Rapid Diagnostic Assay for Intact Influenza Virus ... - ACS Publications

Subscriber access provided by CORNELL UNIVERSITY LIBRARY

Article

A Rapid Diagnostic Assay for Intact Influenza Virus Using a High Affinity Hemagglutinin Binding Protein Caitlin E. Anderson, Carly A. Holstein, Eva-Maria Strauch, Steven Bennett, Aaron Chevalier, Jorgen W. Nelson, Elain Fu, David Baker, and Paul Yager Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 12 May 2017 Downloaded from http://pubs.acs.org on May 13, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Analytical Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 14

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry

"3BQJE%JBHOPTUJD"TTBZGPS*OUBDU*OGMVFO[B7JSVT6TJOHB)JHI "GGJOJUZ)FNBHHMVUJOJO#JOEJOH1SPUFJO $BJUMJO&"OEFSTPO $BSMZ")PMTUFJO &WB.BSJB4USBVDI 4UFWFO#FOOFUU "BSPO$IFWBMJFS +PSHFO /FMTPO &MBJO'V %BWJE#BLFS BOE1BVM:BHFS

%FQBSUNFOUPG#JPFOHJOFFSJOH 6OJWFSTJUZPG8BTIJOHUPO 4FBUUMF 8"

%FQBSUNFOUPG#JPDIFNJTUSZ 6OJWFSTJUZPG8BTIJOHUPO 4FBUUMF 8"

4DIPPMPG$IFNJDBM #JPMPHJDBM BOE&OWJSPONFOUBM&OHJOFFSJOH 0SFHPO4UBUF6OJWFSTJUZ $PSWBMMJT 03

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

*OGMVFO[B DPNNPOMZ LOPXO BT UIF GMV JOGFDUT PG "NFSJDBOT FBDI ZFBS BDDPVOUJOH GPS UP NJMMJPO JOGFDUJPOT BOOVBMMZ 5IFTF JOGFDUJPOT MFBE UP PWFS IPTQJUBMJ[BUJPOT BOEBOBWFSBHFPG EFBUITJOUIF64BMPOFFBDIZFBS NBLJOH JUEFBEMJFSUIBO)*7BOEDFSWJDBMDBODFSDPNCJOFE8IJMFUIFGMVJT FTQFDJBMMZ QSPCMFNBUJD GPS ZPVOH DIJMESFO QSFHOBOU XPNFO UIF FMEFSMZ BOEQFPQMFXJUIPUIFSIFBMUIDPOEJUJPOT UIFGMVDBOBMTPCF B TFWFSF SFTQJSBUPSZ EJTFBTF GPS PUIFSXJTFIFBMUIZ BEVMUT 5IJT JT FTQFDJBMMZ USVF GPS TQFDJGJD GMV TUSBJOT %VSJOH UIF GMV TFBTPO BEVMUT ZFBST PME BDDPVOUFE GPS UIF NBKPSJUZ PG GMV SFMBUFEIPTQJUBMJ[BUJPOTBOEEFBUIT*OBEEJUJPOUPCFJOHBTJHOJGJDBOU IFBMUI QSPCMFN UIF GMV JT BMTP BO FOPSNPVT GJOBODJBM CVSEFO " EFUBJMFETUVEZPOUIFFDPOPNJDCVSEFOPGGMVCZ.PMJOBSJFUBMGPVOE UIBUJOGMVFO[BDPTUTUIF64FDPOPNZ#BOOVBMMZ XJUI#JO EJSFDU NFEJDBM DPTUT # JO MPTU QSPEVDUJWJUZ BOE # JO MPTU FDPOPNJDWBMVFEVFUPFBSMZEFBUI%FTQJUFUIFTFHSJNOVNCFST UIF GMVJTUSFBUBCMF"OUJWJSBMNFEJDBUJPOTTVDIBT5BNJGMV¡ BMTPLOPXO BTPTFMUBNJWJS BSFBWBJMBCMFGPSUSFBUNFOUPGNPTUJOGMVFO[BTUSBJOT BOEIBWFCFFOTIPXOUPSFEVDFUIFTFWFSJUZPGTZNQUPNT TIPSUFO UIF EVSBUJPO PG JMMOFTT BOE EFDSFBTF UIF SJTL DPNQMJDBUJPOTCVU POMZ XIFO QSFTDSJCFE XJUIJO UIF GJSTU IPVST PG TZNQUPNT 5JNFMZBOEBDDVSBUFEJBHOPTJTPGJOGMVFO[BJTUIFSFGPSFUIFGJSTUMJOF PG EFGFOTF BHBJOTU UIF WJSVT BOE LFZ GPS TVDDFTTGVM EJTFBTF NBOBHFNFOU (JWFOUIFJNQPSUBODFPGJOGMVFO[BEJBHOPTJT TFWFSBMMBUFSBMGMPX CBTFESBQJEEJBHOPTUJDUFTUT 3%5T IBWFCFFOEFWFMPQFEGPSQPJOU PGDBSF 10$ EFUFDUJPOPGJOGMVFO[B*UIBTCFFOEFNPOTUSBUFEUIBU

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

ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

)FSFXFSFQPSUUIFEFWFMPQNFOUPGBTFUPGQBQFSCBTFEBTTBZTGPS EFUFDUJPOPGUIFJOGMVFO[BTVSGBDFQSPUFJOIFNBHHMVUJOJO )" )" JTBWBMVBCMFEJBHOPTUJDUBSHFU BTJUJTCPUIBOBOUJHFOUBSHFUFECZUIF IVNBO JNNVOF TZTUFN BOE JT VTFE UP JEFOUJGZ UIF TVCUZQF PG UIF JOGFDUJOH GMV WJSVT FH ) WT ) 8IJMF )" VOEFSHPFT B IJHI EFHSFFPGBOUJHFOJDESJGU ESJWFO JOQBSU CZQSFTTVSFUPFWBEFUIFIPTU JNNVOFTZTUFN IJHIMZDPOTFSWFEBSFBTFYJTUUIBUBSFDSVDJBMGPSWJSBM GVODUJPOBOESFQMJDBUJPO'PSFYBNQMF UIFSFDFQUPSCJOEJOHTJUFPG UIF IFBE SFHJPO VOEFSHPFT MJNJUFE WBSJBUJPO BT JU JT SFRVJSFE GPS BUUBDINFOU PG UIF WJSVT UP UIF IPTU DFMM UISPVHI TJBMJD BDJE NPJFUJFT 5IFTUFNSFHJPOPGUIF)"QSPUFJO XIJDIJTJOWPMWFEJO DPOGPSNBUJPOBMDIBOHFTDSJUJDBMUPFOEPTPNBMFTDBQF IBTBMTPCFFO EFNPOTUSBUFEUPCFIJHIMZDPOTFSWFE$BSFGVMFQJUPQFTFMFDUJPOJT UIFSFGPSFFTTFOUJBMGPSEFTJHOPGBEJBHOPTUJDBTTBZUPUBSHFU)" "TTBZTGPS)"EFTDSJCFEJOUIFMJUFSBUVSFEFNPOTUSBUFTVDDFTTGVM EFUFDUJPO PG UIF SFDPNCJOBOU GPSN PG )" 8IJMF UIF SFDPNCJOBOU WFSTJPO PG )" TFSWFT BT B HPPE NPEFM GPS BTTBZ EFWFMPQNFOU JU JT OPU BO FYBDU SFQMJDB PG UIF OBUJWF GPSN PG )" 4QFDJGJDBMMZ SFDPNCJOBOU )" JT USBEJUJPOBMMZ FYQSFTTFE JO FJUIFS NBNNBMJBO PS JOTFDU MBSWBF DFMMT NFBOJOH UIF SFDPNCJOBOU QSPUFJOTTUSVDUVSF DPOGPSNBUJPO BOENJDSPFOWJSPONFOUNBZEJGGFS GSPNUIFOBUJWFGPSN#FDBVTFPGUIFTFQPUFOUJBMEJGGFSFODFT UIF TVDDFTTPGQSFWJPVTBTTBZTNJHIUOPUUSBOTMBUFUPUIFOBUJWFGPSNPG UIF QSPUFJO "DDPSEJOHMZ XF IBWF EFWFMPQFE B TFU PG BTTBZT UIBU UBSHFUOBUJWFQSPUFJOGSPNUIFDMJOJDBMMZSFMFWBOU JOUBDUGPSNPGUIF WJSVT 5IF BTTBZT SFQPSUFE IFSF VTF B DPNCJOBUJPO PG BOUJCPEJFT BOE OPWFM DPNQVUBUJPOBMMZ EFTJHOFE BGGJOJUZ QSPUFJOT UIBU CJOE UP UIF IFBESFHJPOPG)" 'JHVSF "OUJCPEJFTBSFUSBEJUJPOBMMZVTFEGPS QSPUFJO DBQUVSF BOE EFUFDUJPO JO MBUFSBM GMPX BTTBZT EVF UP UIFJS BGGJOJUZ TQFDJGJDJUZ BOEBCJMJUZUPBETPSCTUSPOHMZUPOJUSPDFMMVMPTF )PXFWFS UIFJS VUJMJUZ JT MJNJUFE CZ UIF MBSHF CBUDIUPCBUDI WBSJBCJMJUZ BOE IJHI QSPEVDUJPO BOE QVSJGJDBUJPO DPTUT BTTPDJBUFE XJUIDVSSFOUDPNNFSDJBMBOUJCPEJFT4JNJMBSMZ BQUBNFSTIBWFCFFO EFNPOTUSBUFE GPS UIF EFUFDUJPO PG )" IPXFWFS UIF QSPDFTT GPS TFMFDUJPO TZTUFNBUJD FWPMVUJPO PG MJHBOET CZ FYQPOFOUJBM FOSJDINFOU4&-&9 SFRVJSFTTJHOJGJDBOUSFTPVSDFTBOEPGUFOGBJMTUP ZJFMENPMFDVMFTXJUITVJUBCMFCJOEJOHBGGJOJUJFT "O BMUFSOBUJWF BQQSPBDI GPS TBOEXJDI JNNVOP BTTBZ EFWFMPQNFOU JOWPMWFT UIF VTF PG DPNQVUBUJPOBMMZ EFTJHOFE EF OPWP BGGJOJUZ SFBHFOUT %F OPWP EFTJHO PG SFDPNCJOBOU BGGJOJUZ QSPUFJOT FOBCMFT UIF EFWFMPQNFOU PG CJOEJOH QSPUFJOT UP UBSHFU IJHIMZTQFDJGJDFQJUPQFTPOBOBOUJHFO*OBEEJUJPOUPUIJTFYRVJTJUF DPOUSPM PWFS UIF UBSHFUCJOEJOH TJUF XIJDI QSPWJEFT DVTUPNJ[BCMF BOUJCPEZMJLF BGGJOJUZ UIFTF SFDPNCJOBOU BGGJOJUZ QSPUFJOT DBO CF QSPEVDFE JO & DPMJ XIJDI NBLFT UIFN NVDI MFTT FYQFOTJWF UP QSPEVDF UIBO NPOPDMPOBM BOUJCPEJFT *O UIF DBTF PG MBUFSBM GMPX UFTUTEFWFMPQNFOU BOUJCPEJFTDPTU POBWFSBHF 64%QFSUFTU XIFSFBT SFDPNCJOBOU BGGJOJUZ QSPUFJOT DPTU BQQSPYJNBUFMZ 64% $PNQVUBUJPOBMMZ EFTJHOFE SFDPNCJOBOU BGGJOJUZ QSPUFJOT UIBU CJOE UP UIF TUFNSFHJPO PG SFDPNCJOBOU )"IBWF CFFO QSFWJPVTMZ EFNPOTUSBUFE $PODVSSFOUMZ TUSBUFHJFTIBWFCFFOEFWFMPQFEGPS UIF JNNPCJMJ[BUJPO PG SFDPNCJOBOU BGGJOJUZ QSPUFJOT UP OJUSPDFMMVMPTF GVSUIFSFOBCMJOHVTFPGUIFTFQSPUFJOTJOQBQFSCBTFE BTTBZT"GVODUJPOBMJOGMVFO[BBTTBZXBTTVCTFRVFOUMZDPOTUSVDUFEJO B QBQFSCBTFE GPSNBU GSPN B SFDPNCJOBOU BGGJOJUZ QSPUFJO UIBU UBSHFUFE UIF TUFNSFHJPO PG UIF )" QSPUFJO 8F TJODF IBWF CVJMU VQPOUIJTXPSLCZEFWFMPQJOHBTTBZTCBTFEPOBSFDPNCJOBOUBGGJOJUZ QSPUFJOUPUBSHFUTUIFTJBMJDBDJESFDFQUPSCJOEJOHTJUFPOUIFSFBEJMZ

Page 2 of 14

BDDFTTJCMF IFBE SFHJPO PG UIF )" QSPUFJO *OGMVFO[B WJSJPOT BSF EFOTFMZ DPWFSFE XJUI )" USJNFST UIBU QSPWJEF NBOZ TJUFT GPS BUUBDINFOUPGUIJTiIFBESFHJPOCJOEFSu5IFIFBESFHJPOCJOEFSJT EFTJHOFEUPCFBUSJNFSXJUIUIFTBNF$TZNNFUSZBOETQBDJOHBT UIF )" USJNFS NBYJNJ[JOH UIF BWJEJUZ PG CJOEJOH UISPVHI BTTPDJBUJPOXJUIBMMUISFFFQJUPQFT 'JHVSF 5IF BTTBZT QSFTFOUFE IFSF SFUBJO UIF QSFWJPVTMZEFTDSJCFE BEWBOUBHFT PG DPNQVUBUJPOBMMZ EFTJHOFE SFDPNCJOBOU BGGJOJUZ QSPUFJOT JODMVEJOHUIFBCJMJUZUPDVTUPNJ[FUIFCJOEFSQSPUFJOBUUIF MFWFMPGJUTHFOFUJDTFRVFODF5IFBWJEJUZPGUIFIFBESFHJPOCJOEJOH USJNFS SFTVMUT JO TUSPOH CJOEJOH UP )" BOE SFTVMUJOH QJDPNPMBS TFOTJUJWJUZJOPVSQBQFSCBTFEBTTBZTGPS)"QSPUFJO8FDPNQBSFE DPNCJOBUJPOT PG BOUJCPEJFT BOE UIF IFBE SFHJPO CJOEFS UP EFNPOTUSBUF UIBU PVS QBQFSCBTFE GMV BTTBZ JT DBQBCMF PG JOUBDU JOGMVFO[B WJSJPO DBQUVSF XJUI DMJOJDBMMZ SFMFWBOU TFOTJUJWJUZ 5IF SFDPNCJOBOUBGGJOJUZQSPUFJOBTTBZGPS)"PVUQFSGPSNTBUSBEJUJPOBM BOUJCPEZCBTFEMBUFSBMGMPXUFTUXIFOVTFEGPSBOBMZUFDBQUVSF5IJT

XPSLSFQSFTFOUTUIFGJSTULOPXOEFNPOTUSBUJPOPGXIPMFJOGMVFO[B WJSVTEFUFDUJPOPOQBQFS

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ö.

BOE DBQUVSF BOUJCPEZ *OGMVFO[B 3FBHFOU 3FTPVSDF *33 '3 NPVTF NPOPDMPOBM BOUJJOGMVFO[B IFNBHHMVUJOJO BOUJCPEZ XBT VTFE BU JUT TUPDL DPODFOUSBUJPO PG NHN- ö. "MM QSPUFJO TPMVUJPOT XFSF QSFQBSFEJOQIPTQIBUFCVGGFSFETBMJOF 1#4 Q) 4JHNB"MESJDI 4U -PVJT .0 5P BWPJE DMPHHJOH UIF QSJOUFS XJUI QBSUJDVMBUF NBUUFSGSPNUIFTBNQMFT UIFQSPUFJOTPMVUJPOTXFSFGJMUFSFEUISPVHI B DFOUSJGVHBM GJMUFS EFWJDF XJUI öN OZMPO NFNCSBOF 783 3BEOPS 1" BUÉHGPS NJOVUFTQSJPSUPTQPUUJOH5FTU MJOFT DPOTJTUFEPGTQPUTTQBDFEöNBQBSU XJUIESPQMFUTQFSTQPU BOEUXPQBTTFTXFSFQSJOUFEGPSFBDIUFTUMJOFJOPSEFSUPTBUVSBUFUIF NFNCSBOF SFHJPO &BDI ESPQMFU XBT Q- SFTVMUJOH JO UFTU MJOFTDPNQSJTJOH_O-PGQSPUFJOTPMVUJPOJOUPUBM


Page 3 of 14

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


'JHVSF*MMVTUSBUJPOTPGUIFOPWFMJOGMVFO[B)"iBTTBZTUBDLTuEFNPOTUSBUFEIFSFJO " "TTBZTUBDLTGPSSFDPNCJOBOU)"EFUFDUJPO JODMVEJOH BMM BOUJCPEZ EFUFDUJPO UPQ BOE IFBE SFHJPO CJOEFS DBQUVSF XJUI BOUJCPEZ EFUFDUJPO CPUUPN # "TTBZ TUBDLT GPS JOUBDU JOGMVFO[B WJSVT JODMVEJOHBMMBOUJCPEZEFUFDUJPO MFGU

UIFNJYFEBTTBZTUBDLXJUIIFBESFHJPOCJOEFSDBQUVSFBOEBOUJCPEZEFUFDUJPO NJEEMF

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ÉH GPS NJOVUFT UP SFNPWF VODPOKVHBUFE CJOEFS5IFBOUJCPEJFTVTFEGPSEFUFDUJPOJOUIFBMMBOUJCPEZBTTBZ XFSF DPOKVHBUFE VTJOH UIF TBNF DPOKVHBUJPO LJU 5IFZ XFSF JODVCBUFE XJUI UIF NBMFJNJEFBDUJWBUFE HPME OBOPQBSUJDMFT GPS NJOVUFTBU$CFGPSFQVSJGJDBUJPOCZDFOUSJGVHBUJPOBU ÉHGPS NJOVUFTUPSFNPWFVODPOKVHBUFEBOUJCPEZ"MMHPMEDPOKVHBUFT XFSFTUPSFEBU$VOUJMVTF

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i"V u"VHPBUBOUJNPVTF*H( "SJTUB#JPMPHJDBMT *OD "MMFOUPXO 1" XBTQVSDIBTFEDPNNFSDJBMMZ'PSUIFBMMBOUJCPEZ BTTBZ EFUFDUJPO BOUJCPEZ XBT DPOKVHBUFE EJSFDUMZ UP HPME MBCFM BT EFTDSJCFEJOUIFQSFWJPVTTFDUJPO 5IFIFBESFHJPOCJOEFSBTTBZDPOTJTUFEPGUIFGPMMPXJOHTUFQT öMWJSVT SFDPNCJOBOU)" PSOFHBUJWFDPOUSPM öMXBTI öM NPVTF BOUJ)" EFUFDUJPO BOUJCPEZ öM XBTI öM HPMEDPOKVHBUFEHPBUBOUJNPVTF*H(BOUJCPEZ BOE öMXBTI 3FDPNCJOBOU)"XBTUFTUFEBUDPODFOUSBUJPOTPGO. O. O. Q. Q. Q. BOEQ.*OGMVFO[BWJSVTXBTUFTUFE BUY Y Y Y Y Y BOE YDIJDLFOFNCSZPJOGFDUJPVTEPTF $&*%N-

XJUIGPVS SFQMJDBUFT QFS DPODFOUSBUJPO #VGGFSPOMZ 1#45 #4" OFHBUJWF DPOUSPMTXFSFBMTPUFTUFE5IFEFUBJMTGPSUIFBOUJCPEZCBTFEBTTBZ BSF HJWFO JO UIF 4VQQMFNFOUBM .FUIPET "GUFS FBDI BTTBZ XBT DPNQMFUF BMMEFWJDFTXFSFUBQFEUPBTUBOEBSEQJFDFPGQSJOUFSQBQFS BOE JNBHFE BU CJU )%3 DPMPS EQJ ê VTJOH B EFTLUPQ TDBOOFS 1FSGFDUJPO71IPUP4DBOOFS &QTPO -POH#FBDI $"

*OGMVFO[B)"%FUFDUJPO"TTBZT &BDIBTTBZXBTQFSGPSNFEJOUIF EJQTUJDLMBUFSBMGMPXGPSNBUVTJOHBXFMMQMBUFQSFMPBEFEXJUIUIF HJWFO BTTBZ SFBHFOUT TFF 4VQQMFNFOUBM .FUIPET 4VQQMFNFOUBM 'JHVSF 5IF OPWFM iBTTBZ TUBDLu EFNPOTUSBUFE IFSFJO VTFE UIF IFBE SFHJPO CJOEFS GPS DBQUVSF BOE NPOPDMPOBM BOUJCPEZ GPS EFUFDUJPO5IJTTUBDLJTJMMVTUSBUFEJO'JHVSFGPSCPUISFDPNCJOBOU )" BOE XIPMF WJSVT EFUFDUJPO 5IJT IFBE SFHJPO CJOEFS TUBDL XBT DPNQBSFEUPBUSBEJUJPOBMBMMBOUJCPEZBTTBZ XIJDIJTJMMVTUSBUFEJO 4JHOBM 2VBOUJGJDBUJPO "MM TJHOBM JOUFOTJUJFT GSPN UFTU MJOFT XFSF 4VQQMFNFOUBM'JHVSF3FDPNCJOBOU)" WJSVT BOEBOUJJOGMVFO[B RVBOUJGJFEVTJOHBDVTUPNTDSJQUJO."5-"# .BUI8PSLT /BUJDL BOUJCPEJFTXFSFBDRVJSFEGSPNUIF*OUFSOBUJPOBM3FBHFOU3FTPVSDF ." 6TJOH UIJT QSPHSBN B SFHJPO PG JOUFSFTU 30* XBT ESBXO QSFWJPVTMZ *OGMVFO[B 3FBHFOU 3FTPVSDF

8)0 $PMMBCPSBUJOH BSPVOEUIFUFTUMJOFPGJOUFSFTU BOEUIFBWFSBHFQJYFMJOUFOTJUZJOTJEF $FOUFS GPS 4VSWFJMMBODF &QJEFNJPMPHZ BOE $POUSPM PG *OGMVFO[B UFTU XBTDPNQVUFE $FOUFST GPS %JTFBTF $POUSPM BOE 1SFWFOUJPO "UMBOUB (" 64"PlusUIJTUFTU30* * ACS Paragon Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 14

'JHVSF 3FDPNCJOBOU 'MV )" BTTBZT XJUI FJUIFS BOUJCPEZ PS OPWFM IFBE SFHJPO CJOEFS BT DBQUVSF BHFOU XJUI BOUJCPEZ HPME SFBEPVU 3FQSFTFOUBUJWFUFTUMJOFTBOEUIFGVMMBTTBZSFTQPOTFDVSWFTBSFQSPWJEFE&BDIDPOEJUJPOXBTUFTUFEXJUIOSFQMJDBUFT " "OUJCPEZDBQUVSF BTTBZSVOXJUISFDPNCJOBOU)" # )FBESFHJPOCJOEFSDBQUVSFBTTBZSVOXJUISFDPNCJOBOU)" 5IJT WBMVF XBT UIFO CBDLHSPVOETVCUSBDUFE VTJOH UIF BWFSBHF QJYFMJOUFOTJUZJOTJEFBMPDBMCBDLHSPVOESFHJPO *CLHE BOEOPSNBMJ[FE POBTDBMFGSPNUPUPHFOFSBUFUIFOPSNBMJ[FEQJYFMJOUFOTJUZPG UIFTQPU *OPSN VTJOH&RO

+JKNI L

+PAOP F +>GC@ r F +>GC@

&RO

'PSUIFBTTBZEBUBIFSFJO BMMDPMPSJNFUSJDTJHOBMTXFSFHFOFSBUFE VTJOH HPME OBOPQBSUJDMFT XIJDI BSF SFE 5IFSFGPSF UIF HSFFO DIBOOFMPGUIF3(#JNBHFXBTVTFEGPSBOBMZTJT CFDBVTFJUXBTUIF NPTUTFOTJUJWFUPUIFDPMPSPGUIFUFTUMJOFT0WFSBMM UIFOPSNBMJ[FE QJYFMJOUFOTJUJFTSFQSFTFOUFEBSBOHFGSPNOPTJHOBM UPNBYJNVN GVMMZ TBUVSBUFE HSFFO DIBOOFM TJHOBM 'PS TJHOJGJDBODF UFTUJOH CFUXFFO UXP TFUT PG EBUB 8FMDIT UUFTU XBT VTFE BMTP LOPXO BT 4UVEFOUTUUFTUXJUIVOFRVBMWBSJBODFT 6OMFTTPUIFSXJTFTQFDJGJFE B TJHOJGJDBODF UISFTIPME PG è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m UIFMBSHFSIFBESFHJPOCJOEFS L%B DBOCFFGGFDUJWFMZ JNNPCJMJ[FE CZ EJSFDU BETPSQUJPO UP OJUSPDFMMVMPTF 4VQQMFNFOUBM 'JHVSF %JMVUJPO TFSJFT PG SFDPNCJOBOU )" BOUJHFO BOE WJSVT DPODFOUSBUJPOTZJFMEFETUBUJTUJDBM-0%TGPSUIJTBTTBZPGQ.)" BOEY$&*%N-MJWFWJSVT5IFTFSFTVMUTBSFTVNNBSJ[FE JO5BCMF/PUFUIBU XIJMFUIFWJSVTTJHOBMJOUFOTJUJFTXFSFMPX XF

XFSFMJNJUFEUPUIFTFUFTUDPODFOUSBUJPOTEVFUPUIFDPODFOUSBUJPOPG UIF WJSVT TUPDL TPMVUJPO Y $&*%N- BOE UIF OFFE UP EJMVUFJOSVOOJOHCVGGFSCZBUMFBTU6OGPSUVOBUFMZ UIFSFJTOP EJSFDU SFMBUJPOTIJQ CFUXFFO NPMBS DPODFOUSBUJPO PG )" BOE UIF JOGFDUJPVT EPTF PG FJUIFS DVMUVSFE WJSVT PS WJSVT GSPN B DMJOJDBM TBNQMF 0VS BQQSPYJNBUJPOT CBTFE PO WBMVFT QVCMJTIFE JO UIF MJUFSBUVSF 4VQQMFNFOUBM.FUIPET

TVHHFTUFEUIBUPVSBTTBZTCBTFE PO UIF EF OPWP CJOEFST IBE -0%T UIBU XFSF XJUIJO UIF DMJOJDBMMZ SFMFWBOUSBOHFPG)"DPODFOUSBUJPOEVSJOHJOGMVFO[BJOGFDUJPO 5P RVBOUJGZ UIF CFOFGJU PG VTJOH UIF IFBE SFHJPO CJOEFS BT B DBQUVSFBHFOUBTDPNQBSFEUPUIFUZQJDBM*H(BOUJCPEZ BUSBEJUJPOBM BMM BOUJCPEZCBTFE MBUFSBM GMPX UFTU XBT FWBMVBUFE VTJOH B EJMVUJPO TFSJFTPGUIFTBNFSFDPNCJOBOU)"BOEXIPMFWJSVTBTBCPWF5IF BTTBZSFTQPOTFDVSWFTBOESFQSFTFOUBUJWFJNBHFTPGUFTUMJOFTBUFBDI DPODFOUSBUJPOBSFQSPWJEFEJO'JHVSFTBOEGPSSFDPNCJOBOU)" BOE XIPMF WJSVT SFTQFDUJWFMZ 5IF TUBOEBSE FSSPS PG UIF -0% FTUJNBUF GPS WJSVT XBT FYUSFNFMZ IJHI PXJOH UP UIF OPJTZ BOE MPX TJHOBM JO UIJT SFHJNF SFTVMUJOH JO BO VOQSPEVDUJWF $* PG 5IJTMBSHFDPOGJEFODFJOUFSWBMSFJUFSBUFEUIFBCTFODFPGBO PCTFSWBCMFTJHOBMJOUIJTDPODFOUSBUJPOSBOHF XIJDIXBTMJNJUFECZ UIF TUPDL DPODFOUSBUJPO PG UIF WJSVT GSPN XIJDI UP HFOFSBUF BO FGGFDUJWF-0%FTUJNBUF)PXFWFS UIFTFSFTVMUTDMFBSMZJOEJDBUFEUIBU UIF BOUJCPEZ BTTBZ XBT MFTT TFOTJUJWF UIBO UIF OPWFM BTTBZ XJUI SFDPNCJOBOUIFBESFHJPOCJOEFS XIJDIQSPEVDFECPUITJHOBMBOEB SFMJBCMF-0%FTUJNBUFGPSUIJTDPODFOUSBUJPOSBOHF *O DPNQBSJTPO UP UIF QSFWJPVT TUFN SFHJPO CJOEFS BTTBZ 4VQQMFNFOUBM'JHVSF"

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


Page 5 of 14

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


UIFBTTBZ JODPOUSBTUUPUIFIFBESFHJPOCJOEFSBTTBZXIJDIXBTBCMF UPEFUFDUUIFOBUJWFGPSNPGUIFQSPUFJO *O BO BUUFNQU UP GVSUIFS EFNPOTUSBUF UIF DBQBCJMJUJFT PG UIF SFDPNCJOBOUIFBESFHJPO BGGJOJUZ QSPUFJOT UIFZ XFSF VTFEBTCPUI DBQUVSFBOEEFUFDUJPOBHFOUT*OUIJTBTTBZ UIFSFDPNCJOBOUDBQUVSF BOEEFUFDUJPOBHFOUTCJOEUIFTBNFFQJUPQFPOUIF)"QSPUFJO TP UIFTJHOBMBUUIFUFTUMJOFJTEFQFOEFOUPOUIFQSFTFODFPGBUMFBTUUXP BWBJMBCMF)"QSPUFJOT5IFSFGPSF SFDPNCJOBOU)" XIJDIDPOUBJOT POMZ POF TJBMJD BDJE CJOEJOH TJUF XBT OPU DBQBCMF PG TJNVMUBOFPVT DBQUVSF BOE EFUFDUJPO )PXFWFS VTJOH UIF IFBE SFHJPO CJOEFS BT CPUI DBQUVSF BOE EFUFDUJPO BHFOUT XF XFSF BCMF UP PCTFSWF EFUFDUJPO PG JOUBDU JOGMVFO[B WJSVT 'JHVSF $ 5IF -0% GPS UIF BTTBZ VTJOH TPMFMZ EF OPWP CJOEFST XBT GPVOE UP CF Y $&*%N- XIJDIXBTTVCTUBOUJBMMZCFUUFSUIBOUIF-0%GPSUIFBMM BOUJCPEZBTTBZ Y$&*%N- XJUITUBUJTUJDBMTJHOJGJDBODF 5IF-0%PGUIFBMMEFOPWPCJOEFSBTTBZXBTGPMECFUUFSUIBOUIF -0%PGUIFBTTBZXJUICJOEFSDBQUVSFBOEBOUJCPEZEFUFDUJPO Y $&*%N-

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

'JHVSF 'MV )" BTTBZ XJUI OPWFM IFBE SFHJPO CJOEFS BT CPUI DBQUVSFBOEEFUFDUJPOPGJOGMVFO[BWJSVT " "OUJCPEZDBQUVSFBOE EFUFDUJPOPGJOUBDUJOGMVFO[BWJSVT # )FBESFHJPOCJOEFSDBQUVSF BOE BOUJCPEZ EFUFDUJPO PG JOUBDU JOGMVFO[B WJSVT $ )FBE SFHJPO CJOEFS DBQUVSF BOE EFUFDUJPO PG JOUBDU JOGMVFO[B WJSVT "T UIF BOUJCPEJFTBSFSFQMBDFEXJUIUIFIFBESFHJPOCJOEFST BSFEVDUJPOJO UIFMPXFSMJNJUPGEFUFDUJPODBOCFTFFO XJUIUIFMPXFTU-0%TIPXO JO QBOFM $ 3FQSFTFOUBUJWF UFTU MJOFT BOE UIF GVMM BTTBZ SFTQPOTF DVSWFTBSFQSPWJEFE&BDIDPOEJUJPOXBTUFTUFEXJUIOSFQMJDBUFT



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

5BCMF4VNNBSZPGBTTBZSFTVMUT5IF-0%XJUI$*GPSFBDI BTTBZ JT MJTUFE BOE DPNQBSFE UP UIF DMJOJDBMMZ SFMFWBOU SBOHF PG JOGMVFO[B*OGMVFO[BWJSVTJOGFDUJWJUZJTSFQPSUFEVTJOHUXPEJGGFSFOU VOJUT UJTTVFDVMUVSFJOGFDUJPVTEPTFFOEQPJOU 5$*%N- PS DIJDLFO FNCSZP JOGFDUJPVT EPTF FOEQPJOU $&*%N- EFQFOEJOHPOXIJDIDFMMMJOFJTVTFEUPEFUFSNJOFUIFJOGFDUJPVTUJUFS 5IF DMJOJDBMMZ SFMFWBOU SBOHF PG WJSVT JO 5$*%N- PG OBTPQIBSZOHFBMXBTIJTLOPXOGSPNUIFMJUFSBUVSF*UJTJNQPSUBOUUP OPUF UIBU UIFTF DPODFOUSBUJPOT XJMM EJGGFS GSPN EJGGFSFOU TBNQMJOH UFDIOJRVFT5IFSBOHFTJOUFSNTPG)"BOEWJSVT BSFFTUJNBUFEBTEFTDSJCFEJOUIF4VQQMFNFOUBM.FUIPET

$MJOJDBMMZ3FMFWBOU3BOHF

-PX

.JE

)JHI

7JSVT

Y

Y

Y

7JSVT

Y

Y

Y

)"

G.

Q.

O.

*OGMVFO[B)"%FUFDUJPO"TTBZ-0%T -PXFS $*

6QQFS

-0%

$*

)FBESFHJPO CJOEFS )"

)FBESFHJPO CJOEFS WJSVT

Y

Y

Y

"MMCJOEFSTUBDL WJSVT

Y

Y

Y

"OUJCPEZ )"

"OUJCPEZ WJSVT

OB

Y

OB

5IF IFBE SFHJPO CJOEFS VTFE JO UIF BTTBZT QSFTFOUFE IFSF XBT EFTJHOFEVTJOHUIFDSZTUBMTUSVDUVSFPGUIF)/4PMPNPO*TMBOET TUSBJOPGUIFJOGMVFO[BWJSVT4USBJOTPGCPUI)/BOE)/ DJSDVMBUF TFBTPOBMMZ BOE IBWF CFFO SFTQPOTJCMF GPS GPVS NBKPS JOGMVFO[B QBOEFNJDT 5IF NVUBHFOJD OBUVSF PG UIF IFBE SFHJPO QSFWFOUT BOUJ)" BOUJCPEJFT BHBJOTU POF TUSBJO GSPN QSPUFDUJOH JOEJWJEVBMT GSPN B EJGGFSFOU TUSBJO PG UIF WJSVT "T B DPOTFRVFODF IJHIBGGJOJUZBHBJOTUPOFTUSBJODPVMEBMTPJODSFBTFTUSBJOTQFDJGJDJUZ POFCJOEFSNBZIBWFMFTTEJGGFSFOUBGGJOJUZUP)"PGBOPUIFSTUSBJO 5BSHFUFE CJOEJOH FOBCMFT HSFBUFS TQFDJGJDJUZ BOE EJGGFSFOUJBUJPO CFUXFFO UIF DJSDVMBUJOH TUSBJOT PG UIF WJSVT 'PS DPNQMFUF DMJOJDBM DPWFSBHF DBSFGVMEFTJHOPGBEEJUJPOBMCJOEFSTUPPUIFSTUSBJOTPGUIF WJSVTGPSVTFJOBNVMUJQMFYFEEFWJDFXJMMCFSFRVJSFE #BTFE PO UIFTF SFTVMUT CPUI SFDPNCJOBOU IFBE SFHJPO CJOEFS CBTFE BTTBZT ZJFME TVQFSJPS BOBMZUJDBM TFOTJUJWJUZ UP UIF MBUFSBM GMPX JNNVOPBTTBZEFWFMPQFEVTJOHDPNNFSDJBMMZBWBJMBCMFNPOPDMPOBM BOUJCPEJFTGPSDBQUVSFBOEEFUFDUJPOPG)"&WFOUIPVHIUIF-0% JT OPU JNQSPWFE BT DPNQBSFE UP UIF DPNNFSDJBMMZ BWBJMBCMF OVDMFPQSPUFJOCBTFESBQJE EJBHOPTUJDT UIFTFBGGJOJUZQSPUFJOTPGGFS UIF QPUFOUJBM UP CFUUFS JOGPSN QBUJFOU NBOBHFNFOU CZ PGGFSJOH

Page 6 of 14

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m *O BEEJUJPO UP BDIJFWJOH B NPSF TFOTJUJWF BOE JOGPSNBUJPOSJDI JOGMVFO[B EFUFDUJPO BTTBZ UIF VTF PG UIF SFDPNCJOBOU BGGJOJUZ QSPUFJOTPQFOTUIFEPPSGPSDPNQBOJPOUIFSBQFVUJDT CFDBVTFUIFTF JOGMVFO[BCJOEFSTIBWFTIPXOUIFQPUFOUJBMGPSUIFSBQFVUJDVTF *O TVDIBDPNQBOJPOTZTUFN UIFJOGMVFO[BCJOEFSCBTFEEJBHOPTUJDUFTU XPVMEOPUPOMZEJBHOPTFUIFGMV CVUJUXPVMEBMTPJOEJDBUFXIJDIGMV CJOEFSTIPVMECFHJWFOGPSUSFBUNFOU CBTFEPOXIJDICJOEFSJTBCMF UPSFDPHOJ[FUIFTQFDJGJDTUSBJOBUIBOENPTUTFOTJUJWFMZ


Page 7 of 14

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


0WFSBMM UIJTXPSLEFNPOTUSBUFTUIFQPXFSPGSFDPNCJOBOUBGGJOJUZ QSPUFJOTGPSBOVNCFSPGEJBHOPTUJDBQQMJDBUJPOT'VUVSFEJSFDUJPOT JODMVEFJODPSQPSBUJPOPGUIFEFNPOTUSBUFEBTTBZJOUPBOBVUPNBUFE EFWJDF BOE WBMJEBUJPO PG UIFTF BTTBZT XJUI DMJOJDBM TBNQMFT 5IJT XPSL JT B TJHOJGJDBOU TUFQ UPXBSE UIF EFWFMPQNFOU PG B IJHI TFOTJUJWJUZ MPXDPTU JOGMVFO[B UFTU GPS JNQSPWFE EJBHOPTJT BOE EJTFBTFNBOBHFNFOUBUUIFQPJOUPGDBSF

"440$*"5&%$0/5&/5 4VQQPSUJOH*OGPSNBUJPO "EEJUJPOBMJOGPSNBUJPOBTOPUFEJOUIFUFYU5IJTNBUFSJBMJTBWBJMBCMFGSFF PGDIBSHFPOUIF"$41VCMJDBUJPOTXFCTJUF

"65)03*/'03."5*0/ $PSSFTQPOEJOH"VUIPST &NBJMDFB!VXFEV

"VUIPS$POUSJCVUJPOT

$")BOE$&"DPOUSJCVUFEFRVBMMZUPUIJTXPSL

/PUFT 5IFBVUIPSTEFDMBSFOPDPNQFUJOHGJOBODJBMJOUFSFTU

"$,/08-&%(.&/5 5IF BVUIPST UIBOL (& (MPCBM 3FTFBSDI GPS QSPWJEJOH OJUSPDFMMVMPTF NFNCSBOFTBOE BSFHSBUFGVMGPSIFMQGVMEJTDVTTJPOTXJUIDPMMFBHVFTJO UIF:BHFSBOE#BLFSHSPVQT5IJTNBUFSJBMJTCBTFEVQPOXPSLTVQQPSUFE CZ UIF /BUJPOBM 4DJFODF 'PVOEBUJPO (SBEVBUF 3FTFBSDI 'FMMPXTIJQ 1SPHSBNVOEFS(SBOU/P%(&m CZUIF/BUJPOBM*OTUJUVUFPG "MMFSHZ BOE *OGFDUJPVT %JTFBTFT PG UIF /BUJPOBM *OTUJUVUFT PG )FBMUI VOEFS BXBSE OVNCFS 3"* BOE CZ UIF 6OJWFSTJUZ PG 8BTIJOHUPO %FQBSUNFOU PG #JPFOHJOFFSJOH 5IF DPOUFOU JT TPMFMZ UIF SFTQPOTJCJMJUZ PG UIF BVUIPST BOE EPFT OPU OFDFTTBSJMZ SFQSFTFOU UIF PGGJDJBMWJFXTPGUIF/BUJPOBM*OTUJUVUFTPG)FBMUI

3&'&3&/$&4 $FOUFST GPS %JTFBTF $POUSPM BOE 1SFWFOUJPO 4FBTPOBM *OGMVFO[B .BS "WBJMBCMF IUUQXXXDEDHPWGMV $FOUFST GPS %JTFBTF $POUSPM BOE 1SFWFOUJPO &TUJNBUFT PG %FBUIT "TTPDJBUFE XJUI 4FBTPOBM *OGMVFO[B 6OJUFE 4UBUFT .PSC .PSUBM8LMZ3FQm *OTUJUVUF GPS )FBMUI .FUSJDT BOE &WBMVBUJPO (MPCBM #VSEFO PG %JTFBTF "WBJMBCMFIUUQXXXIFBMUIEBUBPSHHCE 8SJHIU 1' /FVNBOO ( ,BXBPLB : 'JFMET 7JSPMPHZ UI FE 1IJMBEFMQIJB 1"-JQQJODPUU8JMMJBNTBOE8JMLJOT $FOUFST GPS %JTFBTF $POUSPM BOE 1SFWFOUJPO $%$ 3FQPSUT 'MV )JU :PVOHFS 1FPQMF 1BSUJDVMBSMZ )BSE 5IJT 4FBTPO 'FC "WBJMBCMF IUUQXXXDEDHPWNFEJBSFMFBTFTQGMVSFQPSUIUNM .PMJOBSJ/"0SUFHB4BODIF[*3.FTTPOOJFS.-5IPNQTPO88 8PSUMFZ1.8FJOUSBVC&#SJEHFT$#7BDDJOFm $FOUFSTGPS%JTFBTF$POUSPMBOE1SFWFOUJPO$%$3FDPNNFOEBUJPOTGPS *OGMVFO[B "OUJWJSBM .FEJDBUJPOT 3FNBJO 6ODIBOHFE "QS "WBJMBCMF IUUQXXXDEDHPWNFEJBIBWFZPVIFBSETUPSJFT*OGMVFO[B@BOUJWJSBMI UNM #POOFS "# .POSPF ,8 5BMMFZ -* ,MBTOFS "& ,JNCFSMJO %8 1FEJBUSJDTm )VSU "$ "MFYBOEFS 3 )JCCFSU + %FFE / #BSS *( + $MJO 7JSPM m

-BNC 3" (FOFUJDT PG *OGMVFO[B 7JSVTFT "VTUSJB 4QSJOHFS 7FSMBH8JFOQQm 8FJT8#SPXO+)$VTBDL41BVMTPO+$4LFIFM++8JMFZ%$ /BUVSFm 8FCTUFS3(#FBO8+(PSNBO05$IBNCFST5.,BXBPLB: .JDSPCJPM3FW 1BOH:3POH;8BOH+9JBP38BOH4 #JPTFOTBOE#JPFMFDUSPO $FOUFST GPS %JTFBTF $POUSPM BOE 1SFWFOUJPO &WBMVBUJPO PG $PNNFSDJBMMZ"WBJMBCMF3BQJE*OGMVFO[B%JBHOPTUJD5FTUT6OJUFE4UBUFT .PSC.PSUBM8LMZ3FQm $IBO,)-BJ451PPO--.(VBO::VFO,:1FJSJT+4. + $MJO7JSPMm 0$POOFMM ,1 ,PWBMFWB & $BNQCFMM +) "OEFSTPO 1& #SPXO 4(%BWJT%$7BMEFT++8FMDI38#FOUMFZ8&WBO#FFL/".PM #JPUFDIOPM #PMUPWFUT1.1PMJTDIVL0.,PWBMFOLP0(4OPQPL#""OBMZTU $IFOBJM ( #SPXO /& 4IFB " 'FJSF "- %FOH ( "OBMZUJDBM #JPDIFNJTUSZ (VCBMB7)BSSJT-'3JDDP"+5BO.98JMMJBNT%&"OBM$IFN m .BOTGJFME . -BUFSBM 'MPX *NNVOPBTTBZ /FX :PSL /: )VNBOB 1SFTTQQm 0'BSSFMM#-BUFSBM'MPX*NNVOPBTTBZ/FX:PSL /:)VNBOB1SFTT QQm "INBE,.0I44.D$MFMMFO'.9JBP:4PI)5 1-P40OF F 4IJSBUPSJ * "LJUPNJ + #PMU[ %" )PSJJ , 'VSVJDIJ . 8BHB * #JPDIFN#JPQI3FT$P 8BOH3;IBP++JBOH5,XPO:.-V)+JBP1-JBP.-J: + 7JSPM.FUIPET 8PP ). -FF +. :JN 4 +FPOH :+ 1-P4 0OF F %BT3#BLFS%"OOV3FW#JPDIFN 7FSNBTWVPSJ31SPEVDUJPOPG3FDPNCJOBOU1SPUFJOTBOE.POPDMPOBM "OUJCPEJFT m 5FDIOP&DPOPNJDBM &WBMVBUJPO PG UIF 1SPEVDUJPO.FUIPET )FMTJOLJ 6OJWFSTJUZ PG 5FDIOPMPHZ "WBJMBCMF IUUQMJCULLGJ-JDVSOQEG 'MFJTINBO 4+ 8IJUFIFBE 5" &LJFSU %$ %SFZGVT $ $PSO +& 4USBVDI&.8JMTPO*"#BLFS%4DJFODFm 8IJUFIFBE5"$IFWBMJFS"4POH:%SFZGVT$'MFJTINBO4+%F .BUUPT$.ZFST$",BNJTFUUZ)#MBJS18JMTPO*"#BLFS% /BU #JPUFDIOPMm )PMTUFJO $" $IFWBMJFS " #FOOFU 4 "OEFSTPO $& ,FOJTUPO , 0MTFO $ -J # #BMFT # .PPSF %3 'V & #BLFS % :BHFS 1 "OBM #JPBOBM$IFN 4USBVDI&.#FSOBSE4.-B%#PIO"+-FF14"OEFSTPO$& /JFVTNB 5 )PMTUFJO $"(BSDJB /, )PPQFS," 3BWJDIBOESBO 3 /FMTPO+84IFGGMFS8#MPPN+%-FF,,8BSE"#:BHFS1'VMMFS %)8JMTPO*"#BLFS%/BUVSF#JPUFDIOPM*OQSFTT 4UVEJFS '8 .FUIPET.PM#JPM $VSSJF-"1VSF"QQM$IFNm )PMTUFJO$"(SJGGJO.)POH+4BNQTPO1% "OBM$IFN 7FNVMB47;IBP+2-JV+,8BOH9#JTXBT4)FXMFUU*7JSVTFT -JV 9 -J 5 ;IFOH : 8POH ,8 -V 4 -V ) &NFSH .JDSPCFT *OGFDU FEPJFNJ 'V & 3BNTFZ 4" ,BVGGNBO 1 -VU[ # :BHFS 1 .JDSPGMVJE /BOPGMVJEJDTm 'V&,BVGGNBO1-VU[#:BHFS1 4FOT"DUVBUPST#$IFN m 'V&-JBOH5)PVHIUBMJOH+3BNBDIBOESBO43BNTFZ4"-VU[# :BHFS1"OBM$IFNm 'V & -JBOH5 4QJDBS.JIBMJD 1 )PVHIUBMJOH + 3BNBDIBOESBO 4 :BHFS1"OBM$IFNm


Analytical Chemistry 'SJEMFZ(&-F)2'V&:BHFS1-BC$IJQ

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 14

5PMFZ#+8BOH+"(VQUB.#VTFS+3-BGMFVS-,-VU[#3'V &:BHFS1-BC$IJQm


Page 9 of 14

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 1. Illustration of the influenza virus and the targeted sites for binder development. The virus at left shows the trimeric hemagglutinin (HA) protein (in blue) on the viral surface. The images at center and right show the two classes of conserved sites (in yellow) to which the synthetic binders were targeted. Influenza virus image from cdc.gov. 339x171mm (96 x 96 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 2. Illustration of the influenza surface protein HA (purple) bound to the computationally-designed head region binder (orange). Detailed information about the binder proteins is provided in Supplemental Table 1. 193x101mm (96 x 96 DPI)


Page 10 of 14

Page 11 of 14

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 3. Illustrations of the novel influenza HA “assay stacks” demonstrated herein. (A) Assay stacks for recombinant HA detection, including all antibody detection (top) and head region binder capture with antibody detection (bottom) (B) Assay stacks for intact influenza virus, including all antibody detection (left), the mixed assay stack with head region binder capture and antibody detection (middle), and head region binder capture and detection (right). (C) Figure legend describing each molecular component. Note that the molecular components are not to scale. 464x236mm (96 x 96 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 4. Recombinant Flu HA assays with either antibody or novel head region binder as capture agent, with antibody gold readout. Representative test lines and the full assay response curves are provided. Each condition was tested with n=4 replicates. (A) Antibody capture assay run with recombinant HA. (B) Head region binder capture assay run with recombinant HA. 281x122mm (96 x 96 DPI)


Page 12 of 14

Page 13 of 14

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 5. Flu HA assay with novel head region binder as both capture and detection of influenza virus. (A) Antibody capture and detection of intact influenza virus. (B) Head region binder capture and antibody detection of intact influenza virus. (C) Head region binder capture and detection of intact influenza virus. As the antibodies are replaced with the head region binders, a reduction in the lower limit of detection can be seen, with the lowest LOD shown in panel C. Representative test lines and the full assay response curves are provided. Each condition was tested with n=3 replicates. 154x346mm (96 x 96 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Table of contents graphic 513x278mm (96 x 96 DPI)


Page 14 of 14

Rapid Diagnostic Assay for Intact Influenza Virus ... - ACS Publications

Recommend Documents