DTIC ADA436056: Next-Generation Recombinant Antibodies and Antigens for the Detection of Biological Threat Agents and Simulants PDF
Preview DTIC ADA436056: Next-Generation Recombinant Antibodies and Antigens for the Detection of Biological Threat Agents and Simulants
TXEN - ONCOEIRTARENEG TN ASNEIIBDMOBITNAA SRENOHEFTG IDTNNA IG ON LOFOIOITBCETED STN NATEAG EALRAHCT DSTNALUMIS llennoC’ O. PniveK n ar.haSK b,knAospm o y.h,oGT*R sedl a .VsJem adJ n,a ym r..,ASUretn elCacigolo ilBacime hdCoow e.g rdoEht uganitneserP* lacigolo idB nraeidloS BSSM A,dnammo ClacimehC - TRR - ,dnu ogrnGiv onrePedr e,bd AakowRahkc a3 l8,B1M5B A S0U10 1d2nalyraM TCARTSBA seidobitnA era yltnerruc eht laitnesse tnenopmoc ni lacigolonummi srosnes tahtt ceted WB lacigoloib( )erafraw ,stnega gnivig meht htob ytivitisnes dna .ytivitceles erehT s i elbaredisnoctol - to - tol ytilibairav ni eht noitcudorp fo .seidobitna ehT ssecorp fog nipoleved seidobitna ni slamina ro ni nailammam llec erutluc si oslaemit - ,gnimusnoc hcihw stimile ht yticapac roftsuj“ - ni - ”emit ro egrus .noitcudorp lufrewoP tnanibmocer AND seigolonhcete ra gniwolla eht tnempoleved dna lautneve tnemecalper fo eseht lanoitidart ytiniffa yassas tnegaer htiw levon stnegaer taht evah devorpmi ecnamrofrep dna esae fo .noitcudorp sihT r epap sebircsed ruo stroffe ni owt hcus ,seigolonhcet tnanibmocer seidobitna dnaytiniffa - detalosi .sr em aetdpiatpep INTRODUCTION secna vndiA eyhttili boat eno lycr e evegdriatl ps deennpiiare at droynbraaip llepsseihdt serutcurts no eht secafrus foiretcab gy nl r itoieset ctese vadehugnalertaiholihsdpioi ra edtncaab seditpep dna snietorp taht nac dnib dna tceted WB stnega dna rieht .stnalumis e sehT tnanibmocer stcudorp nac eb desserpxe dna decudorp ni egral - elacs lairetcab, snoitatnemref gniyfilpmiseht ri noitcudorp revo eht tnerruc dohtem fo nailammam llec erutluca modirbyh( noitcudorp fo lanolconom .)seidobitna esehT tnanibmocer sdohtem osla reffo srotagitsevnie ht oht enmoitulo vrealucel ohmguor httcudo re phgtnivorp mriehtr ud fnganiyfid ofnmooitpo ds. secnavdA ni cite nseegigolonhcet evah delban estsitneics ot enolc eneg stnemgarfg nidocne yr toyifdcoi bfoiittcnneia p ns to,sisosrhsoet rchpecxvuesega hspaoiretcab 1,2,3 ne eyrl cd sidpnaar eht yrarbil fo modnar eneg snoitanibmoc ot yfitnedi esohtf stnemgar fo eht elohwy dobitna negit nnaev i fagnooitingoc ecrifice pss’ydobit n naeataid etma hetlucelom 4,5 . SEI DTONBAINTINBAMOCER A tnecer ecnavda ni ydobitna noitcudorp ygolonhcet si eht gninolc fo ydobitna senegd na rieht noisserpxe ni lairetcab ref .snoitatnem sihT ygolonhcet sah neeb nevorp elbapac f o sei drgoo nbfnii o ctW itunBttadanch oeetergtrp aea yd yt frtimoei mohldrr ganoftiuafohqiln uot s e.it doteolnr b aa yieonr ltvsi elnilbstaasa ms sinedoeoatecncltfnpuae exdRedteornoriappuqca ni ;ytitnauq ,erofereht gnihsilbatse a ssecorp rof rieht noitcudorp dluow evorpmi e ht y tyitliilb iadbnnai atanf rtoo. onisdpitmepacedumetlstseeyidsfo ib e Webirc seerdeh ehtg ninolc 1 Report Documentation Page Form Approved OMB No. 0704-0188 Public reporting burden for the collection of information is estimated to average 1 hour per response, including the time for reviewing instructions, searching existing data sources, gathering and maintaining the data needed, and completing and reviewing the collection of information. Send comments regarding this burden estimate or any other aspect of this collection of information, including suggestions for reducing this burden, to Washington Headquarters Services, Directorate for Information Operations and Reports, 1215 Jefferson Davis Highway, Suite 1204, Arlington VA 22202-4302. Respondents should be aware that notwithstanding any other provision of law, no person shall be subject to a penalty for failing to comply with a collection of information if it does not display a currently valid OMB control number. 1. REPORT DATE 2. REPORT TYPE 3. DATES COVERED 00 JAN 2002 N/A - 4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER Next-Generation Recombinant Antibodies And Antigens For The 5b. GRANT NUMBER Detection Of Biological Threat Agents And Simulants 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) 5d. PROJECT NUMBER 5e. TASK NUMBER 5f. WORK UNIT NUMBER 7. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) 8. PERFORMING ORGANIZATION Edgewood Chemical Biological Center, U. S. Army Soldier and Biological REPORT NUMBER Chemical Command, AMSSB-RRT-BM, Aberdeen Proving Ground, Maryland 21010 USA 9. SPONSORING/MONITORING AGENCY NAME(S) AND ADDRESS(ES) 10. SPONSOR/MONITOR’S ACRONYM(S) 11. SPONSOR/MONITOR’S REPORT NUMBER(S) 12. DISTRIBUTION/AVAILABILITY STATEMENT Approved for public release, distribution unlimited 13. SUPPLEMENTARY NOTES This article is from ADA409494 Proceedings of the 2001 ECBC Scientific Conference on Chemical and Biological Defense Research, 6-8 March , Marriott’s Hunt Valley Inn, Hunt Valley, MD., The original document contains color images. 14. ABSTRACT 15. SUBJECT TERMS 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF 18. NUMBER 19a. NAME OF ABSTRACT OF PAGES RESPONSIBLE PERSON a. REPORT b. ABSTRACT c. THIS PAGE UU 6 unclassified unclassified unclassified Standard Form 298 (Rev. 8-98) Prescribed by ANSI Std Z39-18 dna no iltaaiztiirneitc afroa hsceidobitn ataht dnib eht taerhttoniablumis s egahpoiretcab 2SMa ( non - cinegohtap suriv fo eht muiretcab aihcirehcsEiloc , hcihw si desu ot etalum i)ssesurivd na n i nm h iuncaebgi (t lih,os a nw srinvhed npiocb eeieu.stx ts)uaosoonltatriupcm iirs o Tte eemht deen rofhgih - ,ytilauq evisnepxeniitna ydob ,stnegaer ew evah desu egahp yalpsid ote talosi ydobitna seneg morf dezinummi .ecim ehT gnitluser ydobitna selucelom era dellac, sbaF gnitacidni taht yeht era desirpmoc fo yvaeh dna thgil niahc ydobitna secneuqes taht mrofe ht negitna -baira vgnidnib , n eotliugb e ornd .i tnateootnhni natgGotegcsrInoc ,noitazinummI ydobitna eneg ,noitacifilpma dna .gninolc ydobitnA seneg r of nilubolgonummi yrarbil noitcurtsnoc erew deniatbo morf eht sneelps fo c/BLAB e cim dezinummi htiw 2SM dna .nimublavo ANDcaw s dezisehtnys morf latot neelps ,ANRd na deifilpma yb RCP ot etalosi laudividni stes fo nilubolgonumm i.seneg RCP - deifilpy mvaaeh n isaehn cee grnawoh sni .giF .1 yva ed Hnt ahngi ia elhdsncete lngeRbe rCmmyePegbwsasraf noit a)g2iSlM( ro yb RCPlbmessa )n iymu bdd lnen aatosvs ieoehtm(gtcyiiizdwrnteser toN I dna epS I rof gninolc otni eht egahp yalpsid .rotcev ehT yramirp ydobitna yrarbil sawd emrofsnart otni E.coli dna desserpxe no suotnemalif egahp selcitrap yb noitcefni fo eht E. coli tsohs llec htiw . ), Aa Cl ,laeoLnJegatar t3S1(M SeCg Varhepp leehht VII IbI Ic/aI Ib IaI giF ure .1 noitacifilpmA fo yvaeh niahc.seneg Each ylimaf fo esuom nilubolgonummi yvaeh dna thgiln iahc saw deifilpma yletarapes gnisu RCP sremirp cificepso t that family. Shown are amplified gene fr stnemga fox is heavy chain families (Roman numerals). The desired gene fragments are 700 bp long. Other bands represent PCR 700bp artifacts. Right lane, DNA size marker. erugiF .2 citamehcS foydobitna - displaying particle. A siht sedocne… phage particle in the dis yalp yrarbil sniatnoc A ND ereh ydobitna gnidocne na ,ydobitna dna syalpsid taht emasy dobitna no sti .ecafrus ehT eritne eriotreper fo seidobitna denolc morf eht esuom si detrevnoc ot a mrof taht nace b deneercs yb gniylppa eht noitalupop ot a ecafrus detaoc in the ant igen of interest. ...ereh AND sihT ytiniffA tnemhcirne )”gninnapoib“( fo ydobitna .senolc At ndaeniif bimmrrouocpferf o eht 2SM taoc nietorp saw desu ot neercs eht ydobitna yrarbil rof senolc taht dnib . 2S M nimublavO rof htob noitazinummi dna gninnapoib si sawiatbo den yllaicremmoc ecreiP(, oC ,drof k)cLoIR ni ydlehig fi.ihmrruopf e hyTalpsi dfo s etin daeonhbit ibntmonoacer e hetcafrus fo eht egahp dewolla rof eht tnemhcirne fo negitna cificeps senolc hguorht gninnapoibt sniaga 2 tnanibmocer 2SM taoc nietorplibommi dezi ni eht sllew fo retitorcim .setalp egahPs elcitrap gniyalpsiditna - 2SM ro itna - nimublavo seidobitna dna( gniniatnoc eht gnidnopserrocd enolc .”gninnapoib “della cnoitacifiru pytiniff af omro fay bdeniatb oere w)sene gydobitna gninolcbuS dnaerpxe ssion of the antibody genes. gn i ew ,ho tgtldsnlnaiouln Fonfraopoib laudi veigdanhi p sgennionlicat neohct dseeriidsoebd i etdrneeaiwfitne dyi bgl naiurduitvliudcni senolc dna gnineercs rof gnidnib fo e hettair pnoergpiptana yb .ASILE deif ierveiVtisop senolc erew decneuqes ylluf dna morf eht ,ecneuqes eht GgI ssalcbus fo hcae enolc saw. d enimreted seneG rof hcae ba Fydobit nearew neh tdevome rmorf eht ega hypal pssriodtcev dna denolc otni noisserpxe srotcev taht etaroprocni a x6 eniditsih gat no hcae h yvae niahc roft neuqesbus fsonoitatnemr e nfnioitacifir udp nnaoisserpxe E. coli . noisserpxE dna noitacifirup fo tnanibmoceritna - MS2 antibody. ehT noitcudortnif o ydob istennaeg otni neohi tsrso et1rc.pe1xvse idHepsuf eht yvae hniah cseneg htiw a6 s i.Hgxat ehT 1n.o1issi sHre porgtpncxieeyvrra ce(htitna - 2 SyMdo b)istennaeg dseacwud oorttnnii na E. coli n idaerttcsurtsno cot ezimi tnpi oentooirspser pPxPeOT( ,.I)enegatartS slleC erew nworg ni a02 - retil rotnemref dna eht dnuopmoc GTPI sawdedda ot ecudni noisserpxe fo ehty dobitna .seneg retfA ,noitatnemref sllec erew detpursid htiw a rotacinos ot esaeler a edurce tasyl gniniatn oetchntanibmoictenra - 2SM .seidobitna ehTc edur ll eectasy ls adweilpp aot a nmuloc dekcap htiw nolaT latemtiniffa y niser gnidrocca ot eht s’rerutcafuna m,snoitcurtsni dnad etule .erl eohfztfaiudw bi mdietiutlnEa - 2ySdMobi ts n aradweehitfriur fuy pbegas srxaeepvdoahpeS leg noitartlif snmuloc ot retaerg naht %09 .ytirup snoitcarF erew dezylana yb muidosl ycedod us etafl - edimalyrcaylop leg siserohportceleSDS( - )EGAP dna CLPH rof ytirup dna n ietorp .tnetnoc snietorP erew detceted yb gniniats htiw eissamooC tnaillirB eulb dna y b gnittolbonummi erugiF( .)4 ralimiS stluser erew deniatbo nehw gniyifirupitna - nimublavo . ) a nsttw eaooidnhd(sobitna erugiF .4 deifiruP tnanibmoceritna - 2SM.ydobitna retfA noitacifirup sa debircsed ni noitces ,7.2itna - 1 2 3 4 2SM ydobitna senal( 2 dna ,4 sworra ) saw% 09> kdal erup sa denimreted ybSDS - EGAP dna gniniatsh tiw eissamooC tnaillirB ulb e tfel( .)egami enaL , 1 97.4 ralu cte hl;godimreawdn aetnsal ,2 deifiruiptna - MS2 66.2 .y dloabc iiotstttnennenaemad il i,rteehgpgaximeRi 45 1 dna g,n2it a yertthtuitsbtnno emfdeoid enhitetorp sa esuom ydobitna yb lacigolonummi. noitceted 31 enaL ,3 ralucelomiew thg ;rekram enal ,4d eifirup itna - 2SM .ydobitna ehT itna - 2SM ydobitna s aw derrefsnart ot a esolullecortin enarbmem retfaSDS - 21.5 EGAP dna detceted htiw taogitna - esuomy dobitna detag u,j eno sotea cntg ianPlhiIapsCksuBlo/ahTpBN 14.4 as substrate. 3 5 5 3.84 3.836 3.843 4 A 4 B 3 3 1.879 2 2 1.04 1 1 0.06 0.012 0.008 0 0.11 0.008 0 0 0 Optical density (405 nm) 25000 2500 250 25 2.5 No Optical density (405 nm) 18650 1865 186 18.6 1.86 No antigen antigen MS2 virions (protein equivalent, ng) Recombinant MS2 coat protein (ng) e.r5ugiF deifir ugpni snuieto rtpa o2c StMnanibmoc e)r Bd( nsanoir i2v S)M A f(nooitcet eAdSILE itna o oandtezi-lamr o ,n f2noa eem httneserp esreu l. ayVdobit nba a2FSM -.tnemirepx elortno cnegitna Detection of MS2 and ovalbumin with recombinant antibodi .se tnanibmiotcneaR - 2SM dnaitna - nim usbeliadvoobitn aerew desu niemyzne - deknil tnebrosonummi syassa )sASILE(o t tceted rieht gnidnopserroc .snegitna ytisneD - tneidarg - deifirup egahpoiretcab 2SM d na nimub l)aavmogiS( erew yllai rdeestulid dna dnuob yba noitprosd ot sllew fo retitorcim. se talp deifiruP 2SM saw elbatceted nwod ot a level fo 052 gn nietor p)(tnelaviuqe ni sihtt nemirepxe erugiF( .)5 ehT tnanibmocer taoc nietorp desu rof gninnapoib dna ,gnineercs ,revewohs aw detceted ot a level foixorppa ytln ea0ytn 2tai.rimb govmenfiohtc tiersre ntteans ee rrsgaiphpTa taoc nietorp revo eht tcatni suriv yam tcelfer eh tnoitalosi fo eht ydobit nganisu ethntanibmocer nietorp sa eht gninnapoib .tegrat esU fo tcatni snoiriv sa eht ,tegrat sa llew safrep gnimroe ht ytiniffa erutpac foitna - ydob - gniyalpsi degahp ni noitulos lliw wolla eht noitalosi fo sbaFh tiw neve retaerg ytiniffa rof tcatni .2SM itnA - 2SM did ton dnib egahp ,31M ,ASB ron imublavo atad( ton .)nwohs gnisU yllaitrap deifirup tnanibmocer a seidobitnAVO - 3 dna AVO - 4, . )nwo ht soanta dm(argor c fli1oem v oneatw loedlbatcet es danwimublavo PEPTIDE - BASED BIODETECTION r ehtruF msinoitcuder ni gninifed eht ralucelom stnanimreted fo ydobitna yticificepsr of negitna sah neeb deusrup ybisu gn lairotanibmo cmodnar editpep syalpsid ot pam ehtc ificeps onima dica secneuqes fo eht elbairavrepyh noiger fo eht ydobitna taht yllautca sdnib ote ht n e.geiptontaipe tI sah neeb nwohs taht elha tigtn nisednsnisiaebmod fo n aydobit nyaam eb sa llamsa s 5 - 51 onimasdica 6,7,8,9 . modnaR editpep yalpsid seirarbil nac eb deilppa ot o wt tnacifingis seussi ni eht stnemeriuqer rof :noitcetedoib )1 eht ngised fo ralucelomn oitingocer stnemele taht era rellams naht elohw seidobitna dna ydobitna ,stnemgarf dna2 ) eht ngisedf o editpep secneuqes taht nac cimim eht epotipe gnidnib setis no snegitna dna suht eb desus a s leo vrritot nf inoe.sechnogtopi itn tniyaa adsisla a ev eddW ieetyvpoael hppamamocedendaord yrarbil ot neercs rof edit pseepcneuqes taht yamimim c eht gnidnib fo ydobitna ot eht nicird na 4 lacc oncioxloythopraettsne B )BES( snixot dna ot lanol csoeniodmob idtentaarene gtsniagan icir dna.BES modnaR editpeP.yrarbiL ehT xrTilF modnar editpep yrarbil saw deniay tlbloaicremmoc morf .negortivnI ehT yrarbil si desopmoc fo 77.1 X01 8 yramirp senolc fo E. coli htiwe ht remacedo deditpep ecneuqe sdetresni nihtiw eht nixoderoi h)TAxrT( evitca etis .pool ehT AxrT editpep noisuf si denolc otni eht laitnessenon niamod fo eht rojam lairetcab nillegalfrp nieto )CilF (red nluortnoc fo aP retomorp morfahpoiretcab e g e.cën ee,ud d qiee ethcnsptueedhpnWi L si desserpxe no eht ecafrus fo eht E. coli a l hlte igewahltfN - dnaC - de nyiba r ltsasdnnnioemcret . ).6gi Fd(n oebdiflus iad editpep 5 ’ 3 ’ e.r6ugiF f ocitameh c S allegalf r tnemalif yrarb ielditpep .yalps idd nnaoitcurtsnoc xrTilFp N - - C niamod gnidnib nietorp deniartsnoc editpep S --- S nillegalf nillegalf eP editp yrarbil noisserpxe dna .gninnap A kcots erutluc fo ehtllec - deyalpside ditpep yrarbil saw nworg gnidrocca ot eht s’rerutcaf u,nsanmoitcurtsni dna noisserpxe fo ehts editpep erew decudni yb noitidda fo .nahpotpyrt ehT yrarbil saw dennap tsniaga owt snix onticir(d na ,BES deniatbo morf .)amgiS snegitnA erew detulid dna debrosda otno eussit erutluc, setalp desn i.drdneakcol b g nrie tt, nfngeaAonhciite tkd eucdhloetolcsb u ldelnreiuct lsuac wdedda ot eht erutluc .setalp slleC dnuob ot eht etalprew e detule otni 01 slm hserf erutluc aidemy b gnicalp eht etalp no a xetrov ot raehs eht allegalf dna esaeler eht sllec ot eht .aidem ehTs llec erew neht derutluc thginrevo dna eht etelpmoc erudecorp detaeper rof evif sdnuor fo. gn in nap retfA eht htfifuor dn fo ,gni nenrauptluc dna e d,into pis etegpcrnuui dtssnlseiuedcriptxpee ptaht dnu o dseb eethn;vi eteAifeggSt irr iIiyteasy tLtbnwtddrnEee nooapdeabmi rgiodeitcewlndada ot etepmoc htiw eht gnidnib fo eht editpep .senolc laudividnI senolc tahtdetset eviti snoip eht edit peddehneptatce le erAseSwILE - . decneuq ed sndaetalo ss iaA wNgDnidocne seditpeP taht dnib BES ro .nicir mo rdfeniat bsotlus egrnineer cAsSI Le Ehstw o eh8rsugiF w oshesn o n. lerenfc hutiot ox BF oegEothnSttid neidbitpep e hhtt iswnoitcaret ntinacifingis lanolcon o fameocnese rep h ntginidn iebditp e fpnooitibih nei h ytdberusa e sm,anix oBtES wo hnsic itrsnia gganinn arpet fdaetalo ssieno lecditp en pee . th fBr toEuo ySotdFobitna 5 eh tgnitibihn in iytivitc atnacifingis e .h yTdobitn alanolcono madn anici rneewte bgnidni b . )nwo ht soanta de(cneuq essusnes n yoaaclps it dod niddetalo ssieno le chfstoecneuq eesditpep 520.0 30.0 20.0 20.0 510.0 10.0 10.0 OD405 500.0 OD405 0 0 1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 10 pBeApntSEit ide clones Anti-ricin peptide clones erugiF.7 AS IgLnEineercs fo editp espeno ltcsniaga BES nixot )tfel( dna nic i.r)thgir( tepmocnoN eviti sASILE edreatacid nyib eht deda h.ssrab diloS strnaebse r npeoehirttibi hfnoieditpep - snoitca rneitxnoit itn af oecneser peh tni - i BtrEnoSa .ylevit-cepse r,ydobitn alanolcono mnicir SNNOOII SSDSUNULACCSNIODC A tnanibmocer stcudorp hcaorppat o esnefedoi btnega etrnempoleved sah lareves, lacinhcet ,lacitsigol dna lamina esu segatnavda revo lanoitidart sdohtem fo gnitalosi dna g nicudorp .seidobitna ehT lacinhcet segatnavda edulcni eht laitnetop ot ecudorp ydobitna ytisrevidr etaerg than that obt elbania yb eht enummi smetsys fo .slammam ehT gninnapo iebrudecorp ybs ti erutan swolla eht yrarbil otfles - tceles yb gniwol lsaelci tgrnaipyalpsid a elbaris eyddobitnar o editpep ot dnib ot na dezilibommi .tegrat ,yllacitsigoL gnicudorp seditpep dnatna seidobin i la inroeittcaatbne msrreefffo lar e.vseesgatnavda airetcaB era ylevitaler ysae ot ,worg dnae lacs pu ni .noitcudorp riehT dipar htworg sedivorp eht yticapac rof egrus .noitcudorp , yltsaL tnanibmocer tnegaer noitcudorp ni airetcab seziminimna lami esu yb gnisu ylno esohts lamina ydlelzaiintuimn minii e h.tssecorp no itltnla eAleu urqdpuenitsa ncbsaauemfskuan taemcalpn i lairetcab ,sllec gnicuder eht srebmun fo slamina desu morf sderdnuh ot rewef naht netr ep . deno lycdobitna REFERENCES 1. 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