surface plasmon resonance (spr) as a tool for antibody conjugate

8/10/2019 Surface Plasmon Resonance (SPR) as a Tool for Antibody Conjugate

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Surface Plasmon Resonance (SPR) as a Tool for Antibody ConjugateAnalysis

Maciej Adamczyk,* Phillip G. Mattingly, Kevin Shreder, and Zhiguang Yu

Abbott La bora tories, Dia gnostics Division, Department of Chemistry D9NM, Building AP 20, 100 Abbott P ar kRoad, Abbott Park, Illinois, 60064-6016. Received May 20, 1999; Revised Manuscript R eceived August 12, 1999

Surfa ce plasmon resonance (SP R) ana lysis wa s used to a ssess the immunoreactivi ty of an ti-biotin (4)and anti-fluorescein (5) monoclonal a ntibody af ter conjugation with the N-hydr oxysuccinimide esterof a cridinium-9-carb oxamide 1. Only minor cha nges in the a pparent equilibrium dissociat ion consta ntsof the antibody conjugates for their ligands resulted from the conjugation process. However, comparisonof the initial binding rate of the conjugates with their ligands with those of the unmodified antibodiesover a range of concentrations showed that the antibody conjugates were partial ly inactivated. Thea n t i -f lu or e s ce in c on ju g a t e s r e t a i n ed a t l ea s t 9 0% o f t h e ir i m m u n or e a ct i v it y o ve r t h e r a n g e o fmodification test ed, while a nti-biotin conjuga tes showed a progressive loss of reactivity w ith increasedsubsti tution by the label .

INTRODUCTION

Surfa ce plasmon resona nce (SP R) is a phenomenont h a t i s m e a s u r e d b y t h e ch a n g e i n t h e r e f r a ct i v e in d e xa t a s u r f a c e a s a f u n c t i o n o f t h e m a s s o f t h e m a t e r i a lbound to th e surfa ce. As commercia lized on t he B IAcoreinstrument, SPR al lows for the real-t ime measurementof the kinetics of noncovalent interactions between al i g a n d a n d a b i n d i n g p a r t n e r (1-3). We have recentlyhighlighted surface plasmon resonance on the BI Acoresystem a s a va luable tool tha t can be used to chara cterizeimmunoreagents uti l ized in immunoassays (4-8). Thep r ev i ou s r e po rt s h a v e d e t a i l ed t h e u t i li t y of S P R i nevaluating how the structural variat ions in a l ibrary ofsmall molecule haptens af fected the kinetics and ther-modynamics of binding to an ti-ha pten a ntibodies or Fab

fragment s. In one study, each ana logue in the libra ry wa simmobilized on a separate biosensor chip and evaluatedindividua lly with the a ntibody or Fa b (7). The associationan d dissociat ion ra tes for ea ch ana logue were determinedf r om t h e g e n e r a t e d s e n s or g r a m s , a n d t h e a f f in i t y c on -sta nts w ere calculated a nd compared. In this directS P Rmethod, it was necessary to optimize the surface densityof t h e l ig a n d a t a l ow l ev el o n t h e b i os en s or ch i p t om i n i m i z e r eb in d i n g e v en t s a n d m a s s t r a n s p or t l im i t a -tions. Other st udies ha ve ta ken a di f ferent a pproach inw h i ch a s i n gl e b i os e n sor w a s u s ed t o d e t e r m in e t h eaf f ini ty consta nts of each member of the l ibrary w ith th eFab in solution at equil ibrium (5, 8). The technique isb a s ed on t h e ob se rv a t i on t h a t u n d er m a s s t r a n s por tlimited conditions th e initial binding ra te of the ant ibody

with the biosensor is proportiona l t o the concentra tiono f t h e a n t i b o d y a n d i n d e p e n d e n t o f t h e a f f i n i t y o f t h ean tibody for the ligan d on th e biosensor chip (9). P lott inginit ial binding rate versus antibody concentration pro-duces a cal ibration curve from which concentra tions offree an tibody in a n unknown sa mple at equilibrium canbe determined. For this experiment, the surface densityof the l igand on the biosensor must be h i gh enough toensuremass tra nsport l imited kinetics.

Both types of studies were particular ly applicable to

c h a r a c t e r i z i n g t h e r e a g e n t s u s e d i n a s s a y f o r m a t s i nwhich th e a ntibody would be unmodified by labeling orimmobil ization. I t is of ten the case, however, that thean tibody component of the immunoassa y is modified byconjugation to a detecta ble ta g, l igand, or solid support[see, for example, Abraham et al . (10) ] . S P R h a s o n l yrecently been reported to st udy the ef fect of the biotin-ylat ion of ant i-LH mAbs on binding t o LH in a n immu-nometric forma t (11). The degree of biotinylation of themAbs was not determined, but the authors of fered thequali tat ive observation that the conjugation procedureh a d a m a r k ed e ff ect on t h e s en s or -b ou n d a n t i b od ybinding to LH. In general, chemical modifications to anya n t i b o d y m a y v a r y f r o m l o t t o l o t a n d u n p r e d i c t a b l ychange the conjugates binding properties w ith t he a na -

l y t e l i g a n d , w h i c h i n t u r n w o u l d a f f e c t a s s a y p e r f o r -mance. SPR provides a convenient tool for assessing thechanges in antibody binding characterist ics in a repro-ducible, automated fashion using a minimal amount ofthe a ntibody conjugat e.

I n t h i s r e po rt , a s e r ie s o f c on ju g a t e s b et w e e n a n t i -biotin (4) and a nti-fluorescein (5) monoclonal a ntibodiesa n d a n e w ch e m il u m in e s ce n t N-sulfonylacridinium-9-carboxamide derivat ive (1) w a s p r ep a r e d a n d e va l u a t e db y S P R on t h e B I A cor e s y st e m u s in g t h e s ol ut i oncompetition m ethod.

MATERIALS AND METHODS

Anti-biotin (4, IgG2a) and anti-f luorescein (5, I g G 1 )

monoclonal an tibodies, obtained from the Abbott cel lculture faci li ty (Abbott La borat ories, Abbott P ark, IL),were dialyzed exhaust ively aga inst P BS (100 mM sodiumphosphate and 150 mM sodium chloride, pH 7.2) usinga 10 000 MW cutof f membra ne dia lysis cassette (Slide-A-Lyzer, Pierce Chemical Co., Rockford, IL). The acri-dinium-9-carboxam ide label 1 (12, 13) a n d O-(6-fluores-ceinylmethyl)hydroxylamine2 (6-OFMHA) (14, 1 5) wereprepared a s previously described. 5-(B iotinam ido)penty-lamine 3 was obtained from Pierce Chemical Co. UV-vis spectroscopy w as performed on a Beckman D U 640spectrophotometer (Fullerton, CA). Preparative HPLC

* Au t h or t o wh om c or r e s pon d en c e s h ou l d b e ad d r e s s ed .P hone: (847) 938-8927. E-mail: ma ciej.a da mczyk@abbot t.com.

1032 Bioconjugate Chem. 1999,10, 10321037

10.1021/bc990057e CCC: $18.00 1999 American Chemical SocietyPublished on Web 10/14/1999


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w a s p er f or m e d u s in g a Wa t e r s (M i lf or d , M A) 6 0 0Esystem controller and pumps and a model 486 tunable

a b s o rb a n c e d e t e ct o r s e t t o 2 80 n m . S u r f a c e p l a s m onresona nce measurements w ere carr ied out a t 25 C on aB IAcore 2000 (B IAcore, Inc., Piscat aw a y, NJ ) au toma tedsystem using research-grade CM-5 four-channel sensorchips. Reagents for the BIAcore instrument consisted ofHBS running buffer [10 mM Hepes (pH 7.4), 150 mMNaC l, 3.4 mM ED TA, an d 0.05% surfactan t P -20], abiosensor chip a ctivation kit conta ining N-hydroxysuc-cinimide (0.5 M, NHS), N-ethyl-N -(3-dimethylamino-propyl)carbodiimide hydrochloride (0.2 M, EDC), and acapping solution of ethanolamine hydrochloride (1 M, pH8.5), a ll from B IAcore, Inc.

Preparation of Antibody-acridinium-9-carboxa-mide Conjugates.General Procedure.To each antibody(3.0 mg mAb in 1.4 mL of PBS, pH 8.0) was added (a) 4,(b) 8, or (c) 16 molar equivalents of th e acridinium-9-carboxam ide a ctive ester 1 (Figure 1) in DMF (0.2 mL).Upon addition, the solutions were rapidly vortexed thenallowed to stand for 18 h in the da rk. Ea ch reaction wa spurified by prepa ra tive HP LC using a B io-Ra d (Hercules,CA) SEC -250-5 gel permea tion chroma togra phy columneluting w ith P B S [pH 6.3, cont a ining C HAPS (0.1%), 1.0mL/min] and su bsequent ly stored a t 2-8 C i n t h e d a r k .L a b e le d a n t i b od y (Rt 8 .3 m i n ) a n d u n con ju g a t e dacridinium-9-carboxamide label (Rt ) 11.1 min) werereadily separa ted under these conditions.

The concentration of antibody conjugate was deter-m in ed b y U V-vis spectroscopy using the fol lowingformula:

w h e r e A 280 a n d A 369 a r e t h e a b s o rb a n c e r e a d i n gs of t h eacridinium-9-carboxam ide-a ntibody conjugates at thecorresponding wa velength s; 4.1 is the A 369/A 280of the freecarboxylic acid form of 1, a n d 280 (210 000 M-1 cm-1) isthe extinction coefficient of the IgG antibody at 280 nm.The ratio of acridinium label to antibody (r) conjugatew a s d e t e r m i n e d b y U V-vis spectroscopy using the fol-lowing formula:

w h e r e 369 (14 700 M-1 cm-1) is the extinction coefficientof the la bel free ca rboxylic a cid form of 1 at 369 nm. Forthe a cridinium-9-carboxam ide-a nti-biotin conjugat es4a-c, l a b e l-t o -a n t i b od y r a t i o s of 2 .1 , 4 .0 , a n d 7 .5 w e r eobtained, respectively. For the acridinium-9-carboxam-ide-anti-fluorescein conjugates 5a-c, label-to-a ntibodyratios of 2.2, 3.6, and 7.4 were obtained, respectively.

Preparation of Biotin and F luorescein Biosen-sors. Biosensors were prepared under the conditionslisted in Table 1. In general , CM-5 chips were equil i-

brated with running buffer (10 L/min), then a ctivat edwit h N HS /ED C. A solution of 5-(biotinam ido)pentyla mine(3) or 6-OFMHA (2) was injected over the cell. Reactivesites rema ining on th e biosensor chip were blocked w ithethan olam ine (70 L, 1.0 M). The binding capacities ofthe resulting biosensors were estimated using unlabeleda nt i-biotin mAb (4) or un labeled a nt i-fluorescein mAb (5).Control surfaces for each sensor were generated at thesam e conditions, omitting the ligand immobilization step.

Antibody Conjugate Analysis by the SolutionCompetition Method. (A ) C a l i b r a t i on C u r v e. Stocksolutions of the native antibodies (4 a n d 5) an d a ntibodyconjugates (4a-c a nd 5a-c) in PB S w ere serially dilutedinto HBS buffer to obtain 11 solutions in concentrationsr a n gi ng 2-2 2 n M . Th e d i lu t e d s ol u t io n s of k n ow n

concentration were injected over the corresponding bio-sensors (5 L/min, 2.0 min). The biotin biosensor w a sr e ge n er a t e d a f t e r e a c h r u n w i t h f or m i c a c id (1 .0 M , 1min), then guanidineHCl (6.0 M, 1 min; 1.5 M, 1 min).The f luorescein biosensor surface was regenerated byconsecutive pulses of guanidineHCl (6.0 M, 2 1 min;1.5 M, 1 min). The initial binding ra te w as obtained frome a ch s en s or g ra m b y l in ea r f it t i n g o f a 1 5 s w i n d ow ,s t a r t i n g 2 0 s a f t e r i n j e c t i o n . C a l i b r a t i o n c u r v e s w e r eg en er a t e d f or e it h e r t h e n a t i v e a n t i b od ie s or t h e irconjugates by plott ing the initial binding ra te vs a ntibody(or conjuga te) concentra tion.

( B ) D et er mi nat i on of A ppar ent S ol ut i on E qui l i br i um

Di ssociati on Constants (KD). 5-(Biotinamido)pentylamine(3) and 6-OFMHA (2) w e r e e a c h s e r i a l l y d i l u t e d w i t hHBS buffer to give 11 solutions ranging 8-100 nM for 3a n d 4 0-1000 nM for 2. S t o c k s o l u t i o n s o f t h e n a t i v ean tibodies (4 a n d 5) and a ntibody conjugat es (4a-c a n d5a-c) i n P B S b u f f e r w e r e d i l u t e d i n t o H B S b u f f e r t oobtain 40 nM solutions. Each ligand solution was mixedwith the corresponding antibody or conjugate solution(1:1 v/v). Once equil ibrat ion wa s reached a f ter 1 h atambient temperature, the concentration of free antibodyor antibody conjugat e in each sa mple wa s assa yed usinga biosensor chip a nd t he corresponding ca libration curve.Nonlinear least-squa res r egression an alysis suppliedwith the B IAevalua tion softw are (version 3.0) wa s usedto fit a plot of the concentrations of free antibody versusthe concentra tions of the competi tor a nd determine the

a p p a r e n t s ol u t ion a f f in i t y . Th e f or m u l a u s ed i n t h esof tware is shown below:

w h e r e A F ree i s t h e con c en t r a t i on of f r ee a n t i b od y i nequil ibrated solution with the competi tor l igand, A T ist h e t o t a l con ce n t r a t i on of a n t i b od y , L T is t h e t ot a lconcentra tion of l igand, a nd KD is the a pparent equil ib-rium dissociation constant for the binding between thel ig a n d a n d t h e a n t i b od y .

Determination of Retained Antibody ConjugateActivity. Solutions of antibody conjugates (4a-c a n d

Figure1. Structures of the a ctive ester of the acridinium label(1), O-(fluoresceinylmethyl)hydroxylamine (2, 6-OFMH A), a nd5-(biotinamido)pentylamine (3).

[conjugate] ) [A 280 - (A 369/4. 1)]/280

r)A 369/369

[A 280 - (A 369/4. 1)]/280

A Free ) (A T - L T- KD)/2 +

[(A T + L T+ KD)2/4 - L TA T]

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5a-

c) r a n g i n g 2-

22 nM were injected over the corre-sponding biosensors (5 L/min), and the init ial bind ingrate was determined from each sensorgram. The activityof ea ch conjugat e (4a-c a n d 5a-c) wa s compared to th eunmodif ied an tibody (4 or 5, respectively) using t heformula

k0 is the slope in t he calibra tion curve of the un modifieda n t i b o d y (4 or 5) a n d k is the slope observed for theant ibody conjugates (4a-ca n d 5a-c).

RESULTS

Preparation of Antibody-acridinium-9-carbox-amide Conjugates. The antibodies 4 a n d 5 were con-jugated with the acridinium-9-carboxamide active ester1 at three stoichiometries (4:1, 8:1, and 16:1) in buffer/DMF (7:1, pH 8.0). The antibodies and labeling reagentwere completely soluble during the course of the reactionand throughout purif ication. Purif ication of the conju-gates by gel permeation chromatography removed unre-acted or hydrolyzed 1. The efficiency of t he couplingreaction betw een both an tibodies an d th e active ester1ran ged from 53 to 47%ba sed on incorporat ed 1. Therewas no significant difference in the coupling efficiencybetween the three stoichiometries.

Preparation of Biosensors. Anti -Biotin m Ab Sensor.Biotin containing a pentanediamine l inker (3) w a s i m-

mobil ized on a NHS /ED C-activat ed carboxymethyla tedd ex t r a n s u rf a ce of a C M -5 s en s or ch ip t o a f for d abiosensor. Us ing a ligand concentra tion of 10 mM for 10min produced a biosensor with an anti-biotin antibody(4) binding capa city of 11 000 RU (Ta ble 1).

Ant i-Fl uorescein mA b Sensor. Immobilization of 5(6)-fluoresceinam ine, 4-aminomethylfluorescein or 5(6)-ami-nomethylf luorescein (data not sh own) under t he condi-tions th a t w ere successful for th e biotin series, proved tobe insufficient to generat e the l igand density necessaryt o a c h i e v e m a s s t r a n s p o r t l i m i t a t i o n . I t w a s r e a s o n e dtha t a t pH 8 the nega tively charged fluorescein moleculeis electrostat ically repulsed from th e carboxylated sensorsurface. 6-OFMHA (2) was immobil ized on the sensorch i p u n d er a c i di c c on d i t io ns t h a t c ir cu m v en t e d t h i s

problem. Thus, a 1 mM solution of2 buffered at pH 5.5r e a c t e d w i t h t h e a c t i v a t e d C M - 5 c h i p o v e r 2 0 m i n t oaf ford a suitable l igand surface with a binding capacityfor anti-fluorescein mAb 5 of 30 000 RU (Ta ble 1).

Mass tra nsport l imited conditions w ere reached w henbiotin and f luorescein surfaces were prepared to havean tibody capa cities of 11 000 an d 30 000 resonance units(RU), respectively. Injection of na tive an tibodies onsensor chips with slightly less antibody capacities yieldedthe sa me initia l binding ra tes, which confirmed that ma sstransport limited conditions had been reached. Fluores-cein surfa ces w ere regenerated by t wo consecutive pulsesof 6.0 M gua nidineHC l an d one pulse of 1.5 M guanidineHCl, ea ch lasting for 1.0 min. In contra st , regenerationof biotin surfaces was found to be more difficult. To clean

t h e l a t t e r s u r f a c e t h o r o u g h l y , a 1 m i n p u l s e o f 1 . 0 Mformic acid wa s needed prior to wa shing the surfa ce wit h6.0 an d 1.5 M guan idineHCl (Table 1).

Antibody Conjugate Analysis by the SolutionCompetition Method. The requ ired calibra tion curvesof initial binding ra te vs a ntibody (or conjuga te) concen-tration were constructed using the corresponding biotinor fluorescein biosensors. Thus, t he sensorgra ms obta inedfor different concentra tions of a nti-biotin mAb (4) undermass transport l imiting conditions are typical (Figure2A). Both the initial binding rates and the total responseunits obtained during the binding phase of the sensor-

grams increase proportionally to the increase in bindingprotein concentra tion with in t he ra nge examined (2-22nM). The initial binding rate for each concentration of 4w a s o b t a i n ed b y a n a l y zi n g t h e s e n s or g r a m ov er a 1 5 swind ow beginning 20 s postinjection. Dat a from t he first2 0 s o f e a c h s e n s o r g r a m w a s o m i t t e d d u e t o s a m p l edispersion effects a t t he sta rt of injections. A plot of initia lbinding ra te versus concentra tion of 4 yielded t he cal i-bration curve (Figure 2B).

To determine t he a pparent solution equil ibrium dis-sociation consta nt of an ti-biotin an tibody 4 with biotinligand 3, a stock solution of 3 was serial ly di luted intoHB S buffer to obta in solutions ranging 6.6-67 nM. Thesediluted ligand solutions were mixed with the anti-biotinantibody 4 (40 nM) in equa l volumes. After equilibrium

Table 1. Experimental Conditions for the Preparation and Regeneration of Bi otin and Fluorescein Biosensors

type of sensorsensor a ct ivat ion,

0.5 M NHS /0.2 M ED C (L )ligand immobilizat ion,a

ligan d (vol, concn)sensor surfacecapacity (RU)

sensorregenerat ion

biot in 120 3b (100 L , 10 m M) 11 000 cfluor escein 200 2d (200 L, 1.0 mM) >30 000 e

a Any r eact ive sites remaining on t he sensor chip after ligand imm obilizat ion were rea cted with ethanolamine (70L, 1.0 M). b I n H B Sbuffer, pH 7.4 c 1 min, 1.0 M formic acid; 1 min, 6.0 M gua nidine hyd rochloride; 1 min, 1.5 M guanid ine hydr ochloride. d In a cetat e buffer(10 mM NaOAc and 0.1 M NaCl), pH 5.5. e 2 1 min, 6.0 M gua nidine hydrochloride; 1 min, 1.5 M gua ninine hydrochloride.

%a ctive ant ibody conjugat e ) k/k0

Figure 2. Calibration curve for anti-biotin mAb 4. (A) Sen-s or g r am s (4, 2-22 nM). (B) The initial ra te a t ea ch concentra-

tion wa s calculated using a 15 s window, sta rting 20 s af t er theinjection.

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wa s reached (>1 h), the r esulting solutions w ere injectedover the biotin surface to produce the sensorgrams shownin F igure 3A. The init ial slope of each sensorgram wa sconverted to concentration of free native antibody basedon t h e ca l i b r a t i on cu r v e. Th e a p p a r e n t e q u il ib r iu mdissociation constant of anti-biotin antibody 4 with l igand3 wa s determined a f ter plott ing the free concentra tionsof antibody vs concentrations of the ligand 3 (Figure 3B )

using B IAevalua tion 3.0 softwa re. Conjuga tes 4a-c wereevaluat ed a na logously using the corresponding cal ibra -tion curves. Apparent equilibrium dissociat ion consta ntsof a nt i-fluorescein mAb 5 and its acridinium-9-carbox-a m i d e c o n j u g a t e s (5a-c) w e r e m e a s u re d i n a s im i la rfashion using solutions of 6-OFMHA (2), ranging from40 to 1000 nM a s t he competing l igand.

Table 2 shows the apparent equil ibrium dissociationconsta nts of ant i-biotin an tibody 4, anti-fluorescein an-tibody5, a nd their a cridinium-9-carboxamide conjugat esat di f ferent labeling ratios. Within a set , the af f ini t ieswere very similar . For 4 a n d 4a-c, t h e a p p a r e n t e q u i -l ibrium dissociation constant s ran ged 5.2-6.4 nM. For5 a n d 5a-c, t h e a p p a r e n t e q u il ib r iu m d i ss oci a t i onconstants ranged 72-81 nM.

Determination of Retained Antibody ConjugateActivity.To evaluate the extent to which the conjugationprocedure deactivat ed the an tibodies 4 a nd 5, conjuga tes4a-c a n d 5a-c (2-2 2 n M ) w e r e i n je ct e d ov er t h ecorresponding l igand-bearing biosensors under ma sstra nsfer l imiting conditions. Plott ing t he ini t ial binding

rat e from t he result ing sensorgram s versus th e concen-tration of the antibody (conjugate) produced a series ofcalibra tion curves (Figur e 4). The slope (k) of the ca libra-tion curve of each conjugate w as compared to t he slope(k0) of t h e n a t i v e a n t i b od y a n d t h e r a t i o w a s t a k en a s ameasur e of the immunoreactive fraction of the conjuga te.As shown in Table 2, relat ively l i t t le a nti-f luoresceinan tibody wa s deactivat ed during the conjugation process.E v en a t a h ig h l oa d i n g r a t i o o f a c r id in i um t o a n t i -f lu or e sc ei n a n t i b od y (7 .4 :1 ), m or e t h a n 9 0% of t h eantibody retains activi ty similar to the native antibody.By contrast , a larger percentage of the anti-biotin con-ju g a t e s l os t t h e i r b i n d i n g a b i li t y a t h i g h er l a b e l-t o -an tibody rat ios. Approximat ely 23% of t he an ti-biotinconjugates were ef fectively inactive w hen the couplingrat io of a cridinium to a ntibody reached 7.5.

D I S C U S S I O N

Th er e a r e s e ver a l a d v a n t a ge s t o u si ng S P R a s atechnique to cha ra cterize ant ibody conjugates. First, w iththe advent of commercial ly avai lable instrumentation,characterization of the binding parameters of antibodycon ju g a t e s c a n b e p e rf or m ed i n a n a u t om a t e d a n dreproducible fashion. Because antigen bearing sensorch ip s c a n b e r e g en er a t e d a n d r eu s ed , a s in g le a n dcontrollable surface can be created to test the bindingpara meters a mong dif ferent lots of a ntibody conjugates.This a dva nta ge ca n be especia lly useful wh en compa ringa n t i b od i es u t i li z ed i n d i ff er e n t d i a g n os t i c p la t f o r m s.

Second, SP R is a n efficient method of ana lysis. Real-timemonitoring of the a ntibody-binding event elimina tes time-consuming secondary methods (e.g., enzyme or colori-m et r ic b a s ed a s s a y s ) t o d et e ct t h e b ou n d a n t i b od y .D angerous radioactivi ty, as might be used in immuno-a s s a y -b a s ed a n a l y s es , i s a l s o a v oi de d. B e ca u s e S P Rinstrumentation is accompanied by sof tware capable ofcomma nding a utomat ed routines, once the immobiliza-t i on of l ig a n d t o a s e n so r c h ip h a s b ee n o p t im i z ed ,samples can be analyzed in a high-throughput mannerwith in minutes for a typica l ant ibody. Third, the qua ntityof ma terials needed is minimal using SP R a nalysis. Foreach passover , the amount of antibody required rangesfrom roughly 0.0005 to 0.05 mg, depending on the natureof the ant ibody. This minimal a mount of mat erial al lows

Figure3. Solution competition of ant i-biotin mAb4 and biotinligand 3. (A) Sensorgrams using 4 (20 nM) and 3 (0-33.3 nM).(B) Competition curve derived from A.

Table 2. Effect of Acridinium Conjugation on AntibodyActivity and Apparent Affinity

antibody

incorporationrat io of

acridinium:Ab

activeantibody

(%)

apparentdissociation

constant KD (nM)

anti-biotin (4) 100 5.24a 2.1 95 5.54b 4.0 91 5.54c 7.5 77 6.4

an ti-fluorescein (5) 100 815a 2.2 100 775b 3.6 96 725c 7.4 90 85

Figure 4. Comparison of the ca libration curves of ant i-biotinm Ab 4 and conjugates 4a-c.



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for multiple runs to be performed within an experimentor over time without the large expenditure of conjugate.If a n a ntibody lot is destined for commercial development ,the nonconsumptive nature of the method is a plus forboth reasons of production and cost.

As in a ny a na lysis of an tibody binding, the bivalencyof the a ntibody must be considered in determining trueequilibrium affinity or dissociation constants. Often thestudy can be simplif ied by preparing monovalent Fabf r a g m e n t s o f t h e a n t i b o d y (7). This al ternative is not

practical in the evaluation of antibody conjugates thatwill be used as such in an a ssa y. The added ma nipula tionto form a nd purify the Fa b fragment ma y i tself al ter t hebinding properties of the protein. In previous studiesu s in g t h e B I Acor e , i t h a s b e e n s h ow n t h a t t h e b i n d in gd a t a o bt a i n e d f r om i n t a c t a n t i b od i es a c t u a l ly f it s t h eequation for a one to one binding interaction better thanthe equa tion tha t t akes bivalency into consideration (9).The binding constants f rom both equations were quiteclose in tha t ca se.

The present study utilizes Ka rlssons simplified m odelto determine appar ent equilibrium dissociat ion constant su s ef ul i n t h e com pa r i s on of t h e r el a t iv e a v i di t y ofa n t i b od i es c ov a l e nt l y m od i fi ed w i t h a l ow m ol ecu l a rweight label . For the two antibodies studied here, SPRana lysis demonstrat ed tha t conjugat ion with a chemilu-minescent acridinium label did not significantly affect theantibody conjugates abil i ty to bind l igand in solution(Ta ble 2). With t hese da ta a lone, one w ould conclude tha tthe immunoreactivity of the conjugate w as not compro-mised during th e conjugat ion procedure. H owever, com-p a r i so n of t h e ca l i b ra t i on cu r v es g e n er a t e d f or e a c hconjugate w ith t hose of the na tive an tibodies revealed adiscrepancy.

In the anti-biotin mAb series (Figure 4), the calibrationcurve of each conjugate dif fered from the unmodif iedan tibody by showing a progressively sha llower slope tha tcorrelated w ith t he degree of ha pten substi tution. Con-jugate 4c h a d a s lop e t h a t w a s 23% le ss t h a n t h e

unmodified 4. In the anti-f luorescein mAb series, thisef fect wa s much less pronounced; the most highly sub-stituted anti-fluorescein conjugate, 5c w a s s t i l l w i t h i n10% of t h e n a t i v e m Ab . U n d e r t h e m a s s t r a n s por tl im i t in g con d i t ion s of t h e s e e xp er i m en t s , t h e i n it i a lbinding rate depends only on the concentration of thean tibody (conjuga te), the bivalency of the a ntibody is nota n i ss u e. Th e i n it i a l r a t e s w e r e c a l cu la t e d f or t h eant ibody a nd conjugat es at the sa me protein concentra -t i on s , t h u s , t h e ca l i b r a t i on c ur v es s h ou l d b e n e a r l yidentical . Any deviat ion could be d ue to inactivation ofsome percenta ge of the conjugat e during t he conjugat ionprocess. Because the apparent equilibrium dissociat ionc o n s t a n t s w i t h i n a s e t o f c o n j u g a t e s a n d t h e i r p a r e n tantibody were similar, the deactivation of the conjugates

a ppea rs t o be an a ll-or-nothing effect; conjugat ion eithercom p le t el y d i sr u pt s b i n di n g or d oe s n ot ch a n g e t h ea f f in i t y of t h e a n t i bod y . I n a d d i t i on , a n t i -f lu or e sce ina ntibody w a s clea rly less sensitive to the process of labelconjugation than the anti-biotin antibody. For the anti-b iot i n a n t i bo dy , t h i s d i f f er e n ce m a y p oi n t t o a h i g h ersusceptibility of key residues in the CDR to acylation bythe la bel, a lowered resistance to dena tura tion imposedby the covalent a tt achment of the la bel, or a combina tionof both.

The use of the B IAcore instrument t o measure a ctiveprotein concentration is not new. Indeed, Karlsson et al.have measured the concentra tion of a ctive, unmodifiedan tibodies under ma ss tra nsport limiting conditions (16).Th ey ob s er v ed t h a t t h e i ni t ia l r a t e v er s us a n t i b od y

concentra tion cal ibration curves to be identical for avariety of IgG s (monoclonal , monoclona l m ixtures, a ndpolyclona l) and thus va l id as a general method with th ecaveat that the association rate of the antibodies had tob e a t o r a b o v e 1 05 M-1 s-1. O t h e r s h a v e m o d i f i e d t h eprocedure to mea sure th e concentra tion of lower a ffinity,unmodif ied binding proteins under partial mass trans-port l imiting conditions (17, 18). N ei t h e r g r o u p h a dexploited th eir methods to cha ra cterize a ntibodies tha thave been covalently modified.

I n s u m m a r y , o u r p a s t s t u d i e s h a v e s h o w n t h a t S P Rca n b e u s e fu l i n d e t er m i n in g t h e op t im a l s t r u ct u r e o fsmall molecule l igands for binding to a given antibodyw h e t h e r t h e l ig a n d i s i n s ol u t ion or b ou n d t o a s ol idsupport (4-8). While that was a signif icant advance inthe design of immunoassay components, i t also set thebasis for monitoring lot to lot va riat ions in the bindingproperties of the ant ibody. U nti l now, SP R ha d not beenu s ed t o s y s t e m a t i ca l l y e xa m i n e t h e a f f ect of cov a l e ntmodificat ion on a ntibody imm unoreactivity. The presents t u d y d e m o n s t r a t e d t h a t r a n d o m c o u p l i n g o f t h e a c t i -va ted est er of acridinium-9-carboxam ide1 to a nt i-biotin(4) and anti-fluorescein (5) monoclona l an tibodies w aschemically efficient based on UV analysis. Surface plas-mon resonance of the purified conjugates indicated onlyminor varia tions in the a pparent equilibrium dissociat ionconsta nts of these an tibody conjugates a s a result of thecon ju g a t i on p r oc es s , w h i ch w a s a w e l co me r e su l t . I naddit ion, and more importantly, SPR provided a meansof d e t ect i n g t h e r a t i o of a c t i ve t o i n a ct i v e a n t i b od y -con ju g a t e p r es e nt i n t h e p r ep a r a t i o n . Wh i le t h e a n t i -f luorescein conjugat es retained at least 90% of theirimmunorea ctivity over the extent of modifica tion tested,a nti-biotin conjugat es showed a progressive loss of rea c-t i v i t y . T h e u l t i m a t e g o a l i s t o m a t c h t h e c o n j u g a t i o nprocedure t o the an tibody to m inimize losses of immu-noreactive conjugate. The reasons for the loss of activeantibody is, of course, still speculation, but SPR provedto be a very convenient method for observing the effect.

LITERATURE CITED

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(7) Adam czyk, M., Gebler, J . C., G una sekera , A. H., Ma ttingly,P. G., and Pan, Y. (1997) Immunoassay reagents for thyroidtesting. 2. Binding properties a nd energetic par am eters of aT4 m on oc lon al an t i b od y a n d i t s F a b f r ag m e n t w i t h a l ib r ar yof thyroxine analog biosensors using surface plasmon reso-nance. Bioconjugate Chem. 8, 133-145.

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(9) Ka rlsson, R. (1994) Real-time competitive kinetic a na lysisof i n t e r ac t ion s b e t we en l ow-m ole cu l ar -we i g h t l i gan d s i nsolution and surface-immobilized receptors. An a l . Bi o ch em.22 1, 142-151.

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I. , an d Hellstrom, K. E. (1991) The influence of periodat eoxidation on monoclonal antibody avidity and immunoreac-t i vi t y . J . I m m u n o l . M et h od s 1 44 , 77-86.

(11) Pearson, J . E., Kane, J . W., Petraki-Kallioti , I . , Gill , A.,an d Vadgam a, P . (1998) Surfa ce plasmon resona nce: a s t udyof the effect of biotinylation on the selection of antibodies foru s e i n i m m u n oas s ay s . J . I m m u n o l . M et h od s 2 21 , 87-94.

(12) Adam czyk, M., Fishpaugh, J ., Mat tingly, P . G., a nd Sh re-der, K. (1998) Tra cermer (TM) signa l genera tors: An arbores-cent approach to the incorporation of multiple chemilumi-nescent labels . Bi o org . M ed . Ch e m. L ett. 8 , 3595-3598.

(13) Adam czyk, M., Chen, Y.-Y., Matt ingly, P. G ., and P an , Y.(1998) Neopentyl 3-trif lyloxypropan esulfonat e. A reactivesulfopropylation reagent for the preparation of chemilumi-nescent labels . J . Org . Ch e m. 6 3 , 5636-5639.

(14) Adam czyk, M., Chen, Y.-Y., Gr ote, J . , a nd Ma tt ingly, P .G. (1999)O -(Fluoresceinylmethyl)hydroxylamine (OFMHA):A reagent for the preparation of fluorescent O-(fluoresceinyl-methyl)oxime (FMO)-steroid conjugates. Steroids 64, 283-290.

(15) Adam czyk, M., Mat tingly, P . G ., a nd Moore, J . A. (1998)O-(Fluoresceinylmeth yl)hydroxyla mine (OFMHA): a fluores-cent reagent for detection of dam aged n ucleic acids. Bioorg.M ed . Ch e m. L ett. 8 , 3599-3602.

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(1993) Ana lysis of a ctive an tibody concentra tion. Sepa ra tionof af f inity a nd concentra tion para meters. J . I m m u n o l . M et h - ods 166, 75-84.

(17) Christensen, L. L. (1997) Theoretical an alysis of proteinconcentrat ion determinat ion using biosensor technology un -d e r c on d i t i on s of p ar t i a l m as s t r an s p or t l i m i t at i on . A n a l .Bi ochem. 249, 153-164.

(18) Richa let-Secordel, P . M., Ra uffer-Bru yere, N., C hr isten sen,L. L., Ofenloch-Ha ehnle, B., Seidel, C., an d Van R egenmortel,M . H. (1997) C on ce n t r at i on m e as u r e m en t of u n p u r i f ie dp r ot e i n s u s in g b i os e n sor t e ch n ology u n d e r c on d i t i on s ofp a r t i a l m a s s t r a n s po r t l i m it a t i o n . A n a l . B i o ch em . 2 4 9 ,165-173.

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