CA2179310A1 - Process for labelling acridinium to microparticles and application in an instrument - Google Patents

Abstract

Embodiments of the invention provide methods of preparing an activated aeridinium microparticle. Generally, the methods involve direct covalent coupling or an affinity format. The direct covalent coupling method involves coating a microparticle with a proteinaceous compound. Then, a 10-methyl-N-tosyl-N-(2-carboxyethyl)-9-acridini um carboximide trifluoromethane sulfonate is coupled to the proteinaceous compound. In the affinity format, a microparticle is coated with a biotinylated proteinaceous compound. The microparticle is reacted with an anti-biotin labelled 10-methyl-N-tosyl-N-(2-carboxyethyl)-9-acridinium carboximide trifluoromethane sulfonate. Methods are also provided for using such a microparticle. Those methods of use can estimate transfer efficiency, calibrate optics, and measure membrane pore size of a chemiluminescence based instrument. Test elements for analytical instruments are also provided.

Description

W0 95/220s9 ~ 1 7 ~ 5 1 0 P~ 5 Is~
PROCESS FOR LAsELING ACRIDINIUM
TO MICROPARTICLES AND APPLICATION
IN AN l~ x ~ .~U~I~L~
R~l/'lr~Rt~nNn OF TF~T;! I~VE~TION
The present invention relates to methods and compounds for 5 use with an instrument for performing chemical assays and measurements and, more particularly, to methods and rl _r,lln~q for chem~iluminescence-based tests on an instrument employed in performing chemical assays, such as those performing quantitative and gualitative tests on chemical and biochemical 10 solutions.
A variety of assay methods and compounds are employed in ~uantitative and clualitative analyses of chemical and biochemical mixtures. In some instances, those quantitative and lit~tive analyses are performed by an instrument. Certain of 15 the methods and compounds employed in analyses can be useful in testing the instrument employed in those analyses. Such tests may concern, for example, transfer efficiencies, instrument calibrations, membrane pore size meabul ~q, and the like. A
number of methods and compounds employed in these tests 20 reqarding in~ tq are known. New methods and compounds and J~,~ tq in existing methods and I _ ~q, however, are being developed.
One che~mical property utilized in certain analytical applications, inrll~l;n~ tests regarding instruments is referred 25 to as "rhl~milllm;n~qrPnrel~ h~mi lllm;n~qcence is the emission of light as the result of a chemical reaction. (~hf~m; lllm;nPqrPnre occurs when products of a chemical reaction are excited and emit light .

WO 95/22059 . ~ ~ 5~ / --21 7g31 2 In an exemplary chemical reaction that generates rhPm; 1 ; nl~qcencel a step of the reaction is a chemiexcitation atep (which may be unimolecular or bimolecular) which achieves conversion of chemical energy into electronic excitation energy.
5 In the reaction, a product molecule receives chemical energy and converts it to an excited electronic state. This electronically excited product molecule then produces light or luminesces under reaction ~ tmri; t i ~n q Attempts have been made to automate ~Pm;1llm;n~qcence 10 mea~uL~ q. Some attempts have involved a variety of instruments, such as, for example, the use of photographic film and a densitometer to record a c~emiluminescent signal from a reaction in clear microtitration plates.
To provide accurate and desired results, measurements and 15 other determinations regarding the systems may be made periodically. These determinations may indicate the condition of the system involved.
For instance, ~hf~m; 11 ;n~qcence may be useful in r--qllr;ng transfer efficiencies and/or other aspects of in~iLL, ,q 20 employed to perform assays which are measured or observed by r~Pm;1ll-;n~qcPnt characteristics. Inb~LI tq which are capable Of ~ nf~rm;ng to minute tolerances in these regards have relatively high transfer efficiencies. Instruments that do not yield accurate component measurements on transf er have 25 relatively low transfer efficiencies. To perform accurate tests with some analytical in~LL -'ltC, knowledge of transfer efficiencies (and, thus, the inherent accuracies and inaccuracies of the in~LL, tq) is desirable.
It is known to make certain tests on in~;LL, q used to 30 perform assays and other tests for detPrm;n;ng the accuracy and the like of the instrument. A known method for making such tests performs particular assays on a standard sample of known composition and characteristics. Because the composition and characteristics of the standard sample are known, results of 35 assays and tests performed with the sample should yield expected W095f22059 ~17~310~ . r~ 5 i5~
3 ` ~
results if the instrument were to perform accurately. If expected results were not obtained, inaccuracies of the instrument being utilized may be indicated.
It is known to perform particular tests on the instrument 5 used. In ~so testing in~l LI ' q, acridinium activated particles may be used as a standard sample. Acridinium, when reacted with ,i1k~lin~ peroxides, yields a light producing reaction. Some currently available acridinium activated particles used in testing chemiluminescence-based instruments have certain 10 disadvantages. One such disadvantage involves the method of preparing these particles. In this prior method, latex particles are coated with anti-HBsAg labelled acridinium using passive adsorption. Then, activated acridinium is passively adsorbed onto the particle surfaces.
~he product activated acridinium microparticles obtained from some prior methods may be disadvantareous for use in testing some ~h~m;lllm;nP~:cence-based instruments. Ilhe light emission profiles of particles may not be sufficiently ;(l~ntir~
to or closely resemble the actual light f~m;q~;.ln profiles of 20 rh~milllmin~cence-based i fl~ ys. It is desirable that a standard sample for testing perform substantially the same as an unknown will perform when tested. However, this may not always be the case. A possible reason for difference6 in light emission of some currently available product activated 25 acridinium microparticles may be stearic hindrance effects ~aused by acridinium loading.

W09s/22059 r~ Cls27

2~793~ 4 SnMM~RY OF T~ , NVKh LlC~
The ' ,fl;- c of the present invention provide a number of methods for manufacturing an activated acridinium microparticle as well as methods of :using such a microparticle 5 with an instrument. According to one embodiment, a method of preparing an activated acridinium microparticle includes coating a microparticle with a proteinaceous compound and coupling a 10-methyl-N-tosyl-N-~2-carboxyethyl)-9-acridinium r~rhn~r;m;de trifluJLI ^th~nP c~lfr~n~te to the protP;n~rPr~ compound.
According to another ' ~ofli of the invention, another method of preparing an activated acridinium microparticle comprises coating a microparticle with a biotinylated proteinaceous compound. Then, the microparticle is reacted with an anti-biotin labelled 10-methyl-N-tosyl-N- (2-carboxyethyl) -9-15 acridinium carboximide trifluoromethane~ sulfonate.
Accordin~ to an additional embodiment of the invention, a method of testing an analytical instrument is provided. A
microparticle is coated with a proteinaceous compound. A 10-methyl-N-tosyl-N- (2-carboxyethyl) -9-acridinium carboximide 20 trifluoromethane sulfonate is coupled to the proteinaceous compound. The 10-methyl-N-tosyl-N- (2-carboxyethyl)-9-acridinium carboximide trifluuL, h~n-~ sulfonate is activated to obtain an activated acridinium microparticle. Alternatively, the microparticle is prepared by coating it with a biotinylated 25 proteinaceous compound. The microparticle is reacted with an anti-biotin labelled 10-methyl-N-tosyl-N- (2-carboxyethyl)-9-acridinium rArhrl~rimifl~ trifluuL, -~h~n~ sulfonate. The thusly produced microparticle may be used in a test on the instrument.
The test may determine or estimate transfer PffiriPnry, 30 calibrate instrument optics, measure membrane pore size or other tests. The instrument may be a rhPmilllminpccence based instrument.

WO 95/221~59 Yet further embodiments of the invention provide test elements f or use in an analytical instrument . The test element comprises a microparticle coated with a proteinaceous compound.
Then a 10-methyl-N-tosyl-N- (2-carboxyethyl) -9-acridinium carboximide trifluoromethane sulfonate is coupled to the proteinaceous compound on the microparticle. Alternatively, a microparticle is coated with a biotinylated proteinaceous compound. Then, an anti-biotin labelled 10-methyl-N-tosyl-N- (2-carboxyethyl)-9-acridinium carboximide trifluoromethane sulfonate is reacted with the microparticle ~RTRF DESCRIPTION OF TEIE rlR~WINGS
FIG. 1 is a flow chart of a method of preparing activated acridinium m;croparticles by direct covalent coupling;
FIG. 2 is a flow chart of a method of preparing activated acridinium microparticles by affinity format;
FIGS. 3A-C are scanning electron micrograph depictions of 2.3 llm and 4.0 llm affinity format labelled activated acridinium microparticles, respectively;
FIGS. 4A-C are scanning electron micrograph depictions of

3.5 llm and 6.0 llm affinity format labelled activated acridinium microparticles, respectively;
FIG. 5 is a ~i _ 1 i f; P(l, block diagram illustration of a millisecond read system, in~ in r a microcomputer based automated data ac~uisition system, for detecting photon count inf nrr-t; nn;
FIG. 6 is a ~-~Pm;lllm;n~cence light emission profile illustrating decay rate at varying concentrations of anti-biotin acridinium conjugate loading on covalently coupled (about 0.002%) 2.5 llm biotinylated latex particles;
FIG. 7A is a light emission profile for a HBsAg assay;
FIG. 7B is a light emission profile for a 2.5 llm affinity format labelled activated acridinium microparticle;
FIG. 7C is a comparison of light emission profiles for an affinity format labelled activated acridinium microparticle and Wo95/22059 Q 1~ o~527 2jl79~t 6 for an anti-HBsAg labelled microparticle;
FIG. 8 is a chemill1m;n~ql~~n~~e light emission profile illustrating the effect of varying concentrations of acridinium loading on (about 0.001%) 2.5 ~Lm affinity format labelled 5 particles;
FIG. 9 is a ~h~m; l in~qcence light emission profile of (about 0.002%) 2.5 llm, 3.6 ~Lm, 4.5 llm and 6.0 llm affinity format labelled particles;
FIG. 10 illustrates the use of different ~ t;t~nq of ~.5 10 llm activated acridinium microparticles in instrument optics system calibration;
FIG. 11 illustrates the approximately 2-8 degree C
stability of (about 0.1%) 4.5 llm affinity format activated acridinium microparticles;
FIG. 12 illustrates the approximately 45 degree C stability of 5 ~Lm affinity format activated acridinium microparticles compared with the stability of anti-HBsAg coated activated acridinium microparticles;
FIG. 13 illustrates the use of different activated 20 acridinium microparticles in de~rmining the pore size on an analytical instrument; and Fig. 14 is a table comparing estimated transfer: =
~ffi~i,on~;es between an affinity format labelled microparticle and an anti-HssAg labelled microparticle.
: ~

Wo 95l22059 ~l 7 ~ 3 1 0 r~
DET~TT,T~n D13~rRTPTION OF ppT~T.'ERRT.'n E~RODIM~NTS
Disclosed in the following paragraphs are methods of preparation of compounds and compounds for rhf~mi lllmin~ cen based detF~rminAtir,nq about automated in~L, -Atirn and 5 e~uipment employed in performin~ Ays. Microparticles made according to these methods may be used with a variety of suitable in~i~" c to perform desired tests.
Two methods for preparing activated acridinium microparticles representing embodiments of the present invention 10 are disclosed. One :is referred to herein as "direct covalent coupling~. In direct covalent coupling, activated methyl acridinium is directly coupled by covalent bonding to bovine serum albumin ( "BSA" ) coated microparticles. The second method is referred to herein as ~affinity format~. In affinity format, 15 anti-biotin labelled methyl acridinium is reacted in order to conjugate with biotinylated BSA coated microparticles. Certain specific procedures for these two methods are hereinafter described. In an exemplary embodiment, the microparticles may be made of a suitable polymer, such as latex, polystyrene and 20 the like. It is to be remembered that variations of these methods are also possible. The steps disclosed herein may be performed in any suitable order, and steps of one method may be combined with steps of another method.
Referring now to FIG. 1, a flow diagram of the direct 25 covalent coupling method for preparing activated acridinium microparticles is illustrated. Generally, the direct covalent coupling method in this embodiment comprises three steps. A
first step (not shown in detail) is an activation step in which 10-Methyl-N-tosyl-N- (2-carboxyethyl)-9- acridinium carh~-Yimi~
30 triflu~.L ~ l~An~ sulfonate (abbreviated as ~methyl acridinium~
herein) 8 is activated. In a second step, a coating step 22, microparticles 2 are coated with BSA ~. Finally, in a third step, which is a coupling step 23, amino groups of the sSA
coated on the surface of the microparticles 6 is covalently 35 coupled to the activated methyl acridinium 8.
.

Wo 95/22059 ~ ~ ~ 9 ~ 1 ~? r~l,. 1527 The first (or activation) step is initiated by adding approximately 510~L1 o~ N-Hydroxy s~ cinimi~ solution (about 10mg/1. 74ml dimethylformamide) an~ about 510111 of 1-ethyl-3 (dimethylaminopropyl) carbodiimide ("EDAC") (about 10mg/1.31ml 5 dimethylformamide) to a solution ~-~lntAininS about 10mg of methyl acridinium in about 1.04ml of dimethylformamide ("r~F~). This mixture is stirred at ambient temperature in the absence of=
light for a period of about 12 to about 16 hours. The activation is monitored using thin layer chromatography. When 10 the activated product 6 is obtained, the product is used for coupling to protein without purification.
~ ntiml;n~ to refer to FIG. 1, the coating step 22 begins with centrifugation of particles 2, such as polystyrene particles (for example, 2 . 511m, Spherotech, lml, 5% w/v) and the like. The particles 2 are centrifuged at about 11, 000 x g for approximately 5 minutes and the supernatant is decanted. After centrifu~ation, the packed particles 2 are resuspended in about lml of phosphate buffered saline ("PBS~), which is about 0.01M
sodium phosphate, about 0.15M sodium chloride, a pH of about 7.2, and the centrifugation step ls repeated. Other buffers may be used. The packed particles 2 from this second centrifugation step are then resuspended in about lml of sSA solution 4 (about 2mg/ml in PBS ) and the mixture is tumbled at ambient temperature for period of about 16 hours. The~ suspension obtained from the tumbling is then centrifuged at about 11, 000 x g for about 5 minutes and the supernatant is decanted. Particles obtained from this tumbling are then washed, preferably three times, each with about lml of PBS by centrifugation and resuspension. The BSA coated particles 6 obtained are then resuspended in about 5ml of PBS to give an approximate 1% w/v suspe~sion.
Further still referring to FIG. 1, in the coupling step 23, the BSA coated particles 6 (for example 0.5ml, 196 w/v in an exemplary ~mhofl;-^nt) obtained from the coating step 22 are added to about 72ll1 of conjugate buffer (about 0.1 M PBS, with 35 approximately 0.196 CHAPS, pH of about 6.3) and about 15ll1 of the W0 9s/22059 2 1 7 9 3 1 ~ P~ c77 activated methyl acridinium 8 (about 5mg/1 ml D~F) obtained from the activation step. The mixture is then gently tumbled for about 30 minutes at ambient temperature. The particles obtained 10 are then c~ntr; fll~ed at about 11, 000 x g for about 5 minutes, 5 washed by centrifugation, preferably three times, and then rPq~l~rPn~lPd as before in the second (or coating) step. The particles are resuspended in about lml of PBS to give an approximate 0.5% w/v particles 10.
Now referring to FIG. 2, a flow diagram of the affinity 10 format method for preparing activated acridinium microparticles is illustrated. Generally, the affinity format method includes two steps. The first of the steps, a coating step 41, involves the coating of biotinylated BSA 12 to microparticles 2. The second step, an affinity reaction step 42, includes an affinity 15 reaction wherein anti-biotin labelled methyl acridinium 16 is conjugated to biotinylated BSA coated microparticles 14.
t;n~l;n~ to refer to FIG. 2, details of each of the two steps of the affinity format method may be described in detail.
In the first, or coating, step 41 microparticles 2, preferably 20 polystyrene particles (for example, about 2.5~m, Spherotech, approximately lml of 5% w/v), are centrifuged at about 11, 000 x g for about 5 minutes and the supernatant is decanted. The packed particles 2 from that centrifugation are then resuspended in about 1. 5ml of biotinylated BSA solution 12 (for example, 25 5igma, about lmg/ml in PBS, pH of approximately 7.2) and the mixture is tumbled at ambient temperature for about 20 hours.
The suspension obtained from the tumbling is then centrifuged at about 11, 000 x g for about 5 minutes and the sllr~rnRtRnt is decanted. Particles r~htR;rf~d from this tumbling are then 30 washed, preferably three times, each with about lml of PBS by centr;fll!JRtirn and resuspension. The BSA coated particles 14 obtained are then resuspended in about 5ml of PBS to give an approximate 196 w/v suspension.
Further still referring to FIG. 2, in the affinity reaction 35 step 42 of the affinit~ reaction method, the biotinylated BSA

WO gs/22059 21 7 9 ~1 Q PCTIUS95/01527 coated particles 14 (about 1 ml of about 1% w/v) of the coating step 41 of the affinity reaction method are cer,trifuged at about 11,000 x g for approximately 5 minutes and the supernatant i8 fl~r~ntl~ The packed particles 14 obtained from the 5 centrifugation are then resuspended in about lml of anti-biotin labelled with methyl acridinium 16. This amount of anti-biotin labelled with methyl acridinium 16 is obtained from a solution of about 40~L1 of anti-biotin acridinium (approximately 250 llg/ml, protein concentration, Acridinium/IgG is about 1. 8 ) added 10 to approximately 9 . 960 ml of conjugate diluent, said conjugate diluent being comprised of about 40g6 calf serum, about 10%
normal human plasma, about lOOmM phosphate buffer, with a pH of about 6.3, and about 0.15 M NaCl in an exemplary embodiment.
The mixture of packed particles resuspended in the anti-biotin 15 labelled with methyl acridinium 16 is then tumbled for about 16 hours at ambient temperature in the absence of light. The particles 18 obtained from the tumbling are centrifuged at about 11, 000 x g for about 5 minutes, washed, preferably three times, by centrifugation, and then resuspended as before in this 20 affinity reaction step. The activated acridinium microparticles 18 are resuspended in about lOml of PBS to give approximately 0.1% w/v particles.
Now referring to FIGS. 1 and 2 in conjunction, procedures similar to those described in detail above for preparing 25 activated acridinium micropartlcles according to the direct covalent coupling method and the affinity format method may be employed to prepare activated microparticles 10, 18 of various sizes, ~uch as, for example, about 3.5,Um, 4.511m, 511m and 611m.
In addition, though specific procedures have been described 30 herein with respect to each of the direct covalent coupling method and the affinity format method, those procedures are intended only for clarifying understanding. Though the exemplary ; ~ /1; q are described in detail, it is intended and should be understood that those skilled in the art will 35 understand that various changes, additions, modifications, and Wo9s/22059 ~1 ~9310 r~.,.J,, . 15~/

alternatives are possible in the specific procedures in keeping with the ~ ;~~ q described herein.
Next referring to FIGS. 3A-C and 4A-C, certain scanning electron micrographs of 2 . 311m, 4 . O~m, and 3 . 511m and 6 . O~m 5 activated acridinium microparticles and graphs tracking the size distribution of 4 . 0~1m and 3 . 511m are shown. The activated acridinium microparticles shown in the micrographs were prepared in accordance with the affinity format method. The mi~:L~/yLaphs shown in FIGS. 3A-B and 4A-B are provided, in particular, to 10 show the high monodispersity and uniform size of t~e 2.311m,

4.0,um, 3.5~L and 6.011m microparticles 18, respectively, obtained from the affinity format method. FIGS. 3C and 4C show, in particular, the size distribution of 4.011m and 3.511m activated acridinium microparticles 18, respectively.
Now referring to FIG. 5, a read system 30 for measuring perf ormance of activated acridinium microparticles 18 prepared according to the affinity format method is illustrated. The read system 30 employs a millisecond read tPr~nirl~c~. The read system 30 comprises a microcomputer 32 based automated data 20 acriuisition system. The system is eriuipped with an electronic ;nt~rfArP board 40 capable of ac4uiring photon count infnrr~t;rn, generated by optics, such as a reader 34. ~n interface 36 may also be used to synchronize the start of a data aco,uisition cycle, for example, with the dispensing of activator 25 solution at the read station 38. The read system 30 accumulates a running sum of photon counts acriuired during a read cycle of approximately 2 seconds with temporal acriuisitions taken and recorded at about lOms intervals . Acl 1 Ateti data can be used, for example, to obtâin in~L~ Al counts for every read 30 interval, thus, yielding light emission profiles, generated, for example, at an off-line computer terminal 42, substantially similar to those r,htA;nP~ in a rhPm;ll~min,o.qcence reaction.
PIG . ~ s 6, 7A, 7B, 8 and 9 may be obtained by subtracting subseriuent approximately 10 ms intervals to arrive at the num.ber ,5 o~ events per about lOms interval, for example. The about 10 ms Wogs/22059 ~1 ~g~ r~~ DIS27 events are plotted over time to produce a rate reaction profile.
Still referring to FIG. 5, a procedure for assessing perform~arlce of activated acridinium microparticles 10,18, for example, those prepared according to the affinity format method

5 may be understood. This procedure. is substantially similar to that which may be performed on an analytical instrument. The procedure may be initiated by pipetting about 50111 of activated acridinium microparticle directly on a reaction matrix in one or more reaction vessels. The particles may then be washed by pipetting about 100111 of wash solution directly on the matrix.
The reaction vessels may then be transferred to a read station.
Then, employing the read system 30, ~hPm;lllm;npqcence photon counts may be obtained. About 85 111 of an activator c..l llt; ~ln is dispensed onto the reaction tray matrix thus triggering the ,-hPm;lllm;n~ccence reaction. ~ext, net photon counts and in~:L~ "~ti:ll photon counts are each obtained from the read system 30. The net photon counts are the difference between the total activated counts and the previously recorded dark counts taken over, for example, about a 2 second read time. The net photon counts and in~L, ~l photon counts serve for comparison to chemiluminescence reactions observed by the read system 30 in order to assess performance Qf the activated acridinium particles 18.
Next ref erring to FIG . 6, certain chemiluminescence light emission profiles are shown. These light emission profiles ~have been determined for activated acridinium microparticles prepared according to the direct covalent coupling method. The profiles in FIG. 6 illustrate varying concentrations of anti-bioSin~
acridinium conjugate loading on the covalently coupled (about 0.00296) 2.511m biotinylated microparticles. Note that the profiles vary flPrPnfl~n~ upon loading, in particular, with respect to the decay rate of the ~hPm;lllm;npqcence light emission profile. Stearic hindrance effects caused by acridinium loading may cause light emission profiles of: -35 activated acridinium microparticles to be fl;qc;m;li~r to profiles 21~93iO
W095/220~9 ~ r~ 15 Of rhPm; 1 um; n~qcence reactions .
Now referring to FIG. 7A, a rh~m;lllm;n.oqcence light ~m;qq;rn profile for an anti-HBsAg assay positive sample is shown. t~nt;nll;n~ to refer to FIGS. 7A and 7B, light ~m;~g;~n profiles for the rh~m; lllm;n~qc~onr~-based anti-H~sAg a~say and a 2 . 511m affinity format labelled activated acridinium microparticle may be compared. Note the similarity of the two prof iles .
Drawing attention to FIG. 7C, the r~,om; lllm;nPqcence profiles for an affinity format labelled acridinium microparticle are shown having a decay time of approximately 1.52 seconds. Since the tail of the rh,om;ll1m;nPqcence profile resembles an exponential decay, the last about 20 to 30 ms of the approximately 1. 52 second decay time contains less t_an about 0.02% of the total rh~m;ll-m;n~scPn~-e counts. In comparison, the similar prof iles for the anti-HBsAg coated microparticles have decay times substantia31y larger than about 2 seconds. As much as about 1% of t_e total rh~m;ll1m;n~qcence counts'may be rrntA;n~d in the last about 20 to 30 ms of the same approximately 1.52 second decay time, i.e. the profile has not decayed completely. For optics calibration, for instance, a number, such as four, increasing levels of acridinium activated microparticles may be used and a slope may be obtained for each reader using .-hF~m; lllm;n~rcence counts. If an about 14 error in timing the actual chemiluminescence profile were assumed, then error in estimating the slope for a given reader may be as large as 1.24 for the anti-HBsAg coated microparticle. It is notable that, under similar conditions, the error in estimating reader slope for an affinity format labelled microparticle is only about 0 . 0234.
Next referring to FIG. 8, additional rh~m; 1 n~rcence light emission profiles are shown. These profiles show the effect of varying concentrations of acridinium loading when activated acridinium microparticles are prepared in accordance 35 with the affinity format method. These particular profiles are Wo gs/220s9 ~! 1 7 g ~

for (about 0.001%) 2.511m affinity format labelled particles.
Note that acridinium loading does not 5i(Jn;f;r~ntly affect the r~m;lllm;n~ccence light Pm;q~;r~,n profiles of activated acridinium microparticles. At each loading level (i.e., high, 5 medium, low, and 1_low), the light f~m; c:~; nn profile is approximately the same. For this reason, improved results and sirJnificant advantages are provided by particles E)repared according to the em.bodiments of the methods described in detail herein .
Now referring to FIG. 9, additional chemil~m;naqrr~re light emission profiles are shown. Each of these profiles is for activated acridinium microparticles prepared according to the affinity format method. The profiles differ only because different microparticle sizes are employed in the 15 chemiluminescence light emission measurements. ~ven given that the microparticle sizes vary, note the similarities in the r~Pm;l-lm;n~r~r~nre profiles for each of the labout 0.002%) 2.5~Lm, 3.611m, 4.511m and 6.0,Um affinity format activated acridinium microparticles, respectively These particular characteristics 20 of the activated acridinium microparticles offer significant advantages in the technology.
Now referring to FIG. 10, as previously m~nt;rnP~, the embodiments of the invention are particularly effective for~
calibrating instruments, such as chemill~m;n~cr~nre-based5 analytical instruments and the like. In FIG. 10, a bar graph c.tes the f~q;l~;l;ty of uslng different ~ t;nnc of 4.511m activated acridinium microparticle$ for optics calibration of a read system 30.
Referring now to FIGS. 11 and 12,: ' 1l;m~nt5 of the 30 invention provide advantages of enviI~ t~l and over time stability of activated ~rr;~l;n;l~- microparticles. FIG. 11 shows the approximately 2-8 degrees C stability over time of (about 0.1%) 4.511m affinity ~ormat activated acridinium microparticles.
FIG. 12 also illustrates the envi" t;il and time stability of 35 activated acridinium microparticles. As shown in FIG. 12, about Wo95/22059 2179310 ~ Q1527 45 degrees C stability over time Of 411m affinity format activated acridinium microparticles is compared with the prior art anti-HBsAg coated activated acridinium microparticles. As is readily apparent, embodiments of the invention provide significant advantages over the prior technology with respect to stability of activated acridinium microparticles.
Drawing attention to FIa 13, l~t i 1; 7At; nn of microparticles constructed according to the above-discussed Pmhnfl;r-ntq to determine pore size of an analytical instrument is explored. In an exemplary: '~n~l;~- t, the pore size determined may be physically present on a matrix cell or other read vessel having a capture element, such as a glass fiber matrix and the like.
Feasibility of using rhPm;lllm;nPAcence of 2.511m and 3.511m affinity format labelled acridinium activated microparticles for pore sizing is demonstrated. With increasing mean pore size, a decreasing trend in chPm;lllm;nPccent counts is observed.
In FIG. 14, substantial equivalence between transfer efficiency values or estimates obtained using affinity format microparticles and anti-HBsAg microparticles is demonstrated.
Transfer eff;~;.onny is the ratio of ~hPm;ll~m;n~o~npnre counts obtained by triggerir~g about 100~1 of about 0.0033% activated microparticles dispensed directly on a microparticle capture element, such as a glass fiber matrix and the like, to the nhPm;lllm;nPccence counts obtained by ~;cpPn~;n~ a similar volume of microparticles in a separate area, such as an incubation well on a reaction tray and the like. In an exemplary embodiment, the microparticles are transferred into a reaction well using about 6oo~ll of tran5fer solution, and the microparticles are triggered with about 85111 of an activator solution.
As is clearly seen from the foregoing descriptions and illustrations, the embodiments of the present invention are c;gn; f; nAnt; _ ~v~ e in the art. Those embodiments are believed to be especially effective when pc~ ' and employed as described herein, however, those skilled in the art will readily recognize that numerous variations and substitutions may Wo 95/22059 2 1 7 3 3 l;D~

be made in the described embodiments and their use and performance. Each of those variations is ;ntPnflf~fl t~ be included in the description herein.: For instance, while particular solutions and chemical compositions are disclosed, 5 other equivalent sol~lt;~nq and compositions are also possible.
The foregoing detailed description is, thus, to be clearly understood as being given by way of illustration and example only, the spirit and scol7e of the present invention being limited solely by the appended claims.

Claims (27)

What is Claimed is:

1. A method of preparing an activated acridinium microparticle, comprising the steps of:
(a) coating a microparticle with a proteinaceous compound; and (b) coupling a 10-methyl-N-tosyl-N-(2-carboxyethyl)-9-acridinium carboximide trifluoromethane sulfonate to said proteinaceous compound.

2. The method of claim 1, wherein said proteinaceous compound is bovine serum albumin.

3. The method of claim 1, further comprising the step of:
(c) activating said 10-methyl-N-tosyl-N-(2-carboxyethyl)-9-acridinium carboximide trifluoromethane sulfonate.

4. The method of claim 3, wherein said activating step (c) comprises the steps of:
(i) adding a N-hydroxy succinimide solution to said 10-methyl-N-tosyl-N-(2-carboxyethyl)-9-acridinium carboximide trifluoromethane sulfonate; and (ii) adding a 1-ethyl-3 carbodiimide solution to said 10-methyl-N-tosyl-N-(2-carboxyethyl)-9-acridinium carboximide trifluoromethane sulfonate.

5. The method of claim 3, wherein said coating step (a) comprises the steps of:
(i) suspending said microparticle in said proteinaceous compound; and (ii) centrifuging said microparticle.

6. The method of claim 3, wherein said coupling step (b) comprises the steps of:
(i) adding said coated microparticles to a conjugate buffer; and (ii) adding said 10-methyl-N-tosyl-N-(2-carboxyethyl)-9-acridinium carboximide trifluoromethane sulfonate to said conjugate buffer and said coated microparticle.

7. The method of claim 4, wherein said coating step (a) comprises the steps of:
(i) suspending said microparticle in said proteinaceous compound; and (ii) centrifuging said microparticle.

8. The method of claim 7, wherein said coupling step (b) comprises the steps of:
(i) adding said coated microparticle to a conjugate buffer; and (ii) adding said 10-methyl-N-tosyl-N-(2-carboxyethyl)-9-acridinium carboximide trifluoromethane sulfonate to said conjugate buffer and said coated microparticle.

9. A method of preparing an activated acridinium microparticle, comprising the steps of:
(a) coating a microparticle with a biotinylated proteinaceous compound; and (b) reacting said microparticle with an anti-biotin labelled 10-methyl-N-tosyl-N-(2-carboxyethyl)-9-acridinium carboximide trifluoromethane sulfonate.

10. The method of claim 9, wherein said proteinaceous compound is bovine serum albumin.

11. The method of claim 9, wherein said coating step (a) comprises the steps of:
(i) suspending said microparticle in said biotinylated proteinaceous compound; and (ii) centrifuging said microparticle.

12. The method of claim 9, wherein said reacting step (b) comprises the steps of:
(i) suspending said microparticle coated with said biotinylated proteinaceous compound in an anti-biotin labelled with 10-methyl-N-tosyl-N-(2-carboxyethyl)-9-acridinium carboximide trifluoromethane sulfonate.

13. The method of claim 11, wherein said reacting step comprises the steps of:
(i) suspending said microparticle coated with said biotinylated proteinaceous compound in an anti-biotin labelled with 10-methyl-N-tosyl-N-(2-carboxyethyl)-9-acridinium carboximide trifluoromethane sulfonate.

14. The product activated acridinium microparticle of the method of claim 1.

15. The product activated acridinium microparticle of the method of claim 9.

16. A method of performing a test on an analytical instrument, comprising the steps of:
(a) coating a microparticle with a proteinaceous compound;
(b) coupling a 10-methyl-N-tosyl-N-(2-carboxyethyl)-9-acridinium carboximide trifluoromethane sulfonate to said proteinaceous compound;
(c) activating said 10-methyl-N-tosyl-N-(2-carboxyethyl)-9-acridinium carboximide trifluoromethane sulfonate to obtain an activated acridinium microparticle;
and (d) using said activated acridinium microparticle in the test.

17. A method as defined in claim 16 wherein the using step (d) comprises (i) producing a chemiluminescent reaction.

18. A method as defined in claim 16 wherein the using step (d) comprises (i) calibrating an optical system on the analytical instrument.

19. A method as defined in claim 16 wherein the using step (d) comprises (i) determining a membrane pore size of the analytical instrument.

20. A method as defined in claim 16 wherein the using step (d) comprises (i) estimating transfer efficiency of the analytical instrument.

21. A method of performing a test on a chemiluminescence-based analytical instrument, the method comprising the steps of:
(a) coating a microparticle with a biotinylated proteinaceous compound;
(b) reacting said microparticle with an anti-biotin labelled 10-methyl-N-tosyl-N-(2-carboxyethyl)-9-acridinium carboximide trifluoromethane sulfonate; and (c) using said microparticle in the test.

22. A method as defined in claim 21 wherein the using step (c) comprises (i) producing a chemiluminescent reaction.

23. A method as defined in claim 21 wherein the using step (c) comprises (i) calibrating an optical system on the analytical instrument.

24. A method as defined in claim 21 wherein the using step (c) comprises (i) determining a membrane pore size of the analytical instrument.

25. A method as defined in claim 21 wherein the using step (c) comprises (i) estimating transfer efficiency of the analytical instrument.

26. A test element for use in an analytical instrument, the test element comprising:
(a) a microparticle coated with a proteinaceous compound; and (b) a 10-methyl-N-tosyl-N-(2-carboxyethyl)-9-acridinium carboximide trifluoromethane sulfonate coupled to the proteinaceous compound on the microparticle.

27. A test element for use in an analytical instrument, the test element comprising:
(a) a microparticle coated with a biotinylated proteinaceous compound; and (b) an anti-biotin labelled 10-methyl-N-tosyl-N-(2-carboxyethyl)-9-acridinium carboximide trifluoromethane sulfonate reacted with the microparticle.