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[(hy)20.8(br)15.1(id)20.7(om)19.6(as)-429.4(cu)19.7(lt)20.6(ur)15.1(ed)-429.6(in)-428.5(Hy)20(br)20.9(id)15(om)19.6(a-)19.7(SF)21.2(M)-439.7(m)0(e)18.4(d)0(i)20.7(u)0(m)-429.6(\()0(T)13.9(h)0(e)19.7(r)0(m)19.7(o)]TJ
T*
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[(\()-19.8(G).2(E)-292.8(H)-19.8(ea)-19.8(lt)-19.8(hc)-19.8(ar)-19.8(e)-282.3(U)-19.8(K)-282.9(L)-19.8(td)-19.8(,)-277.8(B)-19.8(u)-5.8(c)-19.8(ki)-19.8(ng)-19.8(ha)-19.8(ms)-19.8(hi)-19.8(r)-4.8(e)-19.8(,)-278.9(E)-19.8(ng)-19.8(la)-19.8(nd)-19.8(\))-4.7(.)]TJ
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0 Tc
[(Flow)-338.7(cytometry)]TJ
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[(Cells)-313(were)-312.8(harvested)-312.8(by)-313.2(brief)-312(exposure)-316.2(to)-307.6(0.25%)-314.2(trypsin/)]TJ
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T*
[(Tesque,)-312.9(Inc.\).)-311.9(After)-312.9(washing)-309.2(with)-316.6(0.1%)-307.3(bovine)-313.2(serum)-307.2(albu-)]TJ
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[(min)-281.1(\(BSA\)/phosph)-9.1(ate-buffered)-288.6(saline,)-283.1(the)-281.1(cells)-290.1(were)-284(treated)]TJ
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(m)Tj
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[(Mab-77\))-188.3(for)-187.5(30)-186.5(minutes)]TJ
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[(at)-372.1(4)]TJ
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1.6012 0 TD
()Tj
/F3 1 Tf
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[(C)-376.5(and)-373.1(then)-374.3(with)-374.2(Oregon)-377.2(green-conjugated)-375(anti-mouse)]TJ
23.5054 67.9242 TD
[(IgG)-320.9(\(1:1000)-318.8(diluted;)-315.8(Thermo)-321.2(Fisher)-318.5(Scientic,)-322.2(Inc.\).)-317.6(Fluor-)]TJ
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[(escence)-405(data)-398.5(were)-399.2(collected)-400.9(using)-402.9(EC800)-393.6(or)-399.6(SA3800)-399(Cell)]TJ
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[(Analyzers)-290.6(\(Sony)-276(Corp.\).)]TJ
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[(Western)-336(blot)-338.1(analysis)]TJ
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9.843 0 0 9.843 323.0361 663.477 Tm
[(Cell)-427.3(lysates)-430.4(\(10)]TJ
/F9 1 Tf
6.7561 0 TD
(m)Tj
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[(g\))-422.6(were)-428(boiled)-422.7(in)-428.5(the)-425.1(sodium)-422.5(dodecyl)]TJ
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[(sulfate)-292.3(\(SDS\))-288.2(sample)-292.3(buffer)-289.9(\(Nacalai)-291(Tesque,)-295.6(Inc.\))-285.4(and)-292.5(pro-)]TJ
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[(teins)-242.9(were)-249.5(then)-241.8(electrophoresed)-247(on)-238.3(5%20%)-244.9(polyacrylamide)]TJ
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[(gels)-402.9(\(Wako)-406.3(Pure)-397(Chemical)-406.5(Industries)-403.6(Ltd.,)-403.2(Osaka,)-403.6(Japan\),)]TJ
T*
[(and)-275.2(were)-278.3(transferred)-280.4(onto)-275.2(polyvinylidene)-277.5(diuoride)-276.1(\(PVDF\))]TJ
T*
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[(Mab-)5.6(77\))-345.8(and)-345.8(a)-1.4(n)5.5(ti-)]TJ
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(b)Tj
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[(-actin)-346.3(\(clon)5(e)-353.2(AC-1)5(5;)]TJ
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[(pero)5.3(xid)5.3(a)-1.6(se-)5.4(c)-1.6(on)5.3(jug)5.3(a)-1.6(ted)-196.2(a)-1.6(nt)5.2(i-mouse)-197.4(I)5.4(gG)-197.1(\(1)5.3(:100)5.3(0)-196.2(d)-.4(il)5.2(uted;)-196.4(A)-1.3(g)5.3(ilent)]TJ
T*
[(Techno)5.3(logi)5.2(es,)-331.1(Inc.,)-331(Santa)-329.9(C)-1.5(lara)]TJ
12.3488 0 TD
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[(,)-331.3(C)-1.6(A\),)-337(a)4(nd)-334.7(were)-330.1(nal)5(ly)-334.7(d)5.1(e)-1.8(vel-)]TJ
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[(oped)-328.3(u)5.7(sin)5.7(g)-328.3(Immuno)5.7(Star)-328.3(LD)-329.2(\(Wako)-328.3(P).2(u)5.7(r)0(e)-329.5(C)-1(h)5.7(emi)5.6(cal)-328.5(I)5.8(ndu)5.7(stri)5.6(es)]TJ
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[(Ltd)5.3(.)-2.7(\))-276.9(u)-.4(si)5.2(ng)-276.9(a)-283.8(S)5.5(ayaca-)5.4(Imager)-276.8(\(DRC)-277.9(Co.)-279.2(L)4.1(td.,)-279.2(Tokyo)5.3(,)-279.2(J)-.2(ap)5.3(an\))5.4(.)]TJ
/F8 1 Tf
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0 Tc
[(Determination)-337.6(of)-336.7(the)-334.8(binding)-332.2(afnity)]TJ
0 -1.2203 TD
[(using)-338.5(ow)-329.2(cytometry)]TJ
/F3 1 Tf
9.843 0 0 9.843 323.0361 470.3243 Tm
[(A431)-529.4(and)-522.8(SK-BR-3)-528.8(\(2)]TJ
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10.0161 0 TD
()Tj
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(10)Tj
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(5)Tj
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[(cells\))-527.3(were)-531.7(suspended)-524.5(in)]TJ
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(100)Tj
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(m)Tj
/F3 1 Tf
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[(L)-273(o)0(f)-272.9(serially)-276.2(diluted)-274.2(mAbs)-271.3(\(0.6)-168(ng/mL10)]TJ
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17.0372 0 TD
(m)Tj
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[(g/mL\),)-276.4(and)]TJ
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[(Alexa)-406.3(Fluor)-402.9(488-conjugated)-405.1(anti-mouse)-406.3(IgG)-401.6(\(1:1000;)-400.7(Cell)]TJ
T*
[(Signaling)-316.6(Technology,)-311.9(Inc.,)-310.8(Danvers,)-319.4(MA\))-310.3(was)-310.4(then)-316.7(added.)]TJ
T*
[(Fluorescenc)-10.8(e)-607(data)-611.6(were)-612.3(collected)-608.2(using)-610.2(a)-612.8(cell)-606(analy)-8(zer)]TJ
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[(\(EC800;)-303.8(Sony)-303.7(Corp.\).)-301.3(The)-299.4(dissociation)-309.4(constants)-300.1(\()]TJ
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(K)Tj
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(D)Tj
9.843 0 0 9.843 526.7904 415.5022 Tm
[(\))-300.6(were)]TJ
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[(calculated)-293.7(by)-284.4(tting)-283.3(the)-281.1(binding)-284.4(isothe)-7.8(rms)-280.8(using)-287.7(the)-281.1(built-in)]TJ
T*
[(one-site)-368.3(binding)-365.1(models)-366(in)-365.2(GraphPad)-368.9(PRISM)-364.2(6)-369.7(\(GraphPad)]TJ
0 -1.1174 TD
[(softwa)-8.5(re,)-277.5(Inc.,)-282(La)-281(Jolla,)-280.9(CA\).)]TJ
/F8 1 Tf
8.9663 0 0 8.9663 313.0582 359.3196 Tm
[(Immunohistochemical)-340.3(analyses)]TJ
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9.843 0 0 9.843 323.0361 342.8787 Tm
[(Histologic)-249.5(sections)-241.4(of)-244.1(4-)]TJ
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9.803 0 TD
(m)Tj
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[(m)-238.5(thickness)-248.3(were)-243.7(deparafnized)]TJ
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[(in)-203.9(xylene)-204.9(and)-200.3(then)-201.5(rehydrated)-204.6(and)-206(autoclaved)-206(in)-198.2(citrate)-207.4(buffer)]TJ
T*
[(\(pH)-217.2(6.0;)-208.5(Agilent)-211.7(Technologies,)-217.2(Inc.\))-210.5(for)-210.6(20)-215.3(minutes.)-209.4(Sections)]TJ
0 -1.1174 TD
[(were)-502.9(then)-495.3(incubated)-497.5(with)-500.9(1)]TJ
/F9 1 Tf
12.0205 0 TD
(m)Tj
/F3 1 Tf
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[(g/mL)-502.3(of)-491.7(primary)-499.7(mAbs)-495.9(for)]TJ
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[(1)-588.6(hour)-586.1(at)-591(room)-587.3(temperature)-588.4(and)-592(then)-587.4(treated)-587.4(using)-587.2(an)]TJ
T*
(Envision)Tj
/F7 1 Tf
3.5653 0 TD
(+)Tj
/F3 1 Tf
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[(kit)-377.9(\(Agilent)-379.8(Technologi)-9(es,)-375.2(Inc.\))-383.3(for)-377.6(30)-382.3(minutes.)]TJ
-4.4983 -1.1174 TD
[(Color)-657.5(was)-650.2(developed)-658.6(using)-650.5(3,3-diaminobenzidine)-658.6(tetra-)]TJ
0 -1.1116 TD
[(hydrochloride)-460.4(\(Agilent)-448.9(Technologi)-9(es,)-450.1(Inc.\))-452.4(for)-452.5(2)-450.4(minutes,)]TJ
T*
[(and)-551.6(sections)-546.7(were)-549(then)-552.9(counterstained)-550.1(with)-546.9(hematoxylin)]TJ
0 -1.1174 TD
[(\(Wako)-556.1(Pure)-546.8(Chemical)-556.2(Industries)-553.4(Ltd.\).)-548.2(The)-547.1(intens)-7.8(ity)-545(of)]TJ
0 -1.1116 TD
[(staini)-8(ng)-278.7(was)-281.6(evaluated)-285.5(as)-279.6(0,)-281(1)]TJ
/F7 1 Tf
12.0723 0 TD
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(,2)Tj
/F7 1 Tf
1.0252 0 TD
0 Tc
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[(,)-279.9(and)-280.9(3)]TJ
/F7 1 Tf
2.7589 0 TD
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(.)Tj
/F5 1 Tf
8.9663 0 0 8.9663 313.0582 209.9905 Tm
(Results)Tj
/F8 1 Tf
0 -1.8336 TD
[(Production)-338.3(of)-330.4(anti-HER2)-341.9(mAbs)]TJ
/F3 1 Tf
9.843 0 0 9.843 323.0361 177.1086 Tm
[(Herein,)-191.9(we)-188.5(immunized)-187.8(mice)-192.4(with)-189.8(LN229/HER2)-189.1(or)-186.5(puried)]TJ
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[(recombinan)-10.1(t)-358.3(HER2ec)-369.1(from)-362.7(culture)-367.4(supernatants)-367.9(of)-359.3(LN229/)]TJ
T*
[(HER2ec)-386.4(cells.)-385.7(A)-382.1(booster)-383.2(i.p.)-383.6(injection)-381.4(of)-382.3(LN229/HER2)-384.9(or)]TJ
0 -1.1174 TD
[(HER2ec)-196.4(was)-195.2(also)-195.6(administered)-196.7(and)-188.8(culture)-194.6(supern)-7.5(atants)-193.3(were)]TJ
0 -1.1116 TD
[(then)-287.9(screened)-289.7(for)-285.5(binding)-284.4(to)-290.3(puried)-285.3(HER2ec)-288.5(using)-287.7(ELISA.)]TJ
T*
[(As)-302.4(a)-296(second)-296.7(screen,)-301.3(we)-292.2(used)-299.1(ow)-297.8(cytometry)-295.9(analyses)-300(to)-296.1(as-)]TJ
0 -1.1174 TD
[(sess)-281.4(reactions)-284.1(with)-282(LN229)-278.3(and)-275.2(LN229/HER)-9.5(2)-277.6(cells.)-276.3(Because)]TJ
0 -1.1116 TD
[(LN229)-307.1(cells)-301.6(express)-309.1(endogenous)-301.1(HER2,)-304.7(a)-301.8(stronger)-303.6(reaction)]TJ
T*
[(again)-8(st)-278.6(LN229/HER2)-281.3(was)-287.4(necessary.)]TJ
1.0137 -1.1174 TD
[(A)-641.3(total)-640.6(of)-641.5(29)-635.8(clones)-642.4(were)-641.1(generated)-642.5(from)-639.1(immuni-)]TJ
-1.0137 -1.1116 TD
[(zations)-511.2(with)-512.4(LN229/HER2,)-509.3(and)-505.6(71)-509.1(were)-508.7(generated)-515.8(from)]TJ
T*
[(immunizat)-9.3(ions)-205.9(with)-212.9(puried)-210.4(HER2ec.)-217.1(Among)-209.2(100)-210.6(clones,)-208.1(96)]TJ
/F5 1 Tf
8.9663 0 0 8.9663 62.022 749.6502 Tm
(144)Tj
49.2112 0 TD
[(ITAI)-331.7(ET)-338.7(AL.)]TJ
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[(FIG.)-451.9(1.)]TJ
/F3 1 Tf
4.2268 0 TD
[(Characterization)-456.5(of)-445.4(H)]TJ
7.3837 0 0 6.5625 190.885 116.9575 Tm
(2)Tj
9.845 0 0 9.845 194.57 118.6015 Tm
(Mab-77.)Tj
/F4 1 Tf
3.864 0 TD
[(\(A,)-449.6(B\))]TJ
/F3 1 Tf
3.196 0 TD
[(Flow)-448.2(cytometry)-444.7(with)-448.5(H)]TJ
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(2)Tj
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[(Mab-77;)-452.5(cells)-445.1(were)-450.5(treated)-448.4(with)-448.5(1)]TJ
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14.6613 0 TD
(m)Tj
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[(g/mL)-444.1(of)]TJ
-46.3045 -1.0077 TD
(H)Tj
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(2)Tj
9.845 0 0 9.845 70.526 108.6803 Tm
[(Mab-77)-416.8(followed)-413.4(by)-416.6(Oregon)-411(green-conjugated)-419.4(anti-mouse)-416.7(IgG;)-413.8(black)-416.3(line,)-412(negative)-414.9(control.)]TJ
/F4 1 Tf
38.9681 0 TD
(\(C\))Tj
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[(Western)-420.3(blot)-413.2(using)]TJ
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(H)Tj
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(2)Tj
9.845 0 0 9.845 70.526 98.7023 Tm
[(Mab-77;)-274(cell)-277.3(lysates)-274.2(\(10)]TJ
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9.9508 0 TD
(m)Tj
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[(g\))-272.6(were)-272(electrophoresed)-280(and)-269(proteins)-274.2(were)-277.8(transferred)-273.5(onto)-274.8(PVDF)-270.1(membranes.)-277.1(After)-272(blocking,)]TJ
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[(membranes)-458(were)-450.5(incubated)-456.2(with)-448.5(1)]TJ
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(m)Tj
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[(g/mL)-449.9(of)-451.1(H)]TJ
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(2)Tj
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[(Mab-77)-457.1(or)-451.1(anti-)]TJ
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6.749 0 TD
(b)Tj
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[(-actin)-452(\(AC-15\),)-449.8(and)-453.3(then)-454.5(incubated)-450.4(with)-454.3(peroxidase-)]TJ
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[(conjugated)-319(anti-mouse)-318.8(IgG.)]TJ
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(\(D\))Tj
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(2)Tj
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[(Mab-77)-313.1(was)-315.8(determined)-320.1(using)-315.9(ow)-314.6(cytometry.)-315.6(A431)-315.7(and)-315.1(SK-BR-3)]TJ
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[(cells)-370.3(were)-369.9(suspended)-367.9(in)-364.9(100)]TJ
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[(L)-365(o)0(f)-364.8(serially)-367.7(diluted)-371.4(H)]TJ
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(2)Tj
9.845 0 0 9.845 278.0787 68.8252 Tm
[(Mab-77)-365(\(0.6)-167.6(ng/mL10)]TJ
/F9 1 Tf
9.6225 0 TD
(m)Tj
/F3 1 Tf
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[(g/mL\))-364.5(and)-366.9(secondary)-369.3(anti-mouse)-370.6(IgG)-360.8(was)]TJ
-32.3515 -1.0135 TD
[(then)-339.3(added.)-333.1(Fluorescence)-340(data)-334.7(were)-335.4(collected)-336.6(using)-333.2(a)-336.2(cell)-334.9(analyzer.)-338.6(PVDF,)-331.1(polyvinylidene)-339.4(diuoride.)]TJ
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(145)Tj
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