-

phere and stirred for 20 min at reaction time after adding 10 �lf tributylamine and 4 �l of isobutylchloroformate (solution A).eanwhile, 70 mg of BSA/BGG was dissolved in 3 ml of dis-

illed water and adjusted to pH 9.0 with 0.1N NaOH. Then theolume was increased to 6 ml with distilled water and 6 ml ofimethylformamide (solution B) was added. Solution A wasdded drop-wise to solution B, which was stirred on ice bydjusting pH 8.0–9.0 with 0.1N NaOH. This solution was mixedor another 3 h on ice. The mixture was dialyzed overnightgainst distilled water and freeze-dried to obtain 70 mg of whiteowder.

.2. Production of monoclonal antibodies (MAb)

Hybridoma was produced as described previously (Schulmant al., 1978; Stanker et al., 1987). In brief, 100 �g of hapten-BSAFig. 1), mixed with an equal volume of Freund’s complete adju-ant, was administered intraperitoneally (i.p.) to BALB/c mice6-week-old females; SLC Inc., Shizuoka, Japan). The animalsere housed in a specific pathogen free environment and allowed

ree access to food and water. The Institution Committee of Ani-al Experiments at St. Marianna Medical University approved

ll animal studies. The same dose of hapten-BSA mixed withncomplete Freund’s adjuvant was administered i.p. 3 and 5eeks later. The increase of the antibody titer in the serumas examined using ELISA with hapten-BGG as an antigen

Fukuya et al., 2002; Takatsu et al., 2005). Mice with the highntibody titer received 100 �g of hapten-BSA intravenouslynd were killed 3 days after to obtain the spleen. The isolatedpleen B cells were fused with five times less PAI myelomaells by PEG methods. Hybridoma cells were selected by HATelection, and antibody-producing cells were selected usingLISA as described previously. After sub-cloning, antibody-roducing hybridoma cells were inoculated into the abdomen ofALB/c mice pretreated with pristane. Ascites were obtained–2 weeks later and MAb was purified by salting out withmmonium sulfate and by protein G column (Amersham Phar-acia Biotech Inc., Piscataway, NJ). The purity was deter-ined using SDS-PAGE and the isotype was determined using

ig. 1. Chemical structure of new hapten-carrier: hapten-BSA was used forroduction of anti-dioxin monoclonal antibody (MAb) and hapten-BGG wasmployed for detection and characterization of MAb.

J.-W. Park et al. / Biosensors and Bio

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ig. 2. Chemical structures of competitors: dioxin-like compounds were usedor evaluation of the purified antibody by competitive assay.

�g/ml hapten-BGG per well, blocked with 25% of Block AceDainippon Pharmaceutical Co. Ltd.), and incubated with serialilutions of MAb solution. Then alkaline phosphatase-labeledoat anti-mouse IgG (H + L) (50 �l; Zymed Laboratories Inc.Invitrogen Corp.), San Francisco, CA) was added, followedy p-nitrophenyl phosphate (Kirkegaard & Perry Laboratories,nc., Gaithersburg, MA). Absorbance at 405 nm was measuredsing a microtiter plate reader (Bio-Rad Laboratories Inc., Rich-ond, CA). The compounds employed for the competition assay

re shown in Fig. 2. Serial dilutions of these compounds start-ng from 25 �g/ml were incubated with 0.25 �g/ml of MAb for5 min at 37 ◦C and examined using ELISA as described.

.3. Production of ScFv

We generated a single-chain fragment (ScFv) composedf heavy-chain and light-chain variable domains connectedy a polypeptide linker (Gly4Ser)3 following the manufac-urer’s protocol (Amersham Pharmacia Biotech Inc., Piscat-way, NJ) (Winter et al., 1994). In brief, mRNA was isolatedrom hybridoma cells producing MAb Dx-�. Heavy and lighthain (VH and VL) genes of antibody were amplified sepa-

ately and assembled into ScFv genes with a linker DNA byCR. The ScFv genes were ligated into the phagemid vectorCANTAB5E and the ligated sample was transformed into com-etent Escherichia coli TG1. Transformants were infected with

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electronics 22 (2006) 409–414 411

13K07 helper phage to yield recombinant phages displayingcFv fragments as a g3p fusion protein on the surface of thelamentous phage M13. The positive clone was selected withLISA (hapten-BGG as an antigen) from the enriched phages.igh-affinity ScFv phages were enriched through affinity selec-

ion in microtiter-wells coated with hapten-BGG conjugate.ecombinant phages were selected after one round of panningnd used directly to re-infect E. coli TG1 cells. The E. coli non-uppressor strain HB2151 was infected with an antigen-positivehage clone, previously screened by ELISA, to express solu-le ScFv fragments. Soluble ScFv was then purified with anti-Eag affinity column chromatography and detected using West-rn blot analysis with anti-E Tag-HRP (Amersham Pharmaciaiotech Inc.).

.4. Instrumentation

All QCMs were AT-cut with a basic resonance frequency ofMHz. The crystal consisted of a quartz wafer that was placedetween two gold electrodes (8 mm × 8 mm × 0.185 mm) thatere purchased from Nihon Dempa Kogyo Co. Ltd. The

aboratory-made transistor–transistor logic (TTL) electronic cir-uit was used as the oscillating circuit (Kurosawa et al., 2000).he oscillator was supplied with 5 V by a dc voltage regulator. Aniversal counter (Iwatsu-SC7201; Iwatsu Co. Ltd.) monitoredhe frequency change from the oscillator. The data collectionnd processing system was programmed using Labview soft-are (Version 6.0.2i; National Instruments Corp., TX, USA). It

ccepted the data from the counter and displayed it in real timen a plot of the frequency shift on the computer monitoring aPIB interface (National Instruments Corp.). The temperature

25 ± 0.1 ◦C) was strictly controlled for all experimental stepssing an incubator (Yamato Scientific Co. Ltd., Tokyo, Japan).

.5. Measurements

All gold surfaces of bare QCMs were cleaned thoroughly formin in a freshly prepared 1:3 mixture of H2O2 and H2SO4efore the thiol compounds were introduced. After rinsing withater, acetone and ethanol, the QCMs were dried sufficientlynder an N2 atmosphere. All bare QCMs were measured forheir initial frequency for 5 min, which was determined as F0.

After each experimental step, such as surface activation toeasure the frequency shift, the QCM was rinsed carefullyith distilled water and dried thoroughly under N2 gas. Thescillating frequency (F1) was measured in the air state afterach experimental step to determine the mass change on theurface. Thereafter, the measured oscillating frequency wasalculated through comparison with the initial frequency (F0)f each QCM. It was translated into the mass change usingauerbray’s equation. Similarly, to calculate the frequency shiftor further steps, such as antibody immobilization or antigen-ntibody binding, the corresponding oscillating frequency (F )

as calculated using the oscillating frequency from the previ-us step (Fn−1). All experimental results were obtained using sixCMs and the standard deviation was expressed as experimental

rror.

412 J.-W. Park et al. / Biosensors and Bioelectronics 22 (2006) 409–414

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Fig. 3. Titration of Dx-� and Dx-M-� MAbs using ELISA method.

.6. Surface activation on gold surface andmmunoreaction

Gold electrode surfaces on the QCMs were chemically acti-ated to immobilize the anti-TCDD antibody. All QCMs weremmersed in a solution of 10 mM cyteamine (Wako Pure Chem-cal Industries Ltd.) for 60 min; they were then placed in a% glutaraldehyde (Wako Pure Chemical Industries Ltd.) solu-ion for 60 min to attach the aldehyde group to the gold elec-rodes. The anti-TCDD antibody solution (30 �l) was injectednto the chemically modified gold electrode of the QCM; itas subsequently immobilized for 90 min by covalent bind-

ng at room temperature. In addition, the remaining alde-yde groups were blocked with 20 mM glycine, which waslso purchased from Wako Pure Chemical Industries Ltd.,fter antibody immobilization. Furthermore, the water-solubleoly[2-methacryloyloxyethyl phosphorylcholine(MPC)-co-n-utyl methacrylate (BMA) (PMB)] as a stabilizer was treatedo avoid non-specific binding from a competitive reaction and tochieve the immunologic activity of the immobilized antibodyPark et al., 2003).

The competitive immunoreaction was performed using mix-ures (30 �l) of various concentrations of TCDD and 100 ng/mlf dioxin-BGG for 60 min at 25 ◦C. For the all-antigen-antibodyeaction, the reaction media used a 25% DMSO mixture withhosphate buffered saline (PBS, 10 mM, pH 7.4).

. Results and discussion

.1. New hapten-carrier and titration of MAbs

Fig. 1 shows a complex of hapten:BSA (25.3:1) as anmmunogen; that of hapten:BGG (5.4:1) was synthesized as aetection antigen. Two kinds of anti-TCDD MAb (Dx-� as IgG1,

x-M-� as IgM) were obtained. Fig. 3 shows the titration of

hese anti-TCDD antibodies. The respective 50% reaction con-entrations of Dx-� and Dx-M-� were 0.125 and 2.00 �g/ml.onsequently, the immunological affinity of Dx-� was higher

han that of Dx-M-�.

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ig. 4. Detection of cross-reactivity of Dx-� with competitive immunoassaysing ELISA method.

.2. Cross reactivities of Dx-α

The competition experiment result is shown in Fig. 4. The Dx-cross-reacted with 5-[(3,7,8-trichlorodibenzo-p-dioxin-2-yl)

arbamoyl] pentanoic acid, 2-amino-3,7,8-trichlorodibenzo-p-ioxin (Dx-NH2) and 2-nitro-3,7,8-trichlorodibenzo-p-dioxinDx-NO2) (50% competition; 1, 4 and 12 �g/ml, respectively),ut not with 2,3,7-trichlorodibenzo-p-dioxin. The result indi-ated that Dx-� might react with position 2 of dioxin-likeompounds. Those data are not shown here, but both Dx-�nd Dx-M-� reacted with 2-adibamide-3,7,8-trichlorodibenzo--dioxin-BGG conjugate (Kennel et al., 1986).

.3. Characterization of ScFv

Soluble pure ScFv No. 12H produced in the bacterialeriplasm is shown using Western blot analysis (Fig. 5). ThecFv is detected as a single band, which indicates that purifiedcFv was obtained.

.4. Antibody immobilization and competitivemmunoreaction

After antibody production, because all antibodies weremmobilized with 50 �g/ml at the QCM surface, dioxin-BGG100 ng/ml) was injected to evaluate the blank response for theompetitive reaction. The frequency responses were indicateds 150 ± 84, 161 ± 47 and 218 ± 52 Hz, respectively, when Dx-

IgG, Dx-M-� IgM and Dx-� ScFv No. 12H were reactedith dioxin-BGG (Table 1). The highest response for dioxin-GG was indicated when ScFv type was used. In addition,

gM type antibody showed a higher response than did IgGype. These results are inferred to occur because of the mannerf antibody immobilization on the QCM. All antibodies weremmobilized via covalent binding between aldehyde groups and

heir amino groups. Although antibodies immobilized stronglyn the QCM surface, the manner of immobilization was not wellrdered. For that reason, the epitopes of antibody were inter-ered after the immobilization step. The results reflect that the

J.-W. Park et al. / Biosensors and Bioelectronics 22 (2006) 409–414 413

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Fig. 6. Calibration curves for 2,3,7,8-TCDD according to the competitive reac-tion between 2,3,7,8-TCDD and dioxin-BGG when Dx-� and ScFv No. 12Hwere, respectively, immobilized on the QCM; the concentrations of dioxin-BGGwere fixed as 100 ng/ml during all competitive reactions. All error bars wereachieved from three experiments.

Table 2Sandwich immunoreaction using IgG and IgM type anti-TCDD antibody after1st immunoreaction using dioxin-BGG

Antibody Frequency shift by sandwich reaction (Hz)

Dx-� (IgG1) Dx-M-� (IgM)

Dx-� (IgG1) 12 ± 9 47 ± 19Dx-M-� (IgM) 34 ± 14 77 ± 14Dx-� ScFv No. 12H 89 ± 16 159 ± 9

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pitopes of ScFv were highly oriented toward the outside aftermmobilization.

According to the blank experiment for the competitive reac-ion, Dx-� ScFv No. 12H and Dx-� IgG were selected to achievehe calibration curve for 2,3,7,8-TCDD via the competitive reac-ion. Fig. 6 shows calibration curves for 2,3,7,8-TCDD using twoifferent types of antibodies. When the concentration of dioxin-GG was fixed at 100 ng/ml, calibration curves were achievedith various concentrations of 2,3,7,8-TCDD (from 0.001 to00 ng/ml). As Fig. 6 shows, 2,3,7,8-TCDD was detected until.1 ng/ml using Dx-� ScFv No. 12H. On the other hand, it had aroblem detecting 2,3,7,8-TCDD when IgG type antibody wasmmobilized on the QCM.

.5. Sandwich immunoreaction using several types ofioxin antibodies

The sandwich immunoreaction was considered to improvehe frequency response of immunosensors as another applica-

ion with three different types of antibody. Following bindingetween the antibody and 100 ng/ml dioxin-BGG, we injectedx-� IgG and Dx-M-� IgM to amplify the detection sensitivity.able 2 shows the respective results of the sandwich immunore-

able 1ifferent responses for three different types of anti-2,3,7,8-TCDD antibodies

or dioxin-BGG

nti-2,3,7,8-TCDD monoclonal antibody Frequency shift (Hz)

x-� (IgG1) 150 ± 84x-M-� (IgM) 161 ± 47x-� ScFv No. 12H 218 ± 52

ll antibodies were immobilized with 50 �g/ml on the QCM surface. Frequencyhifts were achieved, respectively, through binding between respective antibod-es and dioxin-BGG (100 ng/ml).

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ll antibodies were immobilized with 50 �g/ml on the QCM surface. Firstmmunoreaction was achieved, respectively, through the binding betweenespective antibodies and dioxin-BGG (100 ng/ml).

ction. Normally, the sandwich immunoreaction is used to detecthe extremely low concentration of analytes. However, the sen-itivity amplification results using two different antibodies wereot good. We are progressing toward preparing the anti-TCDDntibody to obtain high affinity for 2,3,7,8-TCDD.

. Conclusion

Two types of new hapten-carrier – hapten-BSA and hapten-GG – were synthesized to produce and detect anti-TCDDonoclonal antibody. Both IgG1 and IgM types of anti-TCDDAbs were obtained using hapten-BSA. The immunological

eactivity of these MAbs was characterized using ELISA withapten-BGG and IgG1 Dx-� showed higher affinity to TCDD.esults of the competition assay indicated that Dx-� might reactith position 2 of chlorinated dioxin-like compounds. Pure ScFvas obtained: it was produced by amplification and ligation of

DNAs of heavy chains and light chains of Dx-�-producingybridoma. Based on those results, the possibility of its applica-ion into the immunosensor was evaluated using a QCM device

ith Dx-�, Dx-M-� and Dx-� ScFv No. 12H. After antibodies’

espective immobilization on the QCM via covalent binding,heir effectiveness was evaluated from the calibration curvey the competitive immunoreaction between hapten-BGG and

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,3,7,8-TCDD. Among these three kinds of antibody, ScFv No.2H showed an excellent possibility for dioxin detection usingQCM immunosensor.

cknowledgements

This work was supported financially in part by a Grant-in-id for Scientific Research on Priority Areas (16040217) fromEXT, for “Application of permselective and biocompatibleembranes for the improvement quartz crystal microbalance

iosensors” from the Daiwa Anglo-Japanese Foundation and forStudy on monitoring of environmental risk compounds, such asioxins and endocrine disruptors using sensing systems” fromhe Ministry of the Environment, Japan.

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