Biacore. Concentration Analysis Handbook

Biacore Concentration Analysis Handbook

Biacore Concentration Analysis Handbook :IVWMSR%%(IGIQFIV

&MEGSVI 'SRGIRXVEXMSR%REP]WMW,ERHFSSO )HMXMSR(IGIQFIV:IVWMSR%% 'ST]VMKLX &MEGSVI%& %PPVMKLXWVIWIVZIH2STEVXWSJXLMWTYFPMGEXMSRQE]FIVITVSHYGIH [MXLSYX[VMXXIRTIVQMWWMSRJVSQ&MEGSVI%& &MEGSVI%&VIWIVZIWXLIVMKLXXSQEOIGLERKIWXSXLIHIWMKRERH GSRXIRXSJXLI'SRGIRXVEXMSR%REP]WMW,ERHFSSO[MXLSYXTVMSVRSXMGI &MEGSVIMWEVIKMWXIVIHXVEHIQEVOSJ&MEGSVI%& 7IRH]SYVGSQQIRXWF]IQEMPXSHIZIPSTFME$FMEGSVIGSQ &MEGSVI%& 6ETWKEXER 7)9TTWEPE7[IHIR 8IP *E\

Contents Contents 1. Introduction 1 4VMRGMTPIWSJ&MEGSVI &MEGSVIMRGSRGIRXVEXMSRQIEWYVIQIRX ;L]YWI&MEGSVI# %WWE]HIZIPSTQIRXSZIVZMI[ 2. Terminology 5 &MEGSVIXIVQMRSPSK] 4IVJSVQERGIGVMXIVME 7TIGMJMGMX]WIPIGXMZMX]ERHGVSWWVIEGXMZMX] %GGYVEG] 4VIGMWMSR 0MQMXSJHIXIGXMSR03( 0MQMXWSJUYERXMXEXMSR035 0MRIEVMX] 6ERKI 6SFYWXRIWW 7IRWMXMZMX] 3. Assay formats 15 (MVIGXFMRHMRKEWWE]W 7MRKPIWXITHMVIGXQIEWYVIQIRX 7ERH[MGLQIXLSHW -RHMVIGXEWWE]W -RLMFMXMSREWWE]W 7YVJEGIGSQTIXMXMSR &MRHMRKVEXIEWWE]W 'LSMGISJEWWE]XIGLRMUYI 'LSMGISJPMKERHERHSXLIVVIEKIRXW 4VEGXMGEPETTVSEGLIWXSVIEKIRXWIPIGXMSR (MVIGXFMRHMRKEWWE]W -RLMFMXMSREWWE]W 7YVJEGIGSQTIXMXMSREWWE]W 4. Surface preparation principles 27 7IRWSVWYVJEGITVSTIVXMIW 0MKERHMQQSFMPM^EXMSRQIXLSHW %QMRIGSYTPMRK 8LMSPGSYTPMRK M

Contents %PHIL]HIGSYTPMRK 7XVITXEZMHMRFMSXMRGETXYVI +IRIVEPGETXYVIQIXLSHW -QQSFMPM^MRKWQEPPQSPIGYPIW 'SRHMXMSRWJSVPMKERHMQQSFMPM^EXMSR &YJJIVGSRHMXMSRWERHTVIGSRGIRXVEXMSR 7XVEXIK]JSVWYVJEGITVITEVEXMSR %QSYRXSJMQQSFMPM^IHPMKERH 'LSMGISJMQQSFMPM^EXMSRQIXLSH 5. Surface preparation in practice 37 'SRHMXMSRWJSVMQQSFMPM^EXMSR -QQSFMPM^EXMSRTVSGIHYVI 4VITEVMRKWSPYXMSRW %QMRIGSYTPMRK 7YVJEGIXLMSPGSYTPMRK 0MKERHXLMSPGSYTPMRK %PHIL]HIGSYTPMRK 6IWYPXWSJMQQSFMPM^EXMSR 8IWXMRKXLIWYVJEGI 8VSYFPIWLSSXMRKWYVJEGITVITEVEXMSR 0S[MQQSFMPM^EXMSRPIZIPW 0S[EREP]XIVIWTSRWIW 6. Regeneration 49 6IKIRIVEXMSRWGSYXMRKWXVEXIK] 'LSMGISJVIKIRIVEXMSRWSPYXMSR 8IWXMRKVIKIRIVEXMSRGSRHMXMSRW -RXIVTVIXMRKVIKIRIVEXMSRWGSYXMRK 6ITSVXTSMRXTPEGMRK 8VIRHTPSXW 7. Developing and running concentration assays 55 %WWE]VIUYMVIQIRXWTIGMJMGEXMSR +IRIVEPTVEGXMGEPGSRWMHIVEXMSRW 4VITEVMRKWEQTPIW 6ITSVXTSMRXTPEGMRK %HNYWXMRKXLISTIVEXMRKVERKI (MVIGXFMRHMRKEWWE]W -RLMFMXMSREWWE]W 9R[ERXIHFMRHMRKSJEREP]XISVHIXIGXMRKQSPIGYPI XSXLIWIRWSVWYVJEGI 9R[ERXIHFMRHMRKMRHMVIGXFMRHMRKEWWE]W 9R[ERXIHFMRHMRKMRMRLMFMXMSRERHWYVJEGIGSQTIXMXMSR EWWE]W MM

Contents 8IWXMRKJSVYR[ERXIHFMRHMRK (IEPMRK[MXLYR[ERXIHFMRHMRK 7TIGMJMGMX]ERHGVSWWVIEGXMZMX] 1IEWYVMRKWTIGMJMGMX] 3TXMQM^MRKWTIGMJMGMX] 1EXVM\MRXIVJIVIRGI 2SRWTIGMJMGFMRHMRKXSXLIWIRWSVWYVJEGI 3XLIVQEXVM\MRXIVJIVIRGIIJJIGXW %WWE]WXEFMPMX] 8IWXMRKEWWE]WXEFMPX] (IEPMRK[MXLEWWE]WXEFMPMX]MWWYIW 6YRRMRKVSYXMRIEWWE]W 8. Measuring concentration in GxP environments 73 7IXXMRKYT+\4EWWE]W 7]WXIQTIVJSVQERGI %WWE]WIXXMRKW %WWE]TVSGIHYVIHSGYQIRXEXMSR (EXEWXSVEKI :EPMHEXMRK+\4EWWE]W 7TIGMJMGMX] %GGYVEG] 4VIGMWMSR 0MRIEVMX] 6ERKI 6SFYWXRIWW 6YRRMRK+\4EWWE]W (SGYQIRXXLIVYRWIXXMRKW (EXEWXSVEKI %YHMXXVEMPW A. The SPR detection principle 79 % 7YVJEGITPEWQSRVIWSRERGI % ;LEX746QIEWYVIW % &MEGSVIGSRJMKYVEXMSR B. Using binding rates to measure concentration 83 & *EGXSVWHIXIVQMRMRKFMRHMRKVEXIW & &MSGLIQMGEPMRXIVEGXMSRVEXIW & 1EWWXVERWTSVXTVSGIWWIW & ;LEXPMQMXWXLISFWIVZIHFMRHMRK# & %JJMRMX]MRHITIRHIRXEWWE]W & %HZERXEKIWSJEJJMRMX]MRHITIRHIRXEWWE]W & %JJMRMX]MRHITIRHIRXEWWE]WMRTVEGXMGI & 'EPMFVEXMSRMRHITIRHIRXEWWE]W MMM

Contents MZ

Introduction 1. Introduction 1.1 Principles of Biacore &MEGSVIW]WXIQWI\TPSMXXLITLIRSQIRSRSJWYVJEGITPEWQSRVIWSRERGI 746XSQSRMXSVXLIMRXIVEGXMSRFIX[IIRQSPIGYPIWMRVIEPXMQI8LI XIGLRSPSK][LMGLMWHIWGVMFIHMRQSVIHIXEMPMR%TTIRHM\%MRZSPZIW EXXEGLQIRXSJSRIMRXIVEGXMRKTEVXRIVXSXLIWYVJEGISJEWIRWSVGLMT ERHXLIRTEWWMRKWEQTPIGSRXEMRMRKSXLIVTSXIRXMEPMRXIVEGXMSR TEVXRIVWSZIVXLIWYVJEGI&MRHMRKSJQSPIGYPIWXSXLIWIRWSVWYVJEGI KIRIVEXIWEVIWTSRWI[LMGLMWTVSTSVXMSREPXSXLIFSYRHQEWWERH GLERKIWMREQSYRXFSYRHGERFIHIXIGXIHHS[RXSEJI[TMGSKVEQW TIVWUYEVIQMPPMQIXIVSRXLIWIRWSVWYVJEGI&MRHMRKIZIRXWEVI JSPPS[IHMRVIEPXMQIERHEVERKISJMRXIVEGXMSRGLEVEGXIVMWXMGWGERFI HIXIVQMRIH%QSRKXLIUYIWXMSRXLEXGERFIEHHVIWWIH[MXL&MEGSVI EVI 8LIWTIGMJMGMX]SJFMSQSPIGYPEVMRXIVEGXMSRWMRZIWXMKEXIHF]XIWXMRK XLII\XIRXSJFMRHMRKFIX[IIRHMJJIVIRXTEMVWSJQSPIGYPIW 8LIOMRIXMGWERHEJJMRMX]SJERMRXIVEGXMSRMRZIWXMKEXIHF] EREP]^MRKXLIXMQIGYVZIERHPIZIPSJFMRHMRKMRXIVQWSJQSPIGYPEV MRXIVEGXMSRQSHIPW 8LIGSRGIRXVEXMSRSJWTIGMJMGQSPIGYPIWTVIWIRXMRXLIWEQTPI MRZIWXMKEXIHF]QIEWYVMRKXLIPIZIPSJVIWTSRWISFXEMRIHJVSQXLI WEQTPI 1.2 Biacore in concentration measurement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

Introduction 8LMWLERHFSSOTVSZMHIWKYMHIPMRIWJSVLS[XSHIZIPSTERHYWI GSRGIRXVEXMSREWWE]W[MXL&MEGSVIW]WXIQW8LIMRJSVQEXMSRMWKMZIREW JEVEWTSWWMFPIMRKIRIVEPXIVQW[MXLSYXHMVIGXVIJIVIRGIXSWTIGMJMG &MEGSVIW]WXIQW8LITVMRGMTPIWSJEWWE]HIZIPSTQIRXERH GSRGIRXVEXMSRHIXIVQMREXMSRGEREPWSFIETTPMIH[MXLETTVSTVMEXI EHNYWXQIRXWXSEWWE]WFEWIHSRSXLIVXIGLRSPSKMIW 1.3 Why use Biacore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

Introduction 1.4 Assay development overview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

Introduction

Terminology 2. Terminology 8LMWGLETXIVTVIWIRXWXLIXIVQMRSPSK]XLEXMWYWIHMRXLIGSRXI\XSJ GSRGIRXVEXMSRQIEWYVIQIRXW[MXL&MEGSVI8LIJMVWXWIGXMSRTVIWIRXW XIVQWWTIGMJMGXS&MEGSVIETTPMGEXMSRWERHXLIWIGSRHWIGXMSRHIJMRIW XLIXIVQWYWIHXSHIWGVMFIEWWE]TIVJSVQERGIMRXLIGSRXI\XSJ ZEPMHEXIHEREP]XMGEPTVSGIHYVIW 2.1 Biacore terminology &MEGSVIW]WXIQWQSRMXSVXLIMRXIVEGXMSRFIX[IIRX[SQSPIGYPIWSJ [LMGLSRIMWEXXEGLIHXSXLIWIRWSVWYVJEGIERHXLISXLIVMWJVIIMR WSPYXMSR8LIJSPPS[MRKXIVQWEVIYWIHMRXLIGSRXI\XSJGSRGIRXVEXMSR QIEWYVIQIRX[MXL&MEGSVI 8LITEVXRIVEXXEGLIHXSXLIWYVJEGIMWGEPPIHXLIPMKERH%XXEGLQIRX QE]FIGSZEPIRXSVXLVSYKLLMKLEJJMRMX]FMRHMRKXSERSXLIV QSPIGYPI[LMGLMWMRXYVRGSZEPIRXP]EXXEGLIHXSXLIWYVJEGIWII 'LETXIV-RXLIPEXXIVGEWIXLIQSPIGYPIEXXEGLIHXSXLIWYVJEGIMW VIJIVVIHXSEWXLIGETXYVMRKQSPIGYPI8LIXIVQlPMKERHzMW ETTPMIHLIVIMREREPSK][MXLXIVQMRSPSK]YWIHMREJJMRMX] GLVSQEXSKVETL]GSRXI\XWERHHSIWRSXMQTP]XLEXXLIWYVJEGI EXXEGLIHQSPIGYPIMWEPMKERHJSVEGIPPYPEVVIGITXSV 8LIEREP]XIMWXLIQSPIGYPIJSV[LMGLXLIGSRGIRXVEXMSRMWXSFI QIEWYVIH-RHMVIGXFMRHMRKEWWE]WXLIEREP]XIFMRHWHMVIGXP]XSXLI PMKERH-RMRLMFMXMSREWWE]WXLIGSRGIRXVEXMSRSJEREP]XIMW QIEWYVIHMRHMVIGXP]XLVSYKLFMRHMRKSJEREHHMXMSREPQSPIGYPI analyte analyte ligand ligand capturing molecule Figure 2-1. Ligand, analyte and capturing molecule in relation to the sensor surface. 8LIHIXIGXMRKQSPIGYPIMWXLIQSPIGYPIVIWTSRWMFPIJSVHIXIGXMRKXLI EREP]XI-RHMVIGXFMRHMRKEWWE]WXLIHIXIGXMRKQSPIGYPIMWXLIWEQI EWXLIPMKERH-RMRLMFMXMSREWWE]WXLIHIXIGXMRKQSPIGYPIMWXLI QSPIGYPIXLEXFMRHWXSXLIPMKERHERHKIRIVEXIWEVIWTSRWI[LMGLMW FPSGOIHF]XLITVIWIRGISJEREP]XI

Terminology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bsolute response (RU) Report points Relative response (RU) Buffer Sample Buffer Regeneration Buffer Time Figure 2-2. Schematic illustration of a sensorgram. The bars below the sensorgram curve indicate the solutions that pass over the sensor surface. 8LIWEQTPIQEXVM\MWXLIWSPYXMSRIRZMVSRQIRXMR[LMGLEREP]XIMW TVIWIRXMRWEQTPIW8LMWQE]FIEWMQTPI[IPPHIJMRIHWSPYXMSRWYGL EWEFYJJIVSVEGSQTPI\QM\XYVIWYGLEWEFSH]JPYMHGIPPI\XVEGX SVTVSHYGXJSVQYPEXMSR8LIXIVQQEXVM\MWEPWSYWIHXSVIJIVXS XLIGEVFS\]QIXL]PHI\XVERSRXLIWYVJEGISJXLIWIRWSVGLMTMR &MEGSVIEWHIWGVMFIHMR7IGXMSR8LMWYWEKIMWMRRS[E] GSRRIGXIHXSXLIXIVQWEQTPIQEXVM\

Terminology 2.2 Performance criteria,s[izivgsrgirxvexmsrmwqiewyvihxlivimwewixsjwxerhevhm^ih GVMXIVMEXLEXQIEWYVIXLITIVJSVQERGISJXLIEWWE][MXLMRERHFIX[IIR EWWE]SGGEWMSRW(SGYQIRXIHTIVJSVQERGIMWHIWMVEFPIMREPP GSRGIRXVEXMSRQIEWYVIQIRXWERHMWEVIUYMVIQIRXJSVZEPMHEXIHEWWE]W YWIHMRUYEPMX]GSRXVSPERHSXLIVGSRXI\XW8LIWXERHEVHTIVJSVQERGI GVMXIVMEHIJMRIHMRXLIJSPPS[MRKWIGXMSRWEVIFEWIHSRXLI-', VIGSQQIRHEXMSRW 'SRWMHIVEXMSRWSJLS[XSQIEWYVITIVJSVQERGI GVMXIVMEJSVEKMZIREWWE]EVIGSRWMHIVIHMR'LETXIV 2.2.1 Specificity, selectivity and cross-reactivity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

Terminology 100 Response/MW Compound A 50 Compound B 0 1 100 Concentration Figure 2-3. Cross-reactivity in a direct assay is determined from calibration curves of molecular weight-corrected response against concentration. In this illustration, compound B shows 1% cross-reactivity with compound A. 2.2.2 Accuracy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recision 8LITVIGMWMSRSJEREWWE]HIWGVMFIWXLIEKVIIQIRXHIKVIISJWGEXXIV FIX[IIRVIWYPXSFXEMRIHJVSQQYPXMTPIQIEWYVIQIRXWSRXLIWEQI LSQSKIRISYWWEQTPI4VIGMWMSRQE]FIHIXIVQMRIHEXXLVIIPIZIPW 6ITIEXEFMPMX]MWXLITVIGMWMSRSJXLIEWWE]YRHIVXLIWEQISTIVEXMRK GSRHMXMSRW[MXLXLIWEQIWEQTPISZIVEWLSVXTIVMSHSJXMQI

Terminology X]TMGEPP]VITPMGEXIQIEWYVIQIRXW[MXLMRXLIWEQII\TIVMQIRXEPWS VIJIVVIHXSEWMRXVEEWWE]TVIGMWMSR -RXIVQIHMEXITVIGMWMSRMWXLITVIGMWMSR[MXLMRXLIWEQIPEFSVEXSV] SZIVHMJJIVIRXSGGEWMSRWHMJJIVIRXSTIVEXSVWHMJJIVIRXMRHMZMHYEP EWWE]MRWXVYQIRXWIXG6YKKIHRIWWMWEREPXIVREXMZIXIVQJSV MRXIVQIHMEXITVIGMWMSR 6ITVSHYGMFMPMX]MWXLITVIGMWMSRFIX[IIRHMJJIVIRXPEFSVEXSVMIW YWYEPP]ETTPMIHXSGSPPEFSVEXMZIWXYHMIWMRXLIWXERHEVHM^EXMSRSJ QIXLSHSPSK]-RXIVQIHMEXITVIGMWMSRERHVITVSHYGMFMPMX]EVIX[S HMJJIVIRXEWTIGXWSJMRXIVEWWE]TVIGMWMSR 8LITVIGMWMSRSJEREWWE]MWYWYEPP]I\TVIWWIHMRXIVQWSJXLIZEVMERGI WXERHEVHHIZMEXMSR7(SVGSIJJMGMIRXSJZEVMEXMSR':[MXLMRXLI WIVMIWSJQIEWYVIQIRXW*SVEWIXSJVITPMGEXIQIEWYVIQIRXWXLI WXERHEVHHIZMEXMSRMWKMZIRF] 7( = ( R ) (] ]) M [LIVIR!RYQFIVSJQIEWYVIQIRXW ERH]!VIWTSRWIJSVEKMZIRQIEWYVIQIRX 8LIGSIJJMGMIRXSJZEVMEXMSRMWKMZIRF] 7( ': = QIER 7XEXMWXMGEPTEVEQIXIVWJSVZEVMEXMSRMRQIEWYVIHVIWYPXWQE]FIVIPEXIH XSIMXLIVHSWISVVIWTSRWIZEPYIWXLMWMWTIVLETWQSWXGSQQSRMR GMXEXMSRWSJ':ZEPYIW[LIVIXLIHMWXMRGXMSRMWQEHIFIX[IIR': HSWI ERH': VIWTSRWI 8LI': VIWTSRWI ZEPYIVIJPIGXWXLIGSRWMWXIRG]SJ VIWTSRWIZEPYIWJSVEKMZIRGSRGIRXVEXMSR[LMPI': HSWI VIJPIGXWXLI GSRJMHIRGI[MXL[LMGLEKMZIRVIWTSRWIZEPYIGERFIVIPEXIHXSEREP]XI GSRGIRXVEXMSR-RKIRIVEP': HSWI ZEPYIWEVILMKLEXXLIXSTERH FSXXSQSJXLIQIEWYVIQIRXVERKIEXXLIFSXXSQFIGEYWIPS[ VIWTSRWIWEVIHMJJMGYPXXSHIXIVQMRIEGGYVEXIP]EXXLIXSTFIGEYWIXLI WXERHEVHGYVZIJPEXXIRWSYXEXLMKLGSRGIRXVEXMSRWERHEVIPS[IWXMR XLIQMHHPISJXLIVERKI': VIWTSRWI ZEPYIWSRXLISXLIVLERHEVI JVIUYIRXP]PS[IZIREXXLIXSTSJXLIH]REQMGVERKI': HSWI MWMR KIRIVEPEFIXXIVGVMXIVMSRSJEWWE]TIVJSVQERGIXLER': VIWTSRWI

Terminology Response CV response CV dose Concentration Figure 2-4. CV response is an indication of the variability in the response for a given analyte concentration. CV dose is an indication of the variability in calculated concentration derived from a given set of measurements. %GGYVEG]ERHTVIGMWMSREVIJVIUYIRXP]GSRJYWIHMRGSQQSRTEVPERGI EPXLSYKLXLI]EVIGPIEVP]HMWXMRKYMWLIHMRXLIMVJSVQEPHIJMRMXMSRW%R EWWE]XLEXMWTVIGMWIFYXRSXEGGYVEXI[MPPEP[E]WKMZIXLIWEQI [VSRKERW[IV[LMPISRIXLEXMWEGGYVEXIFYXRSXTVIGMWI[MPPKMZIER ETTVS\MQEXIFYXGSVVIGXERW[IV 2.2.4 Limit of detection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imits of quantitation (LOQ) (IXIVQMREXMSRSJXLI035SJEREWWE]VIUYMVIWXLEXXLITVIGMWMSRERH EGGYVEG]SJXLIEWWE]EVIQIEWYVIHSZIVEVERKISJEREP]XI GSRGIRXVEXMSRWMRSVHIVXSHIXIVQMRIXLIGSRGIRXVEXMSRWEFSZIERH FIPS[[LMGLXLITIVJSVQERGIMWEGGITXEFPI8LIWIGSRGIRXVEXMSRWEVI

Terminology XLIRXLI035WHMJJIVIRXZEPYIWQE]FIETTPMGEFPIEGGSVHMRKXS [LIXLIVTVIGMWMSRMWHIXIVQMRIHEWMRXIVQIHMEXITVIGMWMSRSV VITVSHYGMFMPMX]8LI035WLSYPHRSXFIFEWIHSRVITIEXEFMPMX]SV MRXVEEWWE]TVIGMWMSRWMRGIXLIZEPYILEWPMXXPIQIERMRKMRVIJIVIRGIXS EWMRKPIEWWE]SGGEWMSR 8LIPS[IVPMQMXSJUYERXMXEXMSR0035MWXLIPS[IWXEREP]XI GSRGIRXVEXMSRXLEXGERFIQIEWYVIH[MXLWYMXEFPITVIGMWMSRERH EGGYVEG]8LIPIZIPGSVVIWTSRHMRKXSlWYMXEFPIz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esponse 3xSD acceptable precision and accuracy Limit of detection Limits of quantitation Concentration Figure 2-5. Limits of detection and quantitation.

Terminology 2.2.6 Linearity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ange 8LIVERKISJEREWWE]MWXLIMRXIVZEPFIX[IIRERHMRGPYHMRKXLIYTTIV ERHPS[IVPMQMXWSJUYERXMXEXMSRMIXLIVERKI[MXLMR[LMGLXLI TVIGMWMSREGGYVEG]ERHPMRIEVMX]EVIEGGITXEFPI %TEVEQIXIVSJXIRUYSXIHMRVIPEXMSRXSXLIVERKIMWXLIEREP]XI GSRGIRXVEXMSRXLEXKMZIWSJXLIQE\MQYQVIWTSRWI& JSVE HMVIGXFMRHMRKEWWE]-' JSVERMRLMFMXMSREWWE]2SXIXLEXXLMWMWRSX RIGIWWEVMP]XLIQMHTSMRXSJXLIGSRGIRXVEXMSRVERKI Response/MW 100% Response 100% 50% 50% 0 B 50 Concentration 0 IC 50 Concentration Figure 2-6. B 50 (direct assays, left panel) and IC 50 (inhibition assays, right panel) are the analyte concentration that gives 50% of the maximum response.

Terminology 2.2.8 Robustness 8LIVSFYWXRIWWSJEREWWE]MWEQIEWYVISJMXWGETEGMX]XSVIQEMR YREJJIGXIHF]ZEVMEXMSRWMRQIXLSHTEVEQIXIVW6SFYWXRIWWMWVIPEXIH XSMRXIVQIHMEXITVIGMWMSRSVVYKKIHRIWW;LMPIMRXIVQIHMEXITVIGMWMSR VIJIVWXSXLIIJJIGXSJYRMRXIRXMSREPZEVMEXMSRWFIX[IIREWWE]SGGEWMSRW STIVEXSVIXGWII7IGXMSRVSFYWXRIWWMWHIXIVQMRIHF]QIERWSJ HIPMFIVEXIZEVMEXMSRWMRGLSWIREWWE]TEVEQIXIVW%REWWE]XLEXMW VSFYWX[MXLVIWTIGXXSEPPIWWIRXMEPTEVEQIXIVW[MPPEPWSLEZIELMKL PIZIPSJMRXIVQIHMEXITVIGMWMSR 2.2.9 Sensitivity 8LIWIRWMXMZMX]SJEREWWE]MWRSXMRGPYHIHEQSRKXLIVIGSQQIRHIH TIVJSVQERGIGVMXIVMEJSVZEPMHEXMSRFYXMW[SVXL]SJEHIJMRMXMSRLIVI FIGEYWIXLIXIVQMWSJXIRYWIHMRWIZIVEPHMJJIVIRXERHXSWSQII\XIRX GSRJPMGXMRKWIRWIW*SVQEPP]WIRWMXMZMX]MWHIJMRIHEWXLIEWWE]VIWTSRWI TIVYRMXEREP]XIGSRGIRXVEXMSRIK69TIV KQPSV69 18LMWMW XLIWPSTISJXLIWXERHEVHGYVZIJSVXLIEWWE]*SVEWWE]W[LIVIXLI WXERHEVHGYVZIMWRSXPMRIEVXLIWIRWMXMZMX]ZEVMIW[MXLXLIEREP]XI GSRGIRXVEXMSR8LIXIVQlWIRWMXMZMX]zMWLS[IZIVWSQIXMQIWYWIHEWE W]RSR]QJSV03(SV035XLIPS[IWXGSRGIRXVEXMSRXLEXGERFI HIXIGXIHSVQIEWYVIHSVVIWSPYXMSRXLIWQEPPIWXHMJJIVIRGIMR GSRGIRXVEXMSRXLEXGERFIHIXIVQMRIH[MXLGSRJMHIRGI&MEGSVI VIGSQQIRHWYWEKISJXLIXIVQMRXLIJMVWXWIRWIEWWE]VIWTSRWITIV YRMXEREP]XIGSRGIRXVEXMSR Response Sensitivity = slope Concentration Figure 2-7. Sensitivity is the response per unit analyte concentration, which is the slope of the calibration curve. The sensitivity varies over the range of the assay if the calibration curve is not linear.

Terminology

Assay formats 3. Assay formats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

Assay formats analyte analyte enhancement molecule ligand ligand Direct binding assay 1 2 Direct binding assay with enhancement detecting molecule competing analyte analyte analyte ligand ligand Inhibition assay (solution competition) Surface competition assay Figure 3-1. Schematic illustration of four different approaches to concentration measurement with Biacore. 3.1 Direct binding assays &MRHMRKPIZIPEWWE]WVITVIWIRXXLIQSWXHMVIGXETTVSEGLXSQIEWYVMRK GSRGIRXVEXMSR[MXL&MEGSVI8LIHIXIGXMRKQSPIGYPIMWEXXEGLIHXSXLI WIRWSVGLMTWYVJEGIWEQTPIMWMRNIGXIHERHXLIVIWTSRWISFXEMRIHEJXIV EJM\IHGSRXEGXXMQIMWQIEWYVIH8LIVIWTSRWIMWVIPEXIHXSEREP]XI GSRGIRXVEXMSR[MXLXLILIPTSJEWXERHEVHGYVZITVITEVIHF]EREP]^MRK ORS[RGSRGIRXVEXMSRWSJEREP]XIYRHIVXLIWEQIGSRHMXMSRW 3.1.1 Single step direct measurement (MVIGXQIEWYVIQIRXMWWYMXEFPIJSVQEGVSQSPIGYPEVEREP]XIW QSPIGYPEV[IMKLX"HEPXSRW[LMGLKMZIERIEWMP]QIEWYVIH VIWTSRWIIZIREXPS[QSPEVGSRGIRXVEXMSRW (YVMRKMRNIGXMSRSJWEQTPIXLIVIWTSRWIMRGVIEWIWEWEJYRGXMSRSJ XMQIEWEREP]XIFMRHWXSXLIWYVJEGIVIEGLMRKEWXIEH]WXEXIZEPYIMJXLI GSRXEGXXMQIMWWYJJMGMIRXP]PSRK(ITIRHMRKSRXLIXMQIEX[LMGLXLI

Assay formats VIWTSRWIMWQIEWYVIHXLIWIRWMXMZMX]ERHSTIVEXMRKVERKISJXLIEWWE] [MPPFIHMJJIVIRX*MKYVI Response 2 1 Response 1 2 Time Concentration Figure 3-2. A single step direct assay gives an increasing response with increasing concentration. The range and sensitivity are determined in part by the time at which the response is measured: an interaction that is allowed to approach equilibrium will give a higher sensitivity at low analyte concentrations. The inset shows the sensorgrams from which the calibration curves are derived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andwich methods 7ERH[MGLQIXLSHWEVIERI\XIRWMSRSJXLIWMRKPIWXITHMVIGXETTVSEGL EJXIVEREP]XILEWFSYRHXSXLIWYVJEGIEXXEGLIHPMKERHEWIGSRHEV] MRXIVEGXERXXLIIRLERGIQIRXQSPIGYPIMWMRNIGXIHXSFMRHXSXLI EREP]XIERHIRLERGIXLIEREP]XIVIWTSRWI-JXLIWIGSRHEV]EREP]XIMWE

Assay formats PEVKIVQSPIGYPIXLERXLIEREP]XIXLIWIGSRHEV]VIWTSRWI[MPPFI TVSTSVXMSREPP]LMKLIVXLERXLITVMQEV]VIWTSRWISFXEMRIHJVSQXLI EREP]XIMXWIPJTVSZMHMRKEREQTPMJMGEXMSRSJXLIWMKREP*MKYVI Response 2 1 Response 1 2 Time Concentration Figure 3-3. Sensorgrams (inset) and calibration curves for a direct binding assay with enhancement. : primary response (analyte), : secondary response (enhancement reagent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

Assay formats 3.2 Indirect assays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nhibition assays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esponse Response Concentration log[concentration] Figure 3-4. Inhibition and surface competition assays give a calibration curve where the response is inversely related to the analyte concentration. Calibration curves are often shown on a log[concentration] scale (right panel) to expand the low concentration region. 'PIEVP]MXMWIWWIRXMEPXLEXHIXIGXMRKQSPIGYPIWGEVV]MRKFSYRHEREP]XI MRWSPYXMSRWLSYPHRSXFIEFPIXSFMRHXSXLIWYVJEGIEXXEGLIHPMKERH -HIEPP]XLI,1;HIXIGXMRKQSPIGYPIWLSYPHFIQSRSZEPIRXJSVXLMW

Assay formats VIEWSR,S[IZIVQSRSGPSREPERXMFSHMIWEVIGSQQSRP]YWIHEW HIXIGXMRKQSPIGYPIWMRWTMXISJXLIMVFMZEPIRXFMRHMRKTVSTIVXMIW)ZIR XLSYKLFSXLERXMKIRFMRHMRKWMXIWQYWXFISGGYTMIHXSIJJIGXMZIP] MRLMFMXXLIFMRHMRKSJERXMFSH]XSXLIWYVJEGIXLIMRLMFMXMSREWWE] TVMRGMTPIWXMPP[SVOWVIPMEFP]ERHERXMFSHMIWEVISJXIRXLIQSWXVIEHMP] EZEMPEFPIWSYVGISJHIXIGXMRKQSPIGYPIW 8LIEJJMRMX]SJXLIHIXIGXMRKQSPIGYPIJSVXLIEREP]XIMRWSPYXMSR XSKIXLIV[MXLXLIGSRGIRXVEXMSRSJHIXIGXMRKQSPIGYPIHIXIVQMRIWXLI YWIJYPVERKISJERMRLMFMXMSREWWE],MKLIVEJJMRMXMIWEPPS[ QIEWYVIQIRXEXPS[IVEREP]XIGSRGIRXVEXMSRWFYXEPWSVIWYPXMRE REVVS[IVSTIVEXMRKVERKI*MKYVI-RJSVQEPXIVQWXLI-' SJER MRLMFMXMSREWWE]MWKMZIRF] -' = /( + ' [LIVI/ ( MWXLIIUYMPMFVMYQHMWWSGMEXMSRGSRWXERXJSVXLIMRXIVEGXMSR [MXLEREP]XIERH'MWXLIXSXEPGSRGIRXVEXMSRSJHIXIGXMRKQSPIGYPI-J XLIGSRGIRXVEXMSRMWQYGLWQEPPIVXLERXLIHMWWSGMEXMSRGSRWXERXXLI VERKI[MPPFIHIXIVQMRIHTVMQEVMP]F]XLIEJJMRMX]GSRZIVWIP]MJXLI GSRGIRXVEXMSRMWQYGLLMKLIVXLERXLIHMWWSGMEXMSRGSRWXERXXLIVERKI [MPPFIHIXIVQMRIHTVMQEVMP]F]XLIGSRGIRXVEXMSR Response Increasing affinity Increasing concentration of detecting molecule log[concentration] Figure 3-5. Increasing the affinity of the detecting molecule moves the operating range to lower analyte concentration and also narrows the range. -RLMFMXMSREWWE]WEVIQSWXGSQQSRP]YWIHJSV01;EREP]XIW8LI] QE]LS[IZIVEPWSFIYWIHEWEREPXIVREXMZIXSHMVIGXFMRHMRKPIZIP EWWE]WJSVQEGVSQSPIGYPEVEREP]XIWMRWMXYEXMSRW[LIVI MQQSFMPM^EXMSRSVVIKIRIVEXMSRSJXLIHIXIGXMRKQSPIGYPISRXLIWIRWSV WYVJEGIMWRSXWEXMWJEGXSV]

Assay formats 3.2.2 Surface competition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

Assay formats 3.3 Binding rate assays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esponse Initial binding rate Binding level Figure 3-6. Binding rate and binding level measurements. Time &MRHMRKVEXIQIEWYVIQIRXWERHXLIGSRHMXMSRWVIUYMVIHJSVXLIMVYWI EVIGSRWMHIVIHMR%TTIRHM\&

Assay formats 3.4 Choice of assay technique %WHMWGYWWIHEFSZI&MEGSVIW]WXIQWSJJIVERYQFIVSJHMJJIVIRX ETTVSEGLIWXSQIEWYVMRKGSRGIRXVEXMSR8LIGLSMGISJETTVSEGLMW HMGXEXIHPEVKIP]F]XLIGLEVEGXIVMWXMGWSJXLIEREP]XIFIMRKEWWE]IHERH XSWSQII\XIRXF]XLITYVTSWISJXLIEWWE]ERHXLIX]TISJWEQTPI QEXVM\XLEX[MPPFIYWIH8LIXEFPIFIPS[TVSZMHIWWSQIKIRIVEP KYMHIPMRIW 1EGVSQSPIGYPIW X]TMGEPP]1; "HEPXSRW (MVIGXEWWE][MXLXLIHIXIGXMRKQSPIGYPI EXXEGLIHXSXLIWIRWSVWYVJEGI 7ERH[MGLEWWE]GERLIPTXS reqtpmj]xliviwtsrwijsvqspigypiwexxli PS[IRHSJXLIWM^IVERKI rgsrjmvqxlimhirxmx]sjxlierep]ximr GSQTPI\QM\XYVIW rgsrjmvqxlimrxikvmx]sjqypxmjyrgxmsrep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

Assay formats 3.5 Choice of ligand and other reagents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ractical approaches to reagent selection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irect binding assays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

Assay formats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nhibition assays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

Assay formats 3.5.4 Surface competition assays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

Sensor Chip CM5 Certified Grade Surface preparation principles 4. Surface preparation principles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ensor surface properties 8LIWIRWSVGLMTMWEKPEWWWPMHIGSEXIH[MXLEXLMRPE]IVSJKSPHXS [LMGLEQEXVM\SJGEVFS\]QIXL]PEXIHHI\XVERMWGSZEPIRXP]EXXEGLIH *MKYVI8LIKSPHMWVIUYMVIHJSVKIRIVEXMSRSJXLI746VIWTSRWI %TTIRHM\%0MKERHWGERFIEXXEGLIHXSXLIHI\XVERQEXVM\YWMRKE ZEVMIX]SJGLIQMGEPQIXLSHW Dextran matrix Gold layer Glass Figure 4-1. Schematic illustration of the structure of the sensor chip surface.

Surface preparation principles 8LIKSPHPE]IVERHHI\XVERQEXVM\SRXLIWIRWSVWYVJEGIEVIWXEFPI YRHIVE[MHIVERKISJGSRHMXMSRWMRGPYHMRKI\XVIQIWSJT,ERH QSHIVEXIGSRGIRXVEXMSRWSJQER]SVKERMGWSPZIRXW3RGIXLIPMKERH LEWFIIRMQQSFMPM^IHXLIWXEFMPMX]SJXLIWIRWSVWYVJEGIMWHIXIVQMRIH TVMQEVMP]F]XLIWXEFMPMX]SJXLIEXXEGLIHPMKERH 4.2 Ligand immobilization methods 8LIGEVFS\]QIXL]PEXIHHI\XVERQEXVM\SRXLIWIRWSVWYVJEGIMW EQIREFPIXSEVERKISJGLIQMWXVMIWJSVPMKERHMQQSFMPM^EXMSR I\TPSMXMRKHMJJIVIRXKVSYTWSRXLIPMKERHQSPIGYPI-QQSFMPM^EXMSR ETTVSEGLIWQE]FIHMVIGXIHXS[EVHWEQMRIGEVFS\]PXLMSPSV L]HVS\]PKVSYTWSRXLIPMKERHSVQE]YWIWTIGMJMGXEKWMRXVSHYGIHMRXS XLIPMKERHIMXLIVF]GLIQMGEPQSHMJMGEXMSRSVVIGSQFMRERXXIGLRMUYIW 8[SFEWMGGLIQMGEPETTVSEGLIWEVIVIGSQQIRHIHJSVKIRIVEPYWI %QMRIGSYTPMRKYWMRKTVMQEV]EQMRIKVSYTWSRXLIPMKERH 8LMSPGSYTPMRKYWMRKI\GLERKIFIX[IIRVIEGXMZIHMWYPJMHIWERH REXMZISVMRXVSHYGIHXLMSPKVSYTWSRXLIPMKERH %HHMXMSREPQIXLSHWXLEXGERFIZEPYEFPIMRWMXYEXMSRW[LIVIEQMRISV XLMSPGSYTPMRKMWYRWEXMWJEGXSV]MRGPYHI %PHIL]HIGSYTPMRKYWMRKEPHIL]HIKVSYTWSRXLIPMKERH MRXVSHYGIHXLVSYKLS\MHEXMSRSJGMWHMSPWX]TMGEPP]MR GEVFSL]HVEXIVIWMHYIW,MKLEJJMRMX]GETXYVISJFMSXMR]PEXIHPMKERHSRMQQSFMPM^IH WXVITXEZMHMR,MKLEJJMRMX]GETXYVIF]FMRHMRKXSWTIGMJMGGETXYVMRKQSPIGYPIW IKLMKLEJJMRMX]ERXMFSHMIW 8LIGLIQMWXV]MRZSPZIHMRXLIHMJJIVIRXETTVSEGLIWMWWYQQEVM^IH FIPS[ 4.2.1 Amine coupling %QMRIGSYTPMRKGLIQMWXV]MWXLIQSWX[MHIP]ETTPMGEFPIETTVSEGLJSV EXXEGLMRKFMSQSPIGYPIWGSZEPIRXP]XSXLIWIRWSVWYVJEGI;MXLXLMW QIXLSHXLIHI\XVERQEXVM\SRXLIWIRWSVGLMTWYVJEGIMWJMVWXEGXMZEXIH [MXLEQM\XYVISJIXL]PHMQIXL]PEQMRSTVST]PGEVFSHMMQMHI )('ERH2L]HVS\]WYGGMRMQMHI2,7XSKMZIVIEGXMZIWYGGMRMQMHI IWXIVW0MKERHMWXLIRTEWWIHSZIVXLIWYVJEGIERHXLIIWXIVWVIEGX WTSRXERISYWP][MXLYRGLEVKIHEQMRSKVSYTWSVSXLIVRYGPISTLMPMG KVSYTWXSPMROXLIPMKERHGSZEPIRXP]XSXLIHI\XVERWII*MKYVI

Surface preparation principles Figure 4-2. Amine coupling of ligands to the sensor surface. 1SWXTVSXIMRWGSRXEMRWIZIVEPEQMRIKVSYTWWSXLEXIJJMGMIRX EXXEGLQIRXGERFIEGLMIZIH[MXLSYXWIVMSYWP]EJJIGXMRKXLIFMSPSKMGEP EGXMZMX]SJXLIPMKERH-RWSQIMRWXERGIWLS[IZIVEQMRIGSYTPMRKQE] MRZSPZIKVSYTWEXSVRIEVXLIEGXMZIWMXISVFMRHMRKWMXISJXLIPMKERH [MXLXLIVIWYPXXLEXEXXEGLQIRXMWEGGSQTERMIHF]PSWWSJEGXMZMX]-R WYGLGEWIWXLIPMKERHGERFIEXXEGLIHYWMRKEPXIVREXMZIGSYTPMRK GLIQMWXV]SVEGETXYVMRKETTVSEGL 4.2.2 Thiol coupling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l Figure 4-3. PDEA Thiol coupling reagent, 2-(2-pyridinyldithio)ethaneamine hydrochloride.

Surface preparation principles /LJDQGWKLRO 6XUIDFHWKLRO Figure 4-4. Ligand thiol and surface thiol coupling of ligands to the sensor surface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ldehyde coupling 0MKERHWGSRXEMRMRKEPHIL]HIKVSYTWIMXLIVREXMZISVMRXVSHYGIHF] S\MHEXMSRSJGMWHMSPWGERFIMQQSFMPM^IHEJXIVEGXMZEXMRKXLIWYVJEGI [MXLL]HVE^MRISVGEVFSL]HVE^MHI8LIGLIQMWXV]SJEPHIL]HIGSYTPMRK MWWYQQEVM^IHMR*MKYVI Figure 4-5. Aldehyde coupling of ligands to the sensor surface.

Surface preparation principles %PHIL]HIGSYTPMRKTVSZMHIWEREPXIVREXMZIETTVSEGLJSVMQQSFMPM^MRK KP]GSTVSXIMRWERHSXLIVKP]GSGSRNYKEXIW8LIQIXLSHMWTEVXMGYPEVP] WYMXEFPIJSVPMKERHWGSRXEMRMRKWMEPMGEGMHWMRGIXLIWIVIWMHYIWEVIZIV] IEWMP]S\MHM^IHXSEPHIL]HIW 4.2.4 Streptavidin-biotin capture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eneral capture methods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

Surface preparation principles 8LIFEWMGVIUYMVIQIRXJSVWYGGIWWJYPGETXYVMRKMWEVSFYWXLMKLEJJMRMX] MRXIVEGXMSRFIX[IIRXLIGETXYVMRKQSPIGYPIERHXLIPMKERH 1SRSGPSREPERXMFSHMIWEVIJVIUYIRXP]YWIHEWGETXYVMRKEKIRXW)ZIRMJ XLIEJJMRMX]MWLMKLXLIVI[MPPSJXIRFIEGIVXEMREQSYRXSJHMWWSGMEXMSR SJXLIPMKERHJVSQXLIWYVJEGIHYVMRKXLIGSYVWISJEREREP]WMW7MRGI PMKERHMWVIJVIWLIHJSVIEGLG]GPIXLMWGERKIRIVEPP]FIMKRSVIHMR GSRGIRXVEXMSREWWE]W 8LIGETXYVMRKQSPIGYPIMWEXXEGLIHXSXLIWYVJEGIYWMRKSRISJXLI GSZEPIRXGLIQMGEPETTVSEGLIWHIWGVMFIHMRXLITVIZMSYWWIGXMSRW 4.2.6 Immobilizing small molecules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