Production ofMono(;lonal Antibody agajnst Epstein'Barr virus Early Antigen and
ltsApplicatiQn to Detection of Antitu㎡or Promgting Activity
Nahoko lsHIKAwAAbstracl ｡
Hybridoma ceII line，desig!lated 2D3-E7，producing a monoclonal antibody against 4 8 kDa-early antigen of Epstein-Barr vims was established by the fusion of mouse myeloma cells with spleen cells from a mouse immunized with P3HR-1
cens treated with sodium ，z-butyrate（lmM）and a tumor promoter，12-a-tetl;adecanoylphorbol-13-acetate （TPA，40 ng/ml）.Theantibody was purined from the culture supematant by precipitation with ammonium sulfate and column chromatographies on Sephadex G-2 00， Protein G-Sepharose， and Mono Q. lmmunoblotting analysis showed that the purified antibody corliugated with horseradish peroxidase reacted with TPA （80 ng/inl）-induced p（jlypeptide of 48 kDa in Rii cells and its induction was inhibited by curcumin（10μg/ml），an antitumor promoter from turineric.17hese results indicated that the immunoblotting analysis can be ilsed in a confirmation test for detection of antitumor promoting activity using the monoc!ona1 antibody col!jugated with the peroxidase.Furthermore，adhesion of Rii cells caused by treatment of TPA was inhibited by the curcumin in a dose-dependent manner，suggesting tllatthe inhibition of adhesion can be also
used in a preliminary test for detection of antitlimor promoting activity･
Key word:monoclonal antibody ； Epstein-Bafr virus ； early antigen ； antitumor promoter ； TPA
RI!ji cells and P3HR.1 cells ale-human B lymphoblastoid ce11 1ines cafTying the geno㎡e of Epst｛iin-Barrvims （EBV）. When the cells are treated with tumor promoters，12-θ-tetradecanoyl-phorbol-134cetate（17PA），EBv is produced in P3HR-1 cells but not in R司i cells. ln R咄cells，TPA induces the syntheses of EBv early antigens which divide into difnlse and restricted components based on their stain-ing pattems in EBV-infected cells by immunofh16rescence
using human sera of patients with infbctious mononucleosis. An inhibition assay of the TPA-induced antigen measured by immunofluorescence using human sera has been used for the
firstscreening of inhibitors of tumorpromotion.（1.2）ln our previous paper， we showed that the immunobotting analysis usiniμhe polyclonal antiserum against P3HR-1 cells treated withTPA can be detected the 48-kDa polypeptide iil R司i cells treated with TPA and the assay is useful confirmation test for detecting antitumor promoters. （3’4）However，stable supply of the antiserum is limited.Therefore，we attempt-ed to produce a monoclonal antibody agajnst early antigen of EBV.This paper describes the preparation，purificalion， and some appHcation of the antibody･
sources of materials used in this study were
follows : male mice （BALB/c）fiom
Japan Chafles River
OCO(CH2)12CH3ＯＣＯＣＨ３ ＣＨ２０Ｈ ２Ｃ Ｏ ○ H3CO Ｈ Ｏ ’ OCH3 ＯＨ
Structures of TPA （ A）and
Ti,eh.Bu】I.Fac. Agr. Kagawa univ･ ，vol. 56，2004.
15Q0，hyp6xanthine-aminopterin-th畑id畑e(HAT)and HT media supplements.2，21-azin6-bis(j-ethylbi面thiionne)-6 -sulfonic acid (ABTS)，and enzymi.li噛ejimiuilosorbent assay(ELISA)kit for the measurement of n10uselgG from Roche Diagnostics (M8nilhei“1， Germa4) ，/Sephadexダ G-100，Protein-G Sepharose，biotinylated goat anti-mouse lgG and isotyping kit from Amersham Biosciences (Uppsal， sweden)，peroxidase-colljugated streptavidinfiom zymed
Lab.lnc. cal Co.，
(San Francisco， CA)，TPA fi･om Sign!a Chemi-(St.Louis，Mo)，andsodiuin yl-biltyratdand
chemical struciuresof TPA
and curcumin are
shown in Fig. 1. '
Animal cell culture
恥ii cells and mouse myeloma cells（ Sp2/O）weregrown at 37℃in Dulbecc（i’smodified Eaglels minimum eSsential medium （Dajnippon PhamlaQeutical Co.Ltd.，0saka）
supplemented with 20 mM N-21-hydfoxyethyl-piperazine一炉-2-ethane sulfonic acid （HEPES）and 10％filtal calf serum （FCS）.EBv pro4ucer P3HR-1 cells were maintained at 33 ℃in RPMI-1640 medium （Dainippon Pharmaceutical C9. Ltd.，）supplemQnted with 10％FCS by replacement of one-third 6resh medium once a week.These celHines were cultured in a C02 （5％）incubator.
Pr6tein measuliemeilt and immunological analysis
Protein was measured by the method of Lowry gz al. with bovine serum ibumin as the standardy5)lmmunoblotting analysis for screening of monoclonal antibody in culture supematan包and ELISA fbr detection of mouse lgG in column chromatographies were done，as described previ-ously.(3.6)The R4ji cellsweresohlbihed with an extraction buffbr[about 106 cells/100 μl of Ca2゛- and Mg2゛-11ee phosphate-buffered saline (PBS)contajning l％Tween 20， 1％sodium deoxycholate，and l mM EDTA].The extract (30μg of protein)wasel6ctrophorese(! on a･ 10％poly-acrylamide slab gel with s(jdium dodecyl sulfate (SDS).(7) Proteins in the gel were electrophoretically transferred to a nitrocellulose sheet and then the sheet was immunostained with the culture supematants，biotinylated anti-mouse lgG (1 : 500 dilution)，'and peroxidas1-cor!jugllted streptavidin (1:1，000 dilution).The immunoreactive bands were deVelOped With a.S01UtiOn COntaining O .05％3，31-diaminO-benzidine tetrahydrQchloride，0.01％ H202，and 0.075％ cobalt chloride and stopped by rinsing the sheet With cold
water. ( 8 )
Fordetecti(?110f'mφ一e lgG in columll chio°8to“
graphies by ELISA，microt一ri,eil (Nunc-'lmmunop!8te･ Denmafk)was coaieci iitii eluate frotn the column. The wellswere incubatedwitibiotiliylated anti-mouse lgG (1: 5， 000 dilltioll)・ peroxi4se-c叫jugated strepta｀'idi11(1: 5，000 dilution)，and thenﾚsubstrate bufier conlajning ABTS(1 mg/ml).Absorbancewas measured at 415nm on microplate reader (Model 450レBio-Rad).
Produdon and purincation of monoclonal antibody Hybridoma ce11 1ine producing a monoclonal antibody against 48 kDa'early antigen of Epstein-Barr virus was established by the filsion of mous6 myeloma cells with spleen cells from a mouse immunized with P3HR-1 cells treated.with sodium yzjbutyrate(lmM)and TPA(40 ng/ml).The monoclonal antibody was fraciionated from culture supematant by precipitation with ammonium sulfate (50％ saturation)and by sequential column chromato-graphies on Sephadex G-200，Protein G-Sephafose，and Mono Q (Amersham Biotech).
Production of monoclonal antibody
にP3HR-1 ･cells（about 7パ×107 cells）wert treated with TPA･（40ng/ml）and sodium ，1-butyrate（1mM）in･ the RPMI-1640 medium,（45 ml）at 37℃for 48 hr to increase EBV prQdu6tion.Two mice （4-week old）were immunized
4 timeSISUbCUtaneO一ly With the Ce11S （abOUt 107 Ce11S） emulsified ill Freund’s complet6 a（!juvantand once intra-periloneally with the same cells at 14-day intervals.At three days after the last immunization，the spleen cells were harvested from the mice and then fused with the mouse mydlomacells in a ratio of 5 : 1 by treatm色nt with PEG 1500.（6）Fus色d cells were seeded in 192 wens of 48-we11 culture plates and incubated at 3 7℃in HAT medium. The HAT medium was changed every three days prior to screening. After 3 weeks ,in a C02 incubator，the culture fluids were examined ft）rreactivity with the 48 kl）かearly antigen of EBv in detergent-solubilized extrllct（ifR咄cells treated with TPA（40 ng/ml）and sodium 7z-butyrate（1 mM）at 37℃for 48 hr by immunoblotting analysis with a
Screener Blotter（Sanplatec Corp･
−−-−･− −●sJ∽∼･ ぃJ−･r−−’−−rss ｀゛｀″“｀“/‘｀りUIIUUllla cells f¥om three wells were cloned twice by 】imiting dilution
in HT medium containing 5％BriClone（culture fi11rateof interleukin 6-producing cells，Dainippon Pharmaceutical Co.
K.0KAzAKl gz al，:Monoclonal antibody against early antigen of EB virus
Ltd.，0saka).Finany a hybridoma clone,･ designated 2D3 -E7，was established and the clone cells were passaged serially in HT medium.The lg class，subclass，and light chain type of the monoclonal antibody were identified by use of an isotyping kit.Reactivity of the 2D3-E7 culture supematant with typing stick carrying goat antibodies spe-cific for the different types of peptide chain showed that the clone secreted lgGl with a kappa light ch4in.
Pllrincl･tion of monoclonal antibody
The monoclonal antibody was fractionated from 2D3-E7 culture supematant （325 ml）by precipitation with ammo-nium sulfale（50％ saturation）and by gel filtration on
Sephadex G-200 column （Fig.2）.The fractions containing antibody from the Sephadex G-200 column chromatography were poQled,-concentrated，and put on a afnnity column of Protein G-Sepharose （Fig.3）.The antibody solution-f¥om the Protein G-Sepharose column chromatography was con-centrated and put on a column （5父50 mm） of Mono Q （Amersham Biosciences）equilibrated with 20 mM
Ttis-HCl buff15r（pH 8.0）・ The column was eluted with linear gradient of NaCl in the same buffer at a flow rate 0.5
UXU08Z 49 ．ｓｑＶ ２ １ ０ 0 20 40 60 80 Fraction No･（2m1/tube） Fig.2.Gel Filtrationon Sephadex G-100.
２ uxugllr 41 ．ｓｑＶ １ ０ 100
Sephadex G-100 was equilibrated with 50 mM phos-phate buffer（pH 7.0）and packed in a column （1.6× 66 cm）.Antibody solution from precipitation with ammonium sulfate was put on the column and the col-umn was eluted with the same buffer at a now rate of 6
at 4 15 nm ibr antibody
estimated by ELISA ； estimation of protein content.
absorbance at 280 content nm for UXU08Z ２ １ ’ｓｑＶ ０ ０ １０ ２０ Ｆｒａｃｔｉｏｎ Ｎｏ・（ｌｍ１／ｔｕｂｅ） 25 3 0
Fig.3.Affinity Chromatography on Protein G-Sepharose. The antibody solution （5 ml）was ltlixed with equal volume of binding buffer （0.15 M glycine-NaOH， pH 8.9）.Protein G-Sepharose column was sequential elut- j- A ..‥ ‥ j- ted with water (5㎡1)，tbe binding buffer
. j_之 ゝ ．･．．．
m 1 ) ｈ t t p : / / w w w ．
antibody solution (10 ml)･，thej,liiding bufier (7 ml),･ and ehltion bufi5er (5ml，citrate-NaOH，pH 4.0). vertical arrow indicates monoclonal antibody peak. o ， absorbance at 280 nm for estimation of protein content in eluate from the column.
102,000 78,000 49,500 34,200 28,300 19,900
Ａ: 効 皿 恥 滴 忿 咬 ･ ’
Molecular wei ht markers (A)obtained
and the purifiedmonoclonal antibody
were electrophoresedon a
10％polyacrylamide slab gel with 0.1％SDS.The
was stainedwith Coomassie brnliantblue.
26 Tech.Bu11.Fac.Agr. Kagawa univ.，vol.56，2004
ml/min.The antibody was eluted with about 0.6-0.7M NaCl and antibody solution （0.21 mg of lgﾛ/tnl）was obtained.ln SDS-polyacrylamide gel electrophor6sis，only two bands （50 kDa and 25 kDa） corresponding to heavy and light chains of lgG were detected in the preparation （Fig.4）.The pudfied antibody （0.2 mg） was 9oiugated
with horseradish peroxidase （4 mg， Wako Pure Chemical lnd。Ltd.，0saka）activated by 20 mM NalO4 for 2 hf
at 25℃and the reaction was stopped by incubating with NaBH4（0.4mg）at4℃for 2 hr.（9）The colljugate（0.42
mg of protein/ml）was separated from fiee peroxidase and antibody by gel filtration on a Superose 12 column （Amersham Biosciences）｡
Detection of a皿titumor promoting activity
For application of the puiified m(jnoclonal antibody con-jugated with horseradish to detection of antitumor promoting
activity，we examined the enicts of curcumin，a well-kliown antitumor promoter from turmeric，on the expres-sion of EBV early antigen in Rii cells treated with TPA (80 ng/ml)and sgdium zz7butyrate (2mM).The inducer treat-edRE!ji cells adhered to the bottom of plastic dish and the
２ ３ ４ ５ Ｍ
kDa76 kDa ←49 kDa
M， prestainedmarker proteins f¥om Bio-Rad.
analysis was done， as describedin the
8dheSio･ ゛1s inhibited一by additk）n of curc°｀ill（10μ がml）as same as untreated cells（data not shown）.As shown in rig. 5･two polypeptides of 48 kDa and 54 kDa were detected in inducer.treated cells（lane 2）but not in untreated cells（lane 1）by thQ monoclonal antibody･ .The induction of 48- and 54-kDa p6lypeptides （lane 2）were inhibited by 10μg（lane 5）and 5μg（lane 4）of
curcumin per ml，respectively.
Our previous studies demonstrated that the 4 8-kDa polypeptides induced in TPA-treated R･!ji cells is a diffijse component detectable in both the nucleus and cytoplasm of EBv early antigens and immunobotting analysis using the polyclonal antiserum against P3HR-l ceUs is useful confir-mation test for detecting antitumor promotiers.（3.4）ln this study，we produced a monoclonal antibody against early lmtigen of EBv for stable supply and showed that the antitumor promoting actiψityof curcumiil could be detected within 2 hr by one step immunostaining after blotting be-cause of the labeled monoclonal antibQdy. This study also demonstraled that the 54-kDa polypeptide was detected in TPA-treated RI!ji cells by the monoclonal antibody. This induced antigen may be phosphorylated foml of the 4 8-kDa polypeptide.（lo）No parallelinhibition by curcumin was ob-served 6n the expressions of 4 8-kDa and 54-kDa polypep-tidei･ suggesting that the difference is caused by lower synthesis of the 54’kDa phosphoprotein than that of the 48-kDa polyp6ptide. Therefore，lhe monoclonal antibody againstEBv eafly antigen prepafed in this study will be usefijl as fQr virological research in addition to’detection of antitumor promoting activity in natural products. Further-more，the adhesion of Rii cells caused by treatment of TPA was inhibited by the curcumin in a dose-dependent manner. suggesting that the inhibition of adhesion can be also used in a preliminary test for detection of antitumor promoting activity･
thank Prof. F.Mizuno
of Dept. of Microbiology，
Tokyo Medical College foi supplying P3HR-l
K.0KAzAKl gf α1，:Monoclonal antibody against early alltigen of EB vims
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