Proc. Natl. Acad. Sci. USAVol. 91, pp. 5426-5430, June 1994Biochemistry

Luminescent oxygen channeling immunoassay: Measurement ofparticle binding kinetics by chemiluminescence

(homogeneousIMnoasay/snlet oxygen/latex beads/antenna)

EDWIN F. ULLMAN*, HRAIR KIRAKOSSIAN, SHARAT SINGH, Z. PING Wu, BENJAMIN R. IRVIN,JOHN S. PEASE, ARTHUR C. SWITCHENKO, JENNIFER D. IRVINE, ALAN DAFFORN,CARL N. SKOLD, AND DANIEL B. WAGNERResearch Department, Syva Company, Palo Alto, CA 94303

Communicated by Ronald Breslow, January 18, 1994

ABSTRACT A method for monitoring formation of latexparticle pairs by chemilumnescence is described. Molecularoxygen is excited by a photosensitizer and an antenna dye thatare dissolved in one ofthe particles. 1A02dlffse to the secondparticle and initiates a hi quantum yield chemilu entreaction of an olefin that is dissolved in it. The efficiency of14A02 transfer between particles is =3.5%. The techniquepermits real-time measurement of particle binding kinetics.Second-order rate constants increase with the number ofreceptor binding sites on the particles and approach diffusioncontrol. By using antibody-costed particles, a homogenousimmunoassay capable of detecting =4 amol of thyroid-stimulating hormone in 12 min was demonstrated. Singlemolecules of analyte produce particle beterodimers that aredetected even when-no larger aggregates are formed.

Measurement of ligand-receptor binding events by standardmethods such as radioimmunoassay and ELISA requires aphysical separation of bound and free labeled ligand orreceptor. "Homogeneous" methods avoid this troublesomestep because the signal from the label is modulated by themolecular binding event (1). Among the more sensitive ofthese methods is enzyme channeling immunoassay (2) inwhich a binding event brings two enzymes together at asurface. One enzyme produces a product that is a chromo-genic substrate for the second enzyme. Because of slowdiffusion at the surface, the first product has a higher localconcentration than in the bulk solution and selectively reactswith the proximate second enzyme.An alternative homogeneous method utilizes immuno-

chemical aggregation of ligand- or receptor-labeled particles(latex agglutination) (3). Detection of agglutination by con-ventional light scattering requires formation of large aggre-gates and has limited sensitivity. Low-angle light scatteringmeasurements provide higher sensitivity but require rigor-ously exclusion of adventitious particles (4, 5).

In the present study, nonenzymatic channeling of 1.02 isused to provide exquisitely sensitive real-time monitoring ofparticle-particle interactions. The method, called luminescentoxygen channeling immunoassay (LOCI), exploits the shortdiffusion distance of 1AgO2 to initiate a chemiluminescent re-action near the site that it was formed. Unlike methods that uselight scattering, LOCI is relatively insensitive to interference byparticles or other substances present in biological samples.

MATERIALS AND METHODSChemicas. Dioxene 1, thioxene IV, and the squarate dye VII

were prepared by modifications of literature methods (6-8).Aminodextran having one amine per seven glucose units was

N- N

I X=O vI X=OIV X-S vVx=S

III X=OVI X=S

N +CHAH

-0 0

HOs

VII C4 9 49

prepared by adding concentrated ammonia to 500-kDadextran(Pharmacia) that had been activated by reaction with epichlo-rohydrin and Zn(BF4)2. The product was purified by precipi-tation with methanol. Fluorescein-biotin (Fl-biotin) was pre-pared by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide(EDAC) coupling of Fl-5'-COOH to 1,12-diamino-4,9-dioxadodecane followed by reaction with biotinyl-6-ami-nohexanoic acid N-hydroxysuccinimide ester (biotin-LC-NHS, Pierce). Bis-(6-hydroxyhexyl) disulfide and 6-amino-hexanol 5'-phosphate esters of dA40 and dT40 were fromMidland Certified Reagent (Midland, TX). (dT4o-digoxin wasprepared by periodate oxidation of the vicinal glycol ofdigoxin, reductive amination with 3-alanyl 3-alanine, andEDAC coupling with the 6-aminohexanol oligonucleotide es-ter. Fl-5'-CONH(CH2)6NHCOCH2OCH2CONH(CH2)6NH2(Fl-X22) was prepared from diglycolic acid and excess 1,6-diaminohexane followed by coupling to Fl-5'-COOH. Anti-bodies and digoxigenin-3-one O-carboxymethyloxime (CMO-Dig) were from Syva. CMO-Dig was combined with EDACand Fl-X22 to give Fl-X22-CMO-Dig. Chloroaluminum phthalo-cyanine tetrasulfonic acid [PC(SO3H)4AlCl] and chloroalumi-num tetra-n-decylphthalocyanine [PC(dec)4AlCl] were pur-chased from Ultra Diagnostics (Seattle). All other compoundswere from Pierce, Aldrich, or Sigma except as indicated.

Buffers. The standard buffer was 50 mM sodium phos-phate/0.15 M NaCl, pH 7.0. The assay buffer was 0.1 MTris'HCl/0.3 M NaCi/bovine serum albumin (1 mg/ml), pH8.2.

Antibody Conjugates. Anti-thyroid-stimulating hormone(TSH) antibodies, anti-TSH91 and anti-TSH132, were tononoverlapping epitopes. Anti-TSH91 was derivatized withsulfosuccinimidyl 4-(maleimidomethyl)cyclohexane-1-car-

Abbreviations: TTA, 3-(2-thienoyl)-1,1,1-trifluoroacetone; Phen,1,10-phenanthroline; TOPO, trioctylphosphine oxide; Chl, chloro-phyll a; CMO-Dig, digoxigenin-3-one O-carboxymethyloxime; Fl,fluorescein; PC(SO3H)4AIC1, chloroaluminum phthalocyanine tetra-sulfonic acid; PC(dec)4AlCI, chloroaluminum tetra-n-decylphthalo-cyanine; EDAC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide;SAv, streptavidin; TSH, thyroid-stimulating hormone; LOCI, lumi-nescent oxygen channeling immunoassay; RLU, relative lightunit(s).*To whom reprint requests should be addressed.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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Proc. Natl. Acad. Sci. USA 91 (1994) 5427

boxylate (sulfo-SMCC). The bis-(6-hydroxyhexyl) disulfide5'-phosphate ester of dT40 was reduced with tris-(2-carboxyethyl)phosphine (Molecular Probes) at pH 5.3 andcondensed with the antibody at pH 6.0 at 40C under argon.The conjugate was purified over protein A-Sepharose fol-lowed by DEAE-Sephadex. A dT40/protein molar ratio of 1:1was determined from the absorption at 260 and 280 nm.

Biotinylated anti-TSH,2 and anti-digoxin were preparedfrom biotin-LC-NHS followed by dialysis. The number ofbiotins was determined by titration ofthe protein amines with2,4,6-trinitrobenzenesulfonic acid.

Streptavidin (SAv) and anti-Fl antibodies were thiolatedwith N-succinimidyl S-acetylthioacetate and subsequenttreatment with 0.1 mM hydroxylamine for 1 h under argon.Dyeing of Latex Particles. Polystyrene latex particles (175

nm) having about 8.3 carboxyl groups per nm2 of surface(Bangs Laboratories, Carmel, IN) were stirred with 3.3 mMcompound I or IV with or without 15.5 mM Eu(TTA)3 [whereTTA is 3-(2-thienoyl)-1,1,1-trifluoroacetone] and 15.5 mM1,10-phenanthroline (Phen) or 20 mM trioctylphosphine ox-ide (TOPO) in 8:1:1 (vol/vol) ethylene glycol/benzyl alcohol/water for 8-10 min at 110°C. The particles were diluted withequal volumes of ethanol, centrifuged, washed with water,and sonicated. Chemiluninescer particles contained 10 mMI (©O) or >10 mM I or IV together with =100 mM Eu(TTA)3(©sl/Eu(TTA)r/TOPO or C)Iv/Eu(TTA)3/Phen, respectively). A similarprocedure was used to incorporate chlorophyll a (Chl) andVII into latex particles to give sensitizer particles, *cm and*cm/vu. The absorption spectra of the dyes in polystyreneparticles were nearly the same as in toluene.

Surface Modification of Latex Particles. Maleimide-substituted dextran was prepared from amino-dextran andsulfo-SMCC followed by dialysis. Typically :35% of theamino groups reacted. Dyed 175-nm latex particles werecoupled to the amino-dextran with EDAC followed by cen-trifugation, washing, and sonication. After incubation withexcess thiolated SAv, antibody, or dA40 for 16 h at 20°C, thereactive groups were capped with mercaptoacetic acid fol-lowed by excess iodoacetic acid. Binding capacities weremeasured using PHibiotin, Fl, or Fl-labeled dT40.

Binding of bioTin, FI-X22-, or dT40-labeled antibodies orligands to coated particles was carried out by incubation at4°C in assay buffer with periodic sonication.. Binding wasassumed to be quantitative. Dynamic light scattering mea-surements showed that any bead aggregation was reversed bya final sonication in the presence of the free label.Chemiluminescent Measurements. Standard irradiation

conditions for dioxene I particles included irradiation at 37°Cfor 1 min with a 150-W Dolan-Jenner tungsten/halogen lampequipped with a 0.71-cm-diameter glass fiber optic bundleand a 610-nm cutoff filter. Chemiluminescent emission wasthen monitored for 20 s with a Turner (Palo Alto, CA) TC20eluminometer equipped with a 550-nm cutoff filter. A lightoutput of 1 relative light unit (RLU) was near the minimumreproducible signal. Thioxene IV particles were irradiated for1 s at 37°C, and after 50 ms, the emission was monitored for1 s by using a photomultiplier tube equipped with a 620-nmband pass filter and automated shutter.Chemiluminescence quantum yields were determined rel-

ative to that ofI (4CL = 0.021 at 20°C vs. a 14C standard) (6).The amount of dioxetane II produced during irradiation of Iin 0.1 pM PC(dec)4AlCl in toluene was determined from thefluorescence of its decomposition product HI. A similar yieldwas assumed for the conversion of IV to V corrected for thehigher rate of reaction of IV with 'Ag02 (see below). Thequantum yields were measured in the presence and absenceof added Eu(TTA)3Phen.

Quantum yields of chemiluminescer-dyed particles weredetermined by irradiation of particle suspensions in 0.2 pMPC(SO3H)4AlCl and standard buffer at 250C. The diester HI

was determined after centrifugation of the particles anddissolution in dioxane. It was assumed that the rates ofreaction of 1A.02 with I and IV in particles were the samebecause of the rate-leveling effect of very fast reactions bypolystyrene (9). The total light output was determined byextrapolation to zero irradiation time to account for lightemitted during irradiation. Light intensities were also mea-sured after a steady state of the dioxetanes was reached.Quantum Yield of 1402 Formation. Sensitizer-dyed par-

ticles in standard buffer were irradiated and the rates of1g02formation were monitored by the disappearance of 9,10-bis-(carboxyethyl)anthracene. Quantum yields were calculatedby comparing with the rate of 1AgO2 formation during irra-diation of 2 nM PC(SO3H)4AlCl (4 = 0.4) (10).

Kinetic Measurements. Rates of binding of receptor-labeled particles to ligand-labeled particles were determinedin assay buffer at 370C without stirring. Reactions involv-ing I/EuCrTA)W/TOP were stopped by addition of 0.25 mMbiotin prior to measuring the signal. Reactions involving©pIV/Eu(TTA)3/Phen were measured in real time.

RESULTSChemiluminnt Particles. Dioxene I reacts rapidly with

'A5O2 (k = 7.5 x 107 M-1 sol) (11). The resulting dioxetaneH is reported to undergo nearly quantitative first-order decayto the diester HI in toluene at 250C with chemilununesenceemission at 360 and 400 nm (4CL = 0.021) (6). The quantumyield has now been found to increase to 0.15 in the presenceof 0.5 mM Eu(TTA)3Phen. Only europium emission (pre-dominantly at 613 nm) was observed, and the decay rate ofHI was doubled (Table 1). Thioxene IV reacted even morerapidly with 1A0O2 (k = 1.9 x 108 s-1) but with only weakemission at 370 nm. Added Eu(TTA)3Phen had less effect onthe decay rate but produced strong concentration-dependentemission at 613 nm (4CL = 0.20 at 5 mM chelate and 4CL =

0.37 on extrapolation to infinite chelate concentration).Irradiation of suspensions of ©s/EUCTA oo or CIv/

EuCiTA)3 phcn particles in aqueous solutions ofPC(S03H)4AlClat 370C produced delayed emission at 613 nm that decayedwith multiphasic kinetics (Table 1). The quantum yields werehigh (0.31 and 0.46, respectively) and independent of theirradiation intensity. In contrast, ©I particles emitted at 360nm with 4CL = 0.011.The fraction of 1A02 entering the particles that reacts with

the dioxene I was determined by irradiating suspensions of C)1

in 1 pM PC(S03H)4AlCl. The total amount of diester mformed (MT) was found to increase with the dioxene concen-tration in the particles according to the following equation:

1 1 kd + kout 1

MT (lAgODT kr(1Ag02)T [I]

which is derived from the following approximate kineticmodel:

Table 1. Chemiluminescent quantum yields and decay rates ofdioxetanes produced on irradiation of I and IV

Toluene (250C)

4tCL T1/2, S

Particles (370C)

4CL 1/2, S

I 0.021 2370 0.011 50I Eu(TTA)3Phen 0.15 (0.5) 1280 0.31 (100)* 50IV Very low 2.2IV Eu(TTA)3Phen 0.37t 1.8 0.46 (100) 0.6

x1/2 is the time for 50%o decrease in signal during multiphasic decay.Numbers in parentheses indicate the concentration of Eu in mM.*Eu(TTA)3TOPO used instead of Eu(TTA)3Phen.tExtrapolated to infinite [Eu].

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5428 Biochemistry: Ullman et al.

('AgO2)aq + (I)PS (1AgO2 + Pps

(1AgO2 + I)'S('02 + Pps(1A&gO2 + rps (II)PS,

where ps refers to polystyrene, and the integrated total 1A402that enters the particles, (1AgQ2h, is assumed to be indepen-dent of the concentration of I in the particles. Based on theextrapolated yield at infinite [II, 10 mM I trapped 65% of the14O2 entering the particles.Snsltzer Partiles. The efficiency of 1lgO2 formation was

measured by irradiating suspensions of sensitizer particles instandard buffer containing the 1AgO2 trap, 9,10-bis-(carboxyethyl)anthracene. Chl (8670 = 82,000) was a rela-tively efficient oxygen sensitizer, but significantly greater'AgO2 yield could be achieved by also including the verystrongly absorbing 1,3-bis(NN-dibutyl-4-amino-3-hydroxy-phenyl)squaraine VII (ew = 330,000). This highly fluores-cent compound, which by itself is a poor sensitizer, presum-ably serves as an antenna to collect light and transfer energyfrom its excited singlet state to Chl (Fig. 1).The absolute rate of 1A.02 formation from an optimally

dyed dextran-coated Oct/vu particle under standard irradi-ation conditions was about 11,000 1A402 molecules per s (4= 0.018). This rate increased by about 20%o upon saturatingthe buffer with pure oxygen instead of air.

Partide Coating. Nonspecific binding interactions of par-ticles were greatly reduced by coating with dextran. Theapparent particle diameters usually increased from 175 nm to200-240 nm during the coating process. Generic receptorswere bound to the dextran to permit quantitative function-alization of the particles with antibodies or haptens that wereappropriately labeled with biotin, dT40, or Fl-X22. Typically,the number of receptors per particle was 300-500 anti-FlIgGs, 2000-4000 SAvs, or 400-700 oligonucleotides.LOCI Signal. Chemiluminescent emission at 613 nm was

observed after irradiation (>610 nm) of a suspension of*chtvv(anti-Fl)4s50(Fl-X22-biotin)4so and ©I/Eu(TTA)3/TOPO-(SAv)4=oo. The signal increased linearly with increasing con-centration of the chemiluminescent particles (43 pRLU foreach chemiluminescer particle added) and peaked at equalconcentration of the two particles (32 pRLU). At chemilu-minescer particle excess, it approached a slightly lowerplateau near 29 pRLU for each sensitizer particle (Fig. 2).

Initially, a rosette of sensitizer particles would be expectedto form about each added chemiluminescer particle. Thesignal arising from a single particle pair interaction should

I 12

0 I

0.0 5.0 10.0 15.0 20.0Dye in particles; mM

FIG. 1. Yields of lA02 per particle during irradiation of 200 8gof latex particles containing various amounts of Chl (squares) or 3.5mM Chl and various amounts of VUI (triangles) suspended in 100.uManthracene-9,10-dipropionic acid in standard buffer.

100,000

10,000

1000

0.001 0.01 0.1 1 10 100Equivalents of ©I/EuarTA)31.,

FIG. 2. Suspensions of 22 pM *chl/vn(anti-Fl)4so5(Fl-X22-biotin)45o in 0.5 ml ofassay buffer were incubated for 2 h with variousamounts of C1/Eur(TA),rMPo0(SAv)4o0o. The suspensions were thendiluted 1:5 and irradiated at >610 nm for 1 min, and the signal wasmeasured over 20 s.

then be the observed signal per chemiluminescer particledivided by the number of sensitizer particles in the rosette.When the chemiluminescer particles are in excess theyshould completely surround each sensitizer particle. Thesignal from a single particle pair interaction should then bethe observed signal per sensitizer particle divided by thenumber of chemiluminescer particles in the rosette. A min-imum of six spherical particles bound to a central particle isrequired to block additional binding and a mimum of 12particles can bind if perfectly ordered. Random binding wascalculated by computer modeling to yield an average rosetteof 8.3 particles surrounding the central particle. A particlepair, therefore, gives rise to 3.6-7.2 MRLU (sensitizer par-ticle excess) or 2.4-4.8 p.RLU (chemiluminescer particleexcess). This difference in response may result from com-petition between the chemiluminescer particles for a limitedsupply of 1Ag02 when in chemiluminescer particle excess.

Direct evidence that the LOCI signal could be generatedfrom individual particle pairs was obtained by transmissionelectron microscopy. For this purpose *Chvn(anti-Fl)45oparticles were incubated with ©I/Eu(TTA),/topo(SAv)4ooo par-ticles in the presence of anti-digoxin-(biotin)5 and Fl-X -CMO-Dig. Based on the LOCI signal 1.3-2.6 x 108 particlepairs should have formed. This was near to the electronmicroscopically determined number of 3.3 x 10V particlepairs with almost no larger aggregates.Few compounds could be found that interfere with singlet

oxygen channeling. These were compounds that quench orreact very rapidly with 1AgO2. Thus 1 mM sodium azide or 5mM ascorbate resulted in a 50%6 reduction in the signal fromparticle pairs. Since azide, which quenches 1'A02 in waterwith high efficiency (kq = 5 x 108 M-l s-1) (12) displayedtypical Stern-Volmer quenching of the LOCI signal, chan-neling must be due primarily or exclusively to 'Ag02.To estimate the local concentration and efficiency of

capture of 14A02 within a particle pair, the LOCI chemilu-minescence was compared to the chemiluminescence and theyield of diester m when CI/ TA TOPO particles wereexposed to a known 1A402 concentration. A concentration of7.8 pM 1402 during inadiation of 1 PtM PC(SO3H)4AlCI wascalculated from the ratio of the measured 1A.02 formationrate to its decay rate in water of 5 x 105 s51 (13). Whenuncoated 0I particles were present in this solution, anaverage of 780 molecules per min of the diester III wasformed in each particle. Since the presence of coordinativelysaturated Eu(TTA)3 would not be expected to interfere, thesame rate was assumed for ©I/Er(TTA)3/TOpO particles. [Thisassumption was used because of difficulties in measuring HIin the presence of Eu(TTA)3.] Under these conditions,

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©I/Eu(TTA)3/TOPO particles produced a signal of0.28 pRLU perparticle, which was reduced to 0.17 pRLU after coating withdextran. By using the above LOCI signal of 3.6-7.2 pRLUper particle pair, the local 1Ag02 concentration experiencedby a chemiluminescer particle in a particle pair is calculatedto be 0.17-0.33 nM and 10,000-20,000 molecules per min ofthe diester Im are formed.

Binding Kinetics. LOCI could be used to monitor the rateof particle dimer formation. Various concentrations of ©I/EuCFTA)3/ToPO(SAv)2800 were mixed with a suspension of 4.89pM *Chl/vIi(anti-Fl)450.(Fl-X22-digoxin)464[anti-digoxin-(biotin)5s2 particles at 370C. With equimolar concentrations,typical second-order kinetics (k = 2.3 x 109 M-l s-1) wereobserved up to 78% ofthe signal extrapolated to infinite time.The same rate constant was obtained from initial rates. Theinfinity point (2.8 pRLU per particle pair) was close to thatobtained independently from the titration data (3.6-7.2$ARLU per particle pair).The effect of receptor surface density on particle-particle

binding rates was studied with 2.8 pM IV/Eu(TTA)3/Phen-(SAv)34004[(biotin)s-anti-digoxin]J and 2.8 pM *chi/v(dA40)sWo.(dT4w-digoxin)3oo. Rates were measured as a func-tion of the number of anti-digoxin IgGs (n) that were boundto the chemiluminescer particle. The relative rate constantsinitially increased nearly linearly with n but increased lessrapidly at higher surface densities (Fig. 3).

Assay Response. Highly sensitive detection of digoxin, atypical small molecule analyte, was possible by incubating asuspension ofCiv/E TTAh/pin(SAv)_mwoiotin-anti-dioxin)12and serum, adding Ochi/vu(dA40)54o(dT4o-digoxin)3oo, andmeasuring the signal. The total assay time was only 63 s (Fig.4). Increasing the concentration of either particle resulted inproportional increases in the signal. Since the incubationperiods were substantially shorter than required for completeparticle binding (=3 min), the signal increases primarily reflectincomplete particle binding.Human TSH was selected as a model multiepitopic analyte

because of its exceptionally low concentration in hyperthy-roid patient serum. WIV/Eu(TTA)3/Phen(dA4O)54b0(dT4o-anti-TSH,31)25o particles (9.3 pM) and anti-TSHP2-(biotin)15 (6.7nM) were incubated in a 1:3 dilution of serum in assay bufferfor 8 min. The solution was then diluted 1:6 with 17 pM*chl/vu(SAv)200o particles and incubated an additional 4 mn.The assay response was linear over four orders of magnitude

Anti-digoxin IgG/particle ratio

FIG. 3. Effect of anti-digoxin/particle ratios on the kinetics ofbinding to digoxin-coupled particles. *chI/vn(dA4O)54OW(dT40-digoxin)30o (2.8 pM) was incubated at 37°C in assay buffer withOIv/Eu(rTA)3/Phen(SAv)34wo[(biotin)s-anti-digoxin]" (2.8 pM) havingdifferent amounts ofanti-digoxin IgG. The initial rates, followed over95 s, were normalized (Inset) using the independently measuredsecond-order rate constant of 3.8 x 108 M-1-s-1 for n = 24. Ratiorefers to anti-digoxin IgG/particle ratio.

125r-

.ll 100cww 75ri

S 50

w 25

01 3 5

Digoxin, ng/ml

FIG. 4. Serum (50 A) was incubated with 16 pM C½/EurTA)3/Phen(SAv)340w.[(biotin)s-anti-digoxin]12 (350 A4) in assay buffer at 37Cfor 33 s. The suspension was then diluted with 600 A4 of 30 pM*chl/vu(dA4o)540o(dT4o-digoxin)3oo, incubated for20 s, irradiated, andmeasured. Duplicate points are shown.

and the signal from 4.2 amol of TSH (2.5 x 106 molecules)was fully resolved (-6 ar above background). This limit wasimposed by a background signal attributable to nonspecificbinding between the sensitizer and chemiluminescer particlesthat increased linearly with time. At zeroTSH concentration,there were also smaller time-independent contributions tobackground due to the instrument, the autochemilumines-cence of the chemiluminescer, and 102 migration to unboundchemiluminescer particles. The intensity of the latter wasabout equal to the specific signal at the detection limit.

DISCUSSIONLatex agglutination methods for rapid immunochemical de-tection generally employ relatively insensitive light-scattering measurements (3). Although it is possible to detectimmunochemical formation of particle pairs by low-anglelight scattering or by using flow systems such as the optical-pulse particle analyzer (14), these methods require rigorouslyparticle-free solvents and impractically low particle concen-trations (4, 5). The present study takes advantage ofthe shortdiffusion distance of 14.02 produced in one particle to causepreferential reaction within a particle pair. The ensuingchemiluminescent reaction in an adjacent particle permitsready monitoring of particle pair formation without interfer-ence from particles in the sample. The long wavelengthemission of chemiluminescent reagents, I and IV, in thepresence of a europium chelate minimizes interference bybiological substances, most ofwhich absorb at shorter wave-length. Some precedence for the method can be found in theknown selective cytotoxic effect of 1A402 formed uponirradiation of sensitizers that are immunochemically targetedto cells. Direct antibody-dye conjugates have been used forin vivo photodynamic therapy (15) and dye-labeled liposomesbound to antibodies exhibit selective in vitro killing of Tlymphocytes (16).The LOCI method permits real-time monitoring of particle

binding. The second-order rate constant at 37C for thereaction of biotinylated particles (125 per particle) withparticles heavily labeled with avidin (2800 per particle) was2.3 x 109 M-1 s-1. This approached the calculated bimolec-ular diffusion-controlled rate constant in buffer (9.0 x 109M-1 s-1) (17) and was similar to the rate constant for dimer-ization of oppositely charged latex particles (18). In contrast,digoxin-labeled particles (300 per particle) bind to particleslabeled with anti-digoxin antibodies at a lower rate that isdependent on the antibody surface density. This behavior isconsistent with formation ofparticle-particle encounter com-plexes that dissociate at a rate competitive with particlebinding within the complex. The rate constant is given by

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k = k+konDsAs/(ko.D.As + k4),

where k+ and k- are, respectively, the rate constants forformation and dissociation of the encounter complex; D, andA, are the fractional surface densities of the digoxin and theantibody; and kon is the first order rate constant for bindingwithin the encounter complex. Extrapolation to infinite A,(Fig. 3 Inset) gave k+ = 2 x 109 M-l s-1, which approximatedthe rate constant for avidin particle-biotin particle binding.Since the rate was unaffected by variations in the surfaceconcentration of the digoxin, it is probably close to themaximum achievable. Previously estimated antibody-mediated particle dimerization rate constants were 100-foldlower despite very high antibody surface density (19), pos-sibly because the antibody orientation was not controlled.Based on Fick's First Law, a chemiluminescer particle in

a particle pair experiences a 14g02 concentration at a distancer from the center of an irradiated sensitizer particle of:

Sc(r) =

4Dl ad)expll-(r - a)/d],()4irrD(1 + aid) P

where S is the rate offormation Of1gO2 by the particle (takenhere as 11,000 s-1), a is the radius of the polystyrene portionof the sensitizer particle (87.5 nm), d is the 14802 diffusionlength (D/kd)l/2 (-80 nm), kd is the 'A.02 decay constant inwater, andD is the diffusion coefficient in water. At 370C therelative 1AgO2 concentration is negligible beyond =250 nmfrom the surface (Fig. 5). For particles separated by 50 nm ofdextran, the mean 1A42 concentration at the surface of thechemiluminescer particle would be 0.2-0.3 nM 1AgO2. This isconsistent with the experimentally derived local 1AgO2 con-centration of 0.17-0.33 nM.Data relating to the transfer efficiency of 1AgO2 between

adjacent particles are summarized in Table 2. Since it wasfound that Im was produced at a rate of 10,000-20,000molecules per min in a particle pair and 11,000 1AgO2 per s areformed during the 1-min standard irradiation conditions, the14gO2 capture efficiency is 1.5-3.0%. The 1gO2 transferefficiency between two bound particles is higher (2.3-4.6%)because only 65% ofthe 1AgO2 entering the chemiluminescentparticles reacts with I. Based on the chemiluminescencequantum yield of the dioxetane II in particles (31%6), 4600 ±1500 photons were emitted per binding event of which =10%were emitted during the 20-s monitoring period.The TSH assay data are informative as to the limits of

sensitivity ofLOCI. A linear response toTSH concentrationsdown to <0.1% of the particle concentration illustrates thata single TSH molecule affects particle pair formation and thatparticle pairing is sufficient to obtain a signal. However,detection of TSH was limited to =4 amol (90 fM in serum)despite sufficient instrumental sensitivity. This limit was

2.5 k 1

2.0

1.5

1.0

0.5

0.0

0 100 200 300Distance from bead surface, nm

FIG. 5. Concentration of 'AgO2 calculated as a function ofdistance from a 175-nm particle producing 11,000 1AgO2 per s.

Table 2. Efficiency of 'AgO2 channeling within pairs of *Cv/vnand Ci/,aTTAn poo particles under standard 1-minirradiation conditions

Parameter ValueRate of 140Q2 production by *cb1/vi, s51 11,000Efficiency of 1Ag02 transfer between

particles 0.035 ± 0.012Fraction of 1AgO2 entering a particle that

reacts with I 0.65Chemiluminescence quantum yield of H 0.31Photons emitted per particle pair 4600 ± 1500

primarily due to time-dependent nonspecific binding of theparticles. Drift of 14A02 to unbound particles produced a lesstroublesome time-independent background, which was of amagnitude consistent with the expected steady-state concen-tration of 1AgO2 (1 pM).

It is noteworthy that 102-103 fewer photons are emitted peranalyte molecule in LOCI than in homogenous fluorescenceimmunoassay methods (20). Nevertheless LOCI is >103more sensitive due to the inherently lower background emis-sion of chemiluminescence. The method is, therefore, appli-cable to a broad range of analytes such as nucleic acids,antibodies, and antigens. Because no separation step isnecessary, there may also be an opportunity to make directmeasurements of components of fragile biological structuressuch as organelles and cell surfaces. Additionally, the abilityto follow binding kinetics offers the opportunity to gaingreater understanding of the detailed processes associatedwith particle-particle interactions.We particularly thank Dr. George Danilov, Dr. Dariush Davalian,

Jackie Ishkanian, Dr. Rohan Penes, and Marcel Pirio for specificmeasurements, preparations, and concepts that were not describedin this paper but provided critical insights.1. Miyai, K. (1985) Adv. Clin. Chem. 24, 61-110.2. Litman, D. J., Hanlon, T. M. & Ullman, E. F. (1980) Anal. Rio-

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eff, R. F., Albert, W. H. & Staines, N. A. (VCH, Weinheim,Germany), Vol. 1, pp. 203-214.

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9. Ogilby, P. R., Dillon, M. P., Kristiansen, M. & Clough, R. L. (1992)Macromolecules 25, 3399-3405.

10. Darwent, J. R., Douglas, P., Harriman, A., Porter, G. & Richoux,M. C. (1982) Coord. Chem. Rev. 44, 83-126.

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Murray, R. W. (Academic, New York), p. 167.14. Bowen, M. S., Broide, M. L. & Cowen, R. J. (1985) J. Colloid

Interface Sci. 105, 605-616.15. Mew, D., Wat, C., Towers, G. H. N. & Levy, J. G. (1983) J.

Immunol. 130, 1473-1477.16. Yemul, S., Berger, C., Estabrook, A., Suarez, S., Edelson, R. &

Bayley, H. (1987) Proc. Natl. Acad. Sci. USA 84, 246-250.17. Backstrom, H. L. J. & Sandros, K. (1960) Acta Chem. Scand. 14,

48-62.18. Kitamo, H., Iwai, S., Ise, N. & Okubo, T. (1987) J. Am. Chem. Soc.

109, 6641-6644.19. Kitamo, H., Yan, C., Maeda, Y. & Ise, N. (1989) Biopolymers 28,

693-702.20. Robbins, B. A. & Nakamura, R. M. (1988) J. Clin. Lab. Anal. 2,

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5430 Biochemistry: Ullman et al.

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