CA2098617A1 - Simultaneous determination of multiple analytes using a time-resolved heterogeneous chemiluminescence assay - Google Patents

Abstract

Assays for the determination of multiple analytes which may be present in a test sample by using different short-lived and long-lived chemiluminescent labels and integrating the generated chemiluminescence signal and time-discriminating the short-lived and the long-lived components of the signal generated. Also provided are assay kits which contain these short-lived and long-lived chemiluminescent compounds.

Description

WO 9~/122~5 PCr/lJS91/097l4 ~ 2 ~ L 7 SIMULTANEOUS DETERMINATION OF MULTIPLE ANALYTES USING
A TIMERESOLVED HETEROGENEOUS CHEMIWMINESCENCE ASSAY

This application is a continuation-in-part application of U. S. Serial No.
071636,038, which enjoys common ownership and is incorporated hercin by reference .

BackQ~n~ of the Inv~ntiQrl This application relatss generally to chemilumin~sccnt labels and more particularly, relates to the simultaneous determination of multiple analytes using a heterogeneous chemiluminescent assay method.

The generation of light as a resuit of a chemical reaction is known in the art and has been reviewed by Schuster and Schmidt, ~Chemiluminescence of Organic Compounds~, in V. Gold and D. Bethel, eds., Ad~an~es in ~sa o~:~
18:187-238, Acad~mic Press, Naw York (19 2). The application of Chemiluminescence generation and its usa in immunoassays also is well-known in the art. See, for example, W. Rudolf Seitz "Immunoassay Labels Basa~ on Chemiluminescence and Bioluminescence,~ t~linical Ch~rni~try 17: 120-126 (1 984) The use of acridinium compounds as labels for immunoassays and the subsequent generation of short-lived cherniluminesc~nt signals from these labels has 2 5 been described by 1. Weeks et al., "Acridinium EsSers as High Specific Activity Labels in Irnmunoassays,~ ~lini~L~h~ 29:1474-147~ (1984). The use of stable acridinium sulfonamide esters and phenanthridine compounds have been described in pending United States Patent Application Serial No. 921,979 filed October 22, 1986, which enjoys common ownership and is incorporated herein by reference.
The generation of long-lived luminescent signals has been described in the art as resulting from action of enzymes or nucleophilic agents on dioxetane cornpounds containing an adamantane structure. See, for example, US 4,962,192, published EPO Application No. EP 0-254-051-A2 to A. P. Schapp; published PCT application No. WO 881 00694 (WO 8906650) to 1. Bronstein; I. Bronstein et al., "1,2-Dioxetanes, Novel Chemiluminescent Substrates, Applications to Immunoassays, "InProceedings of the Vlh International Conference on Bioluminescence and Chemiluminescence," Florence-Bologna, Italy, September 25-28, 1988 and also in 1~:t WO 92/1225~ 2 0 ~ 7 PCr/USgl/09714 2 ~
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the Jollrn~ cenc~ and ~emi~m~s~4:99 (1988), and US patent 4,950 ,593.

Triggering chemiluminescent reactions in a porous matrix and the subsequent 5 detection of signals resulting from immobilizsci microparticlas is described in co-pending United States Patent Application Serial No. 206,645, which enjoys commonownership and is incorporated herein by refarence. Thc use of an ion capture separation mathcd with chemiluminescent detection in a porous matrix is described in co-pending United States Patent Application Serial No. 425,643, which enjoys 10 common ownership and is incorporated herein by reference.

The ability to perform simultaneous testing for multiple analytes In the same sample would offer sev~ral advantages. First, it would allow obtaining multiple assay results for the same sample processing time, ~hus it would increase the 15 through-put of automated instruments. Sacond, it would dscrease the cost per test, ber,ause the same position of a disposable test device would b~ used for several test results. Third, it would decrease tha amount of volume of used disposabla test devices and therefore, the amount of biohazard waste material. Fourth, it would allow simultaneous assays for analytes such as viral antigens or antibodies to viral 2 0 antigens, thus offering better screening tests. Finally, it would allow simultaneous assays for differ0nt analytes such as drugs or for a drug and its metabolites, thus increasing the predictive value of th0 assay, which in turn would lead to more accurate diagnosis.

2 5 The simultaneous detection of multiple anaiytes using chemiluminescencecould not be achieved heretofore because of the limited number of molecular species that could be chemically excited; only limited emission wavelengths were available.
Also, the selectivity of excitation wavelengths such as in the case of fluorescence detection, where it is possible to excite two independent species, does not appiy to chemiluminescent measurements. Finally, the majority of light-generating chemical reactions are not compa!ible with each other in terms of reaction conditions.

The present invention overcomes the problems of existing methods by 3 5 allowing the simultaneous determination of analytes in a chemiluminescent assay by using time resolutions of the light emissions generated under the same reaction oonditions. The present invention provides a novel method in which the simultaneous measurement of analytes in a compe~itive or in a sandwich assay are achieved. This WO 9~ 255 PC~/US91/09714 ~, . 20~61~'7 in turn improves the predictive value of the assay and leads to more accurate diagnosis.

5 sum~m.~rY of the lnve~iQn The pressn~ invention provides a method for the datermina~ion of multiple analytes which may be present in a ~est sample comprising (a) incubaRng Ihe tcstsample with a mix~ure of members of specific bindin~ pairs attached ~o a solid phase for a time and under conditions suffici~nt for analyte/antl-analyte spacific binding 10 pairs to form; (b) incubating with the so-forrned specific binding pairs a mixture of labeled members of specific binding pairs for each analyte wherein each analyte is bound to a different chemiluminescent compound (label) capable of ~enerating a different short-lived or iong-lived chemiluminescant signal upon c~ntact with a triggering solution; (c) triggering the signal with a tri~g~ring solution; (d) 15 mcasuring the chemiluminescent signal detected; and (e) determ3ning the presence and the amount of each analyte prssent in th0 test sample by calcu~ating the difference in ~ime-profile of the signals ~eneratQd from the chemiluminescsnt compounds (labels). The solid phasa can includ~ a susp~nsion of microparticles comprising a mixture of groups of particles, each group having attached ther~to a 0 member of a speclfic binding pair for one analyte, a tube coate~ with a mixture of members of specific binding pairs for the analytes, a su~pension of magnetizableparticles c~mprising a mixture of groups of particles wherein each group of particles has attached thereto members of a specific binding pair for an analyte, a plastic baad coated with a mixture of m&mbers of specific binding pairs for the 2 5 analytes and a derivatized membrane having attached th~reto by chamical binding members of specific binding pairs for the analytes which cover all the membrane or discrate regions of the membrane. The melhod can ~urther somprise a separation step. If the solid phase comprises analyte/anti-analyte specific binding pairs attached to microparticles, microparticle separation on a porous element and 3 0 washing said solid phase can be performed. If the solid phase comprises magnetizable particles, the separation step can be performed by magnetic separation.

The present invention also provides a method for performing a simultaneous determination of multiple analytes in a test sample which may contain said analytes 3 5 using a competitive binding chemiluminescence assay comprising (a) incubating the test sample with a known amount of chemilurninescent labeled analytes each capable of generating a short-lived and long-lived chemiluminescent signal and a solid phase which has a limited amount of a mixture of members of specific binding pairs for SUBSTIT~JTJE $H~E~

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said analyte attached thereto for a time and undar conditions sufficien~ for analyte/anti-analyte specific binding member, to form; (b) adding a substrale specific for ona of the labels and incubatin~ to allow a lon~-lived chemiiuminescance-generating reaction to occur; (c) tri~gering the resultant mixture with alkaline peroxide; and (d) inte~ratin0 and time-discriminating the short-lived and the long-lived componen~s of th~ chamiluminesc~nce signal ~nerated. The analytes include haptens, macromolecules, ma1abolites and antibodies. In yet anothsr format for a simultaneous determination of multiple analytes in ~ test sample which may cvntain th~ analytes usin~ a competitive binding 10 chemiluminescsnce assay, the assay oomprises (a) incubating the test sample with a known amount of chemiluminescent labeled specific binding pair membcrs each member capable of generating either a short-lived or a long-liYed chemiluminescent signal and a solid phase which has a mixture of analyta derivatives attached thareto, for a time and under conditions sufficient for analyte/anti-analyte spacific binding 15 pairs to form; (b) adding a substrate specific for one of the labels and incubating to allow a long-lived chemiluminescence-generating reaction to occur; (c) tri~gering the resultant mixturs with a triggering solution specific for the other label; and ~d) integrating and time-discriminating the shorl-livad and the lon~-lived componerlts of the chemiluminescence signal genarated.
The present invention further provides a method for performin~ a simultaneous chemiluminescence assay for any of multiple analytes which may be present in a test sample, comprising: (a) incubating the test sample with a solid phase coated with a mixture of members of specific binding member pairs for the 25 analytes to form analyte/anti-analyte specific binding pairs; (b) separating the solid phase; (c) adding a mixture of members of specific binding pairs for the analytes having attached thereto different chemiluminescent compounds (labels) capable of generating a different short-lived or long-lived chemiluminescent signal upon contact with a triggering solution and incubating same; (d) aWing a substrate to 3 0 one of the labels and incubating same to allow a bng-lived chermiluminescence generating reaction to proceed; (e) triggering the resultant mixture with a triggering soluUon; and (f) integrating !he generated chemiluminescence signal an time discriminating the short-lived and the long-lived components of the signal generated. The analytes which can be tested include infectious dissase antigens,3 5 hormones, cancer markers and DNA probe sequences.

The present invention additionally provides a method for the determination of multiple analytes in a test sample which may contain any of the analytes, SUIBST17'U~E SHE~

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. 5 comprising: (a) incubating the test sample with mixtur~ o~ members of specific binding pairs of aach analyte attached to polymeric ionic molecules; (b) adding a mixture of chemilumin~scent labeled members of specific binding pairs for each analyte wherein sach analyt~ is bound to a differ~nt chemiluminescent compound (labels) capable of generating a different short-lived or long-lived chemiluminescent signal upon contact with a triggering solution and incubating same to form a reaction mixture of analyte/anti-analyte specific binding pairs; (c) transferring the reaction mixture to a porou; membrane treat0d with a polymeric cationic compound; (d~ triggoring a chemiluminescant signal wlth a tri~gering solution; (e) detecting 1he chemiluminescent signal ~enerated; and (f) determining the presence and the amount of each analyte from tho dlfference in time-profile of the signals ~enerated from the chemiluminescent compounds ~labels). Thc analytesinclude haptens, macromolecules, metabolites, anti~odies, Infectious disease antigens, hormones, cancer markers and DNA probe sequences.

The present invention also provides a kit for performing a simultaneous determination of two analytes comprising containers containin~ membsrs of specific binding pairs ~or each analyte wherein each analyte is bound to a different chemiluminesccnt compound (label~ capabla of generatin~ a differ~nt short-lived or long-lived chemiluminescent signal upon contact with a triggering solution. The short-lived chemiluminescent compound (label) in the kit is an acridinium sulfonamide compound (label); and the long-lived chemiluminescent compound is analkaline phosphatase substrate or a 13-galactosidase substrate.

Detail~ Description oflhe~lnyention The chemiluminescent properties of acridinium compounds ~labels) and their use for immunoassays has been described. Immunochemical tracers with acridinium esters or acridinium sulfonamide levels can be triggered with an alkaline peroxide 3 0 solution to produce a chemiluminescent signal that maximizes after approximately two ~2) seconds. I ight emission is completely extinguished after approximately ten (10) seconds. Acridinium sulfonamide labeling chemistry may be amployed a~ording to this invention for making a stable tracer of high quantum yield. Such chemistry is described in co-pending U.S. Patent Application Serial No. 371,763 enti~led ~Chemilllminescent Acridinium Salts fileci June 23, 1989, which enjoys common ownership and is incorporated herein by reference.

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Chemically catalyzed long-lived cherniluminesccnt 1,2-dioxetanes can be generated in a variety of ways. Thus, a siloxy-substituted dioxetan~, 4-(6,tert-butyldimethylsiloxy-2-napthyl)-4-methoxyspiro[1 ,2-dioxetane-3,2 adamantane]
is triggerable with tetrabutylammonium floride solution to produce a 5 chemiluminescent signal lasting for 20 minutes. Also, enzymes such 3s aryl esterase, alkaline phosphatase or B-~alactosidase react with aryl dioxetane derivatives stabilized with an adamantane ca~e to produce similar long-lived chemiluminescent si~nals. Further, lon~-lived emissions from alkaline phosphatase catalyzed reactions of 3(2-spiroadamantane)-4-methoxv-4-(3-10 phosphoryloxy)-phenyl-1,2-dioxetane (AMPPD) and of a similar 13-galactosidasesubstrate have been described in WO 881 00694, along with the use of these compounds (labels) in an immunoassay. Alkaline phosphatase and B-~alactosidase labeling techniques are known, and catalyzed dioxetane chsmiluminescence can be used according to this invention to generate long-lived signals.

According to the invention, a short-lived chemiluminescont labal can be any member of the acridinium and/or ph~nanthridinium compounds (labels) or any chemiluminescent compound (label), as long as it is capable of generating a short-lived signal. Luminol derivatives also can be used with the chemiluminescent 2 0 generating reaction performed. at a triggering condition of pH and catalysts, in such a way that the reac~ion is triggered and decays in a few seconds.

A long-lived chemiluminescent compound (label) according to this invention can be one of the c ass of dioxetane compounds (lab~ls) that can reacS with enzyme 2 5 labels such as alkaline phosphatase or B-yalactosidase to generate chemiluminescent signals of long durations. It is also contemplated that any compound which can be configured to generate a long-lived si~nal, such as luminol, or any compound which generates a long-lived signal can ~e used. Alkaline phosphatase catalyses the decomposition of AMPPD at pH 8-9 to generate a chemiluminescence signal of long 3 0 duration. Increasing the pH to ca. 12 enhances the generation of tha signal. ~-galactosidase ca~aiysis the decomposition of AMPGD at pH 8. The resultant intermediate is rendered chemiluminescent by raising the pH of the solution to ca.12. Under these conditions a lonQ-lived chemiluminescence signal is generated.

3 5 A trigger solution according to this invention is an alkaline peroxide solution.
This trigger solution can be prepared by the addition o~ hydrogen peroxide to a sodium hydroxide solution or by dissolving solid urea-hydrogen peroxide adduct in sodium hydroxide.

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The alkaline peroxide trigger solution, according to this invsntion, acts on th0acridinium label to generate a shorl-lived ch~miluminflscence signal due to the decomposition of a high energy intermediate ~nd the ~eneration of an excited acridone 5 derivative. Alkalina peroxide, also according to this invention, enhances the enolization 9~ the enzyme reaction product and hence, the intonsity of the chemiluminescence signal. This appaars as a pHjum~like change in the signal.
Alkaline peroxide also according to this invention, reacts with tho non-luminescent reaction product of 13~alactosidase with AMPGD to generat0 ~ chemiluminescence 1 û signal of long duration.

A solid phase according to the present invention is a mixture of polymeric microparticles with chemically or physically bound antigens or antibodies.
Microparticles that can be used includ0 polystyrene, carboxylated polystyrene, 15 polymethylacrylate or similar particles with radius in Ihe ran~e of from a~out 0.1 to 20 ,urn. A preferred separation mathod for these particles is the use of microparticle cap2ure on a glass fiber matrix followed by triggering a chemiluminescent reaction on this matrix.

2 0 Another preferred method of separation is that which is described in r,o-pending U.S. Patent Application Serial No. 150,278, and U. S. Patent ApplicationSerial No. 375,02g, both of which enjoy common ownership and both of which are incorporated herein reference. These applications describe the use of ion capture separation, in which the antibodies or antigens for the assay in question are 2 5 chemically attached to polyanionic acids such as polyglutamic acid or polyacrylic acid. The fibrous pad is treated with a cationic polymer to render the fibers positively charged. Separation of the immunochemical reaction products are affected by the electrostatic interaction be2ween the positively charged pad and the negativeiy charged poly-anion/immune complex.
Other solid phases that can be used include a mixture of magnetizable polymeric microparticles with chemically or physically bound antigens or antibodies. Magnetizable microparticles that can be used preferably have ferric oxide or chromiurn oxide cores and pclystyrene, carboxylated polystyrene, 3 5 poiymethylacrylate coating. A preferred separation method for these particles is the use of constant or pulsed magnets, washing said particles, and then susp~nding the separated particles in a vessel where a chemiluminescent reaction can be generated and detected. Yet other solid supports are known 20 those in the art and include the - S~JB~ITUTE: ~;HEET

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WO 92/122S5 PCI'/US91/Og714 2~9"'6 ~7 walls of walls of a reaction tray, test tubes, polystyrene beads, nitrocellulose strips, mernbranes, and others.

Polymeric microparticles with chemically or physically bound anti~ens or 5 antibodies can be used according to the inv0ntion as capture phases in a binding reaction to make use of the fast diffusion rates of thase parlicles in solution to yield rapid results. Microparticles that can be used accordin~ to this invantion include polystyrene, carboxylated polystyrene, polyrnethylacrylate or similar particlas with radius in Ihe range of frorn about 0.1 to 20 ~lrn.

Test samples which can be tested by the nnethods of the present invention described herein includa biological fluids such as human and animal body fluids.Thus, whole blood, serum, plasma, cerebrospinal fluid, urine, as wall as cell culture supernatants, and lha like may b~ used.

"Analyte," as used herein, is the substance to be detscted which may be present in the test sample. The analyte can be any substanc~ for which there exists a naturally occurring specific binding rnember (such as, an antibody), or for which a specific binding membsr can be prepared. Thus, an analyte is a substance that can 2 0 bind to one or more specific bindin~ membars in an assay. nAnalyte" also includes any antigenic substances including infsctious diseasa antigens such as viral, bacterial, rickettsial antigens and also cancer markers such as CEA, also macromolacules, haptens and/or thcir metabolites, antibodies, and combinations thereof. As a mamber of a specific binding pair, the analyte can be detected by means 25 of naturally occurring specific binding partners (pairs~ such as the use of intrinsic factor protein as a member of a specific binding pair for the determination of Vitamin B~2, or the use of lectin as a member of a specific binding pair for thedeterminalion of a carbohydrate. The analyte can includ~ a protsin, a peptide, an amino acid. DNA or RNA probe sequences, a hormone, a steroid, a vitamin, a drug 3 0 including those administered for therapeutic pur,ooses as well as those administered for illicit purposes, a bacterium, a virus, and metabolites of or antibodias to any of the above substances. The details for the preparation of such antibodies and thesuitability for use as specific binding members are well-known to lhose skilled in the art.
A ncapture reagentn, as used herein, refers to an unlabeled specific binding member which is specific either for the analyte as in a sandwich assay, for the indicator reagent or analyte as in a competitive assay, or for an ancillary specific $U~;TITIJTE SIHEE~T

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binding msmber, which itself is specific for the analyte, as in an indirect assay. The capture reagent can be directly or indirectly bound to a solid phase material before th~ performanca of the assay or during the p~rformance of the assay, thereby enablin~ the ssparation of immobilized complexes from ~he test sample .

The "indicator rea~entU comprises a si~nal ~Qnerating compound (la~l) which is capable of ~eneratin~ a maasurable si~nal detectabie by external means conjugated (attached) to a specific bindin~ rnembor for th~ ~naiyta(s). "Specific bindin~ membar~ as usad herein means a mem~r of a specific binding pair. That is, 10 two different molecules where one of the molacules throu~h chemical or physical means speciflcally binds to the second molecule. In addition to boing an antibody member of a specific binding pair for for th~ analyte, the indicator reagent also can be a member of any specific bindin~ pair, including eithsr hapt~n-anti-hapten systems such as biotin or anti-biotin, avidin or biotin, a carbohydrata or a lectin, a 15 complementary nucleotide sequence, an effector or a receptor molecule, an enzyme cofactor and an snzyme, an enzyme inhibitor or an enzyme, and th~ like. An immunoreactive specific binding member can be an antibody, an antig~n, or an an~ibody/antigen complex that i5 capable of bindin~ either to an analyte as in ~sandwich assay, to the capture reagent as in a competitive assay, or to tha ancillary 2 0 specific binding mamber as in an indirect assay.

The various signal generating compounds (labels) contsmplated in the practice of the invention include short-lived chemiluminesc~nce signal yenerating compounds (labels) such as an acridinium sulfonamide, an acridinium ester or a 25 phenanthridine compound (labsl), and lon~-livad chemiluminescenco signal generating compounds (labels) whose signals result from action of enzymes or nucleophi!ic agents on dioxetane compounds containing an adamantane structure.

It is contemplated tha~ the reagent(s) employed for the assay can be provided 3 0 in the form of a kit wilh one or more containers such as via~s or bottles, with ~ach container containing a separate reagent such as a monoclonal anti~ody, or a cocktail of monoclonal antibodies, employed in the assay as capture phases or as indicator reagents which comprise chemiluminescent compounds (labels) as signal generatingoompounds.
According to a method of this invention, a test sample which may contain any or all of the analytes of interest, a mixture of probes for the analytes labeled as described herein and a mixture of capture phases for the analytes are incubated for a S19B5T~TU!TE SlHEEr WO g2/12255 P(:~/US91/09714 2 a 9 3 ~ ~ 7 1 0 ~

periocl of lime and undet conditions sufficient to allow optimal immunochernicalbinding reactions for the analytes to take place. The oapture phase then is separated and washed. A substrate specific to the alkaline phosphatase label then is adcled. This substrate is rendered chemiluminescent by the action of th~ enzyme lab~l. Atter the 5 onzyme/substrate rcaction reaches an end point, the separated reaclion mixture is trig~ered using an alkaline peroxide solution. The signal is collected over a period of about four to ten seconds and is integrated to çlive tha sum of the pHjump 0nhanced alkaline phosphatase catalyzed chamiluminesclsnco and the alkaiine ,oeroxide triggered acridinium sulfonannide chemiluminescerlcfl. The rasidual signal is 10 collected and integrated for an equal time Interval to giv~ the steady state en2yme oatalyzed chemiluminescence. If two analytes arc tesled, tho presence or absence of either of the two analytes is determined from the relative magnitude of the signals collecled at the two time intervals.

In an alternative way of performing the assay method of this invention, a test sample which rnay contain any or all of the analytss of intorest, a mixture of probes for the analytes labeled as describe~ herein and a mixturo of capture phases for the analytes are incubated for a period of time and under conditions sufficient to allow op~imal immunochemical binding reactions for the analytes to take place. The 2 O capture phase then is separated and washed. A substrate specific to the alkaline phosphatase label then is added. This substrate is rendered chemiluminescent by the action of the enzyme label. After the anzyme/substrate reaction reaches an end point, the ~enerated chemiluminescence signal corresponding to the enzyme label is integrated. The separated reaction mixture is then tri~gered usin~ an alkaline 2 5 paroxide solution. The signal is collected over a period of a few hundred milliseconds and corresponds to the alkaline peroxide triggered acridinium sulfonamide chemiluminescence. If two analytes are tested, the presence or absence of either of the two analytes is determined from the relative magnitude of the signals collected at the two time intervals.
Yet in an alternative method of this invention, a test sample which may contain any or all oi the analytes of interest, a mixture of probes for the analytes labeled as described herein and a mixture of capture phases for the analytes areincubated for a period of time and under conditions sufficient to allow optimal 3 5 immunochemical binding reactions for the analyles to take place. The capture phase then is separated and washed. A substrate specific to the B-galactosidase label then is added. This substrate is hydrolized by the action of the enzyme label. After the enzyme/substrate reaction reaches an end point, the separated reaction mixture is . . . -., . - . .

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triggered using an alkaline peroxide soiution. The signai is oollected over a perio~ of about one second and is inte~rated to ~ive the alkaline p~roxide triggered acridinium sulfonamide chemiluminescence. The residLIal signal Is coll~ctQd after a dslay of four to fiYe seconds and integrated for an equal time interval to ~ive the steady state 5 enzyme catalyzed product that is rsndered chemiluminescent by ths action of alkaline peroxide. If two analytes are tested, the presence or absence of either of the two analytes is determined from the relative magnitude of the signals collected at the two time intervals.

A microparticle-based one-step sandwich immunoassay for muitiple analytes is performed according to this invention, as follows: A t~st sample which may contain any or all of the analytes of interest is contacted with a mixture of microparticles comprising two groups of particles, one group coated with a polyclonal antibody to the first analyte of interes1, and the second ~roup of particles 1~ coated with a polyclonal antibody to tha second analyte of interest. A conjugate mixture of labeled antibodies then is added to the reaction vess~l. One antibody in this mixture is specific 7Or the first analyte and is labeled with (bound ~o) ~ short-lived chemiluminescence signal g~nerating compound (label) such as ~n acridiniumsulfonamida, an acridinium ester or a phenan~hridine compound. The second antibody 20 of lhe conjugate mixture is labeled wi~h alkaline phosphatase. The mixture isincubated for a time and under conditions sufficient for analyte-specific complexes, comprising analyte and anti-analyte antibodies, to form. Then, the microparticles are separated and washed. A prsferred method of separation is one which ~mploys a glass fiber pad, using the fluid removal method described in co-pending U. S. Patent Application Serial No. 07/184,726, which anjoys common ownership and is incorporated herain by reference. An alkaline phosphatase substrate, such as that described in published EP 0 254 051 or WO 881 00694, capable of generating a long-lived chemiluminascence signal when reacted with the subslrate, ~hen is added to the separated particles and is allowed to react for a time and under conditions 3 0 sufficient to approach steady state. This generally takes only a few minutes. The microparticles and substrate reaction product then is triggered with an alkalineperoxide solution in an apparatus such as that described in co-pending U. S. Patent Application 07/2G6,645.

3 5 Another method of the invention comprises contacting the test sample which may contain either or both of ~he analytes of interest with a mixture comprising two groups of microparticles. One group of microparticles-is coated with a monoclonal antibody which specifically binds to an epitope of the first analyte, and the second $~Ji3$TlT~JTE SHIE:E~

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WO 92/1225~ P~/US91/09714 203~17 ~2 (~ 1' 0roup of microparticl~s is coated with a monoclonal anti~ody which specincally ~inds to one epitope of the sacond analyte. A conju~ate mixture of label~d antibodies is added into the reaction vessel. One antibody of ths conju~at~ mixtura is specific to the first analyte and is labaled with a short-lived chamilumlnescenca signal ~eneratin~ label such as an acridinium sulfonamide, an acridinium ester, or a phenanthridine compound ~label). The second antibody of tha conjugate mixture isspecific for the second analyte and is labeled with alkaline phosphatase. The mixfure is incubated for a time and under conditions sufficient to form analyte/anti-anaiyte/anti-anti-analyte complexes. Then, th~ micropart~les are separated and 1 0 washed, and the chemiluminescence signals triggered and measured as described hereinabove.

In another variation of the method described hereinabove, the microparticles are coated with polyclonal antibodies and the conjugate is a mixture of labaled 15 polyclonal antibodies.

A microparticle-based two-step sandwich immunoassay for lwo analytas can be perforrned accordin~ to the invention, as follows. The test sample which may contain either or both analytes of interest and a mixture of microparticles 2 0 comprising two groups of particles, one group of particl~s coated with a polyclonal antibody specific to the first analyte, and the second group of particles coated with a polyclonal antibody to the second analyte, are contacted. The test sampls and microparticles are incubated for a tima and under conditions sufficient to allowbinding of the analytes to the particles. Then, the microparticles are separatad and 2 5 washed. A preferred method of ssparation is on a ~lass fibor pad, using the fluid removal method described in co-pending applicalion Serial No. 184,726 previouslyincorporated herein by reference. A conjugate mixture of labeled antibodies is added to the separated particles on the pad. One antibody in the conjugate mixture is specific to the first analyte and is labeled with a short-lived chemiluminescent3 0 generating label such as an acridinium sulfonamide, an acridinium ester or aphenanthridine compouncl (label). The second antibody of the conjugate mixture is specific for the second analyte and is labeled with alkaiine phosphatase. This conjugate mixture is allowed to incubate with the separated microparticles for atime and under conditions sufficient for complexes to form. Th~ excess conjugate3 5 mixtura then is removed by washing. An alkalin~ phosphatase substrate which is capable of producing long-lived chemiluminescence signal when reacted with the enzyme then is added to the separated particles. This mixture is allowed to react for a time and under conditions sufficient to approach steady state and thus to form a SU~SrlTUTE ~ ~

WO 92/12255 P~/US91/09'714 2 ~ 9 ~ 6 ~. ~

substrate reaction product. The amount of time to reach steady state is usually only a few minutes. The microparticle and substrate reaction pr~duct thsn is triggered with an alkaline peroxide solution in a reaction Yessel such as that clescribed in co-panding U. S. Patent Application Serial No. 206,645 previously incorporatsd herein 5 by refersnce.

In yet another embodiment of the invention, th~ t~st sample which may con~ain any of the analytes of interest and a mixture comprisin~ two groups of microparticles, one ~roup of particles coated with a mon~clona3 antibody to an 1 0 epitope of the first analyte and tha second ~roup of particles coated with a monoclonal antibody to an epi~ope of the second analyt~, is contacted to form a mixture. The mixture is incubated for a time and under conditions sufficient for analyte/anti-analyte antibody complexes to form. Then, the microparticles ara separated and washed. A conjugate mixture of labeled antibodies then is added to the so-formed15 complexes. One antibody of the conJugate mixture is specific to the first analyte and is labeled with a short-liYed chemiluminescence-generating label such as an acridinium sulfonamide, an acridinium ester or a phenanthridine compound (label).
The second antibody of the conjugate mixture is speciflc for the ssoond analyte and is labeled with alkaline phosphatase. After incubation for a time and undar conditions 2 0 sufficient for the conjugate to bind to either or both analyte/anti-analy~e complexes, the excess conjugate mixture is washed off and the chemiluminescence signals aregenerated and measured.

Alternatively, the microparticles can be coated with polyclonal antibodies and 2 5 the conjugate can ~e a mixture of labeled polyclonal antibodies.

lon capture procedurPs for immobilizing an immobilizable roaction complex with a negatively charged polymer, described in co-pending U. S. Patent Application Serial No. 150,278 filed January 29, 198~, which enjoys common ownership and 3 0 is incorporated herein by reference, can be employed according to the presenl invention to effect a fast solution-phase immunochemical reaction. An immobilizable immune complex is separated from the rest of the reaction mixture by ionic interactions between the negatively char~ed poly-anion/immune complex and the previously treated, positively charged porous matrix and det~cted by using 3 5 chemiluminescent signal measurements as described in co-pending U.S. Patent Application Serial No. 425,643, previously incorporated herein by reference.

WO 92/12255 P~/US91/09714 20~6~

An ion capture-based competitivs chemiluminescent immunoassay for two haptens can be performed according to this invention, as Follows. The tast sample which may contain any of the analytes of interest is contacted with a mixture oflabeled antibodies. One antibody in the mixture is specific to the first hapten, and is 5 labeled with an acridinium sulfonamide, an acridinium astar or a phenanthridine compound (label). The second antibody of thls mi~tur~ is specific for the secbndhapten and is lab~led with alkaline phosphatase. The test sample and mixtur~ of labeled antibodies are incubated for a tim0 and under conditions sufficient for hapten/anti-hapten complexes to form a reaction mixturo. Th~n, a mixture of 10 capturs phase is acidsd to the reaction mixture. The cap~ure phase mixlure comprises a mixture of two components each of which is a hapten bound to polyglutamic acid residue. The binding can be accomplish~ either directly or through a carrier.

Another alternative way of performing these assays according to the pres0nt invention is to use a combination of microparticle capture and ion capture separation procadures. Thus in a two-step sandwich assay, a sample susp~ted of containing multiple analytes can be incubated with a mixtura of microparticles having boundthereto a member of specific binding pair for one analyte, and polyionic residues 2 O having bound thereto members of speciflc binding pairs for the sr,~cond analyte. The reaction mixture is then transferred to a porous matrix that has been treated tocarry an opposite charge to the polyionic residue. Tho microparticles captured analyte is retalned on the porous matrix by hydrophobic interactions and the poly-ionic captured analyte is retained on the oppositely charged porous matrix by ionic 25 interaction. The retained reaction products are washed and a mixture of conjugates is added on the porous matrix. One of these conjugates is labeied with a label that can be triggered to yield a short-lived chemiluminescence signal, the other is labeled with a label that yields a long lived chemiluminesce signal. The signals are triggered and segregated by time resolution.
The invention will now be described by way of examples, which are meant 2O
illustrate, but not to limit, the spirit and scope of the invention.

~;UB~Tl~uT~ E~7 ; - .. . . , , . , .. ~ . .. .

wO 92/12255 2 ~ 1 7 Pcr/usgl/~g714 EX~MPLES

Example. L ~~

The phencyclidine (PCP) capture raagent was prepared by coupling the free acid form of polyglutamic acid with phenyicyclidine-4-chloroformate. Tha fr0c acid form of polyglutamic acid was prcpared from the sodium salt of polyglu~amic acidaccording to the following procedure: 1 gm of polyglutamic acid sodium salt (Si~ma Chemical Company; St. Louis, MO) was stirred overnight with 7 gms of AG50W-X8 10 cation exchangs resin (Bio-Rad Laboratories, Richmond, CA) suspend~ in 20 mL
water. The ion exchange resin was previously swellad and washed in distilied water.
The sup4rnatant was ssparated from ths resin and Iyophilized to give a~out 0.8 9 of the frse acid form of polyglutamic acid.

PCP-4-chloroformate was prepared by reacting 1.1 mg 4-Hydroxyphenylcyclidine (4.24 x 10-6 moles) in 0.5 mL tetrahydrofuran with 0.5 mL of 10% solution of phosgene in benzen~ (130 mole excess). Th~ reaction was allowed to proceed for 2.5 hours at room temparature. Solvents were evaporated under a stream of nitrogen to yield a residue of PCP-4-chloroformate. Tha residue 2 0 was dissolved in 0.5 mL tetrahydrofuran and 1.7 mg of the fres acid form of polyglutamic acid (MW 40,000) in 0.5 mL of 1-Methyl-2-Pyrrolidinone (Aldrich Chemical Co., Milwaukee, Wl) was aWed to it. The r~action was carried out overnight at room temperature and then the reaction mixture was evaporated to dryness. The dried product was dissolved in 13 mL 0.1 M sodium phosphate buffsr,pH 7.0, and was dialyzed against a volume of ~he same buffer in a 3,500 molecular weight cut-off dialysis bag overnight at room ternperature. The pr~cipitate was filtered. The cloudy aqueous filtrate was extracted with methylene chloride (Fish~r Scientific, Itasca, IL) until it was clear.

3 0 A fluorescence polarization immunoassay confirmed the presence of PCP onthe PGA residue (performed on Abbott TDX~9, available from Abbott Laboratories, Abbott Park, IL 60064). Ths aqueous layer was diluted in a solution containing 1%
fish gelatin, 100 mM sodium chloride, 25 mM Tris, 1 mM magnesium chlorida, 0.1 mM zinc chloride, 0.1% sodium azide, pH 7.2, to yield 1.875 llgm~mL of PCP-PGA
capture reagent.

The morphine capture reagent was prepared by coupling the iso~hiocyanate derivative of ~he free acid form of polyglutamic acid with morphine-ovalbumin. The _.
SU~5TITUTE ~HEET

WO 92~12255 PCr/US91/09714 2 a ~ 7 ~ l free acid form of polyglutamic acid was activated by derivatizing it to an isothiocyanate (ITC-PGAFA). The isothiocyanate deriva~ive was reacted wi~h ovalbumin to form ovalbumin-PGA. Finally, morphine was coupled to ovalbumin-PGA using isobutylchloroformate to yield the capture reagent: morphlne-ovalbumin-PS3A.

The procecure used was as ,'ollows. Ten (10) m~ of tha fras acid form of polyglutamic acid was dissolved in 1 ml dimethylform~mide (DMF). 0.01 ml of proton adsor~ing agent 4-mathylmorpholin~ and 4.8 m~ ot 1,4-phenylane diisothiocyanats (DlTC)(available from ths Aldrich Chemical Co., Milwaukee, Wl) in 0.2 ml of dimethylformamide were added to this solution (100 mole axc~ss). This reaction mixture was stirred at room tsmperature overnight and th~n it was concentrated using a rotary evaporator. Tw0nty-five (25) ml of methylene chloride was added dropwise to precipitate the ITC-PGAFA. The Slocculent precipitate was centrifuged, and methylene chloride and unreacted DiTC were de~anted. The precipitate then was suspended in 1 ml of dimethylformamide. The precipitation/centrifugation/suspension procass was repeatsd until no DITC was detectable in the supernatant using thin-layer chromatography (TLC) on silica slides. The remalning solid was vacuum dried to yield the ITC-PGAFA as a yeilow 2 0 powder.

Ovalbumin-PGA was prepared according to the following procsdure. Ten (t0) mg ovalbumin (available from Sigma Chcmical Co., St. Louis, MO) were dissolved in 0.5 ml of 0.2 M sodium phosphate buffer (pH 8.5) and filtered through a 0.45 ~Lm syringe filter. This solution was reacted with 133.3 mg of ITC-PGA
dissolved by sonication in 3 ml of 0.2 M sodium phosphate buffer at pH 9Ø The pH
then was adjusted to 8.5 with 1 N sodium hydroxide. Th~ mixture was incubated overnight at 37C, and then it was fractionated on an HPLC instrument using a TSK-3000SWG preparative gel filtration column (availabie from Beckman Instruments, 3 0 Arlington Heights, IL) run at S ml per minute with 0.1 M sodium phosphate, 0.3 M
sodium chloride ~pH 6.8). Fractions were monitored using an absorbance detector at 280 nm. Two (2) ml fractions were cu~ and six (6) fractions (12 ml~ were grouped starting with the void volume. 6.5 ml of the second fraction of ovalbumin-PGA containing 77.18 llg/ml ovalbumin were dialyzed against a volume of 0.1 M
sodium bicarbonate, pH 8.5, in a 3,500 rnolecular weight cut-off dialysis tube.

One (1) mg morphine 3-B-D-glucuronide (Sigma Chemical Co., St. Louis, MO) MW 461.5, was activated by reacting it with 100 mole excess $U1~5TJTYTE 8HEET

.. . , . I , WO ~2/1~255 2 0 ~ `3 ~ ~ 7 PCI/US91/09714 ,,.
~ 7 isobutylchloroformate (MW 13~.15, available ftom Sigma Ch0mical Co., St. Louis, MO) and 100 mole excess 4-methylmorpholine in DMF at 0C for one-half hour.
The activated morphine derivative was added to the dialyzed ovalbumin-PGA and k~pt in an ice bath for one (1) hour at 0-4OC. The reac~ion mixture then was kept at 5 room temperature overnight. The procluct solution was dialyze~ a~ainst 0.1 M
sodium phosphate buffar at pH 8.5, in 3,500 molecular woi~ht cut~fl dialysis bagto remove the uncoupled morphine. The reoovered dialysate was run in the fluorescence polarization immunoassay for morphine, usin~ a ~ommercial analyzer (TDX'~91 Abbott Laboratories, North Chica~o, IL), which ~ave 3.5 morphine 10 molecules per ovalbumin-PGA residue. The capture roagent (morphine-ovalbumin-PGA) was diluted in a buffer solution containin~ 1% fish gelatin, 25 mM Tris, 100 mM sodium chloride, 1 mM magnesium chloride, 0.1 mM zinc chlorWe and 0.1%
sodium azide at a pH of 7.2. The final concentration was 249.9 ng ovalbumin/ml.

The simultaneous assay capture reagent was prepared by mixing the two individual assay capture reagants. Solutions were diluted to~ethsr to give 1.875g/ml of phenylcyclidine-poly~lutamic acid and 0.25 Il~/ml of morphine-ovalbumin-polyglutamic acid in a final solution which contained 1% fish ~elatin, 25 mM Tris, 100 mM sodium chloride, 1 mM magnesium chloride, 0.1 mM zinc 2 0 chloride and û.1% sodium azide at a pH of 7.2.

Monoclonal anti-phenyioyclidine antibody was labeled with acridinium sulfonamide using EDAC coupling procedures known in the art. It then was dilutad in a buffer which contained 1% fish ~elatin, 25 mM Tris, 100 mM sodium chloride, 1 2~ mM magnesium chloride, 0.1 mM zinc chloride, 10% normal mouse serum and 0.1 sodium azide at pH 7.2. The final antibody concentration was 118 ng/ml.

Monoclonal anti-morphine antibody was labeled with alkaline phosphatase using procedures known in the art. It was diluted in the same buffer used for the 30 anti-phenylcyclidine-acridinium probe. Its final concentration was 48.2 ny/ml.

The simultaneous assay probe oonsisted of a monoclonal anti-phenylcyclidine antibody conjugated to acridinium (11B nglml) and a monoclonal anti-morphine antibody conjugated to alkaline phosphatase (48.2 ng/ml) diluted in the same buffer.
The alkaline phosphatase substrate solution was 0.4 mM of 3-(2'-spiroadamantane)-4-methoxy-4-(3"-phosphoryloxy) phenyl-1,2,dioxe~ane, SUE~5TITVTE SHEE~

.. . ~ . .. . .. ..... .

WO 92/12255 P~/US91/09714 2 ~

AMPPD (available from Tropix Inc., B~dford, MA) disodium salt, in a solution of 0.05 M sodium bicarbonate buffer containing 1 mM rna~nesium chloride at pH 9.5.

The disposable reaction tray used for this assay was that described in co-pending U. S. Patant Application Serial No. 425,651, which enjoys common ownership and whlch is incorporated herein by referenc~. The device comprised a funnel-like structure, a porous elernent, and an absorbant material, which ware assembled to provide intimate contact betwaen th0 porous el0rnent and the absorben~
material. The porous r;~lement was made of flbrous ~lass material, Product No. 4111 glass fiber filler paper, which had a nominal thickness of 0.05 inches and which is available from Hollin~sworth and Voss Co., East Walpole, MA. It was trsated with a 0.5% soiution of a polymeric quaternary ammonium compound, C~lquat ~L-20 (National Starch and Chemical Co., Bridgewat2r, NJ), to ~ive the solid phase material a positive charge. 80 ~LI of 0.5% Celquat~L-200 in 10 mM sodium 1~ chloride was applied to each porous elemant of the disposable reaction tray.

- ~xamplQ2. ~

Test samples used were phenylcyclidins caltbrators from a commercially 20 available fluorescence polarization kit ~ TDX~9 kit, Abbott Laboratories, North Chicago, IL) which contained 500, 250,120, 60, 25 and 0 n~/ml PCP in human urine. 80 I~J of Tris buffer (Abbott Laboratories, North Chicago, IL) were dispensed on the fibrous glass matrix pad of the detection well of a disposable reaction tray.
This was followed by 80 ~1 of a 0.5% Celquat~L-200 solution on each pad.
2 5 Solutions were disp~nsed using two FMI-RH pumps (Fluid Met~ring Inc., Oyster Bay, NY), and controlled via a triac board by an Intel 310 Deveiopment System (Intel Inc., Sunnyvale, CA). The tray was moved on a linear track usin3 a timing belt and a stepper motor. The stepper motor was controlled by a board employing components known in the art. Afler 4.8 minutes, 50 1ll of test sample was pipetted into the3 0 shallow reaction well of the disposable reaction tray, using an automated pipettor.
50 111 of acridinium-labeled anti-PCP antibodies were dispensed into each incubation well. The mixture was incubated for 9.6 minutes while the disposable reaction tray was moving on the track in a temperature controlled tunnal at 32C.
The timing belt steps at the rate of 0.8 inches per minute, the reac~ion tray was 3 5 stationary for 36 seconds after each step for a reaction step to t~ke place. After 9.6 minutes of incubation on the moving timing belt, 50 ~11 of a solution containing PCP-PGA capture reagent prepared as in Example 1 at a ~ncentration of 118 ng PGA/ml was dispensed into the incubation well through a tip centered on the well and SlJBSTlTlJT~: SHEIE:~I

., . :, .. `
:,, .. , : . , :

WV 92/1~ 2 ~ 7 Pcr/usgl/09714 ,~" 1 9 connected to an FMI pump through a Teflon~ line. The reaction mixturc was further incubated while the disposable tray continu0d movement along the trac~. After 4.8 minutes, each qualernary ammonium polymer-lreated glass fibar rnatrix was rinsedwith 100 ~l of IMX~9 Tris buffer (available from Abbott Laboratories, North 5 Chicago, IL) dispensed from a tip centared on the fibrous pad of tha detection well.
After 4.8 more minutes, the disposable reaction tray was locatcd under the transfer device described in the co-pending Patent Application Serial No. 425,643, previously incorporated herein by reference. The 150 111 assay rnixture then wastransferred from the shallow incubation well onto the prQ-~r~atod cJlass Sib0r matrix in the detection well. Transfer was affected using one 350 111 pulse of IMX ~Tris buffer injected from three adjacent nozzles at a linear rate of 210 cm/sec. The transfer fluid was injacted by a stepper-motor controlled syringe pump. A vaive directed the transfer solution to each side of the disposable reaction tray. Thetransferred mixture then was allowed to drain through the fibrous pad for 12 1~ seconds. Then, 50 ~LI of the AMPPD substrate solution was dispensed in each detection well to salurate the fibrous matrix. The disposable tray was movad on the timing belt to allow subsequent well pairs to be located under the transfer device to effect transfer of the reaction mixture. The disposable device then was movecl to a detection position where a chemiluminescence detector (described in co-pending U.
2 0 S. Patent Application Serial No. 425,643) was located.

The detection head had two photomultiplier tubes (PMT) and light pipes, each centered on a detection well. Each PMT was powered by a Bertan high voltage power supply and connected to a photon counting amplifier board and a counterttimer board 2 5 constructed using components and methods known in the art. The two trigger solution injectors associated with each PMT were connected to an FtAI pump using a black Teflon~1s tube and a manifold. As the tray reached the detection position, the detector head was lowered to create a light-tight seal with the surface feature on lhe disposable. The high voltage to the PMT was gated on, ~he two trigger solution pumps 30 and the counter/timer boards were activated simultaneously by the Intel 310 Development System (previously described). After the substrate was incubated for4.8 minutes, 85 111 of 0.3% alkaline peroxide trigger solution was injected and the resulting chemiluminescence was measured for eight (8) seconds from the onset oftrigger solution injection to give the intensity of the short-lived chemiluminescence 3 5 signal. The results of the assay are shown in Table 1. The chemiluminescenc~ signal in the short-lived winclow followed the PCP concentration in the sample, while ~he long-lived signal had a slowly varying function of PCP concen~ra~ion. A longer time window or a signal deconvolution scheme ean be applied by those skilled in the art to SlJBSTlTllT1E~ SHEIE:~

.- ~ . - . ....................... . . .. ~ .

, . . : ~ . , : : - : . : , WO 92/1225~ Pcr/usgl/09714 2 0 ~ 2 0 improve the dose response curve in ~he first window and eliminate it in the second window. Also, variations to the assay optimization steps such as wash solution volume, wash solution composition, addition of det~r~ants to th~ transfer buffered soiution, pratreatment of the glass fiber pad by aWition of prot~ins such as fish 5 gelatin, casein, etc., can be appli~d by those skilled in th~ art to improve the dose response curve, and these variations lic within the t~achings of the inv~ntion. The cut-off of this assay was considered to be 25 ng/ml. The data from Table 1 indicate that all tes~ samples which containad 25 ng/rrll PCP or hi~her were w~
differentiated from the negative control, which indicated the validity of 1he assay 1 0 procedure.

T~BL~ 1 ACRIDINIU~LABELED PHENYLCYCLIDINE CHEMILUMINESCENCE IMMUNOAS~;AY
1 5 (CLIA) .
[PCPl ng/mlShort-lived Signal % l~Long-liv~d Signal t0-8 seconds) (8-16 seconds) o 188,329 0.0 24,716 2 5 50,347 73.3 20,560 6 0 30,839 83.6 1 B,896 120 23,977 87.3 1 B,632 25 250 20,379 89.2 1 7,3B8 500 19,7~9 8g.5 18,188 ~%I=Percent Inhibi~ion SUI~STITUTE S~T

WO 92/1225~ pcr/usg1/û9714 2 ~

E~ampl~3. ~lkaliD-Q-phos~Qhata~~

Test samples used wers calibrators from a commercially available opiates fluor~scencc polarization kit (TDX~9 kit, Abbott Laboratories, North Chicago, IL) which contained 1000, 600, 350, 200, 100 and 0 n~/ml morphine in human urine.
80 ~LI of IMX~9 Tris buffcr solution (available from Abbott Laboratories, North Chica~o, IL), foilowed by 80 111 of C:elquatlUL-200 solution ware dispensed on the glass fiber matrices of a disposable reaction tray previously d~scribed in Example 2.
Solutions wer~ dispens~d usin~ two FMI-RH pumps and controiled via a triac boardby an Intel 310 Development System ~Intel Inc., Sunnyvale, CA). The tray was moved on a linear track using a timing belt and a stepper mo~or. The steppar motor was controlled by a board employing components known to those skilled in tho art.
After 4.8 minutes, 50 ,~11 of alkaline phosphatase-labal~d anti^morphin~ antibody was dispensed into each incubation well. The mixture was incubated for 9.6 minutes while the disposable reac!ion tray was moving on the track in a temperature controlled ~unnel at 32C. The timing belt st0ps at Ihe rate of 0.8 inches per minute, the raaction tray was stationary for 36 seconds after each step for a reacUon step to take place. A~ter 9.6 minutes of incubation on the moving timing belt, 50 ~11 of a solution containing morphine-ovalbumine-PGA capture reagent as prepared in 2 0 Example 1 a~ a concentration of 0.25 ~g PGA/ml was dispensed into the incubation well through a tip centered on the well. The reaction mixture was further incubat0d while the disposable tray continued movement along the track. ARer 4.8 minutes, each quaternary ammonium polymer-treated glass fiber matrix was rinsed with 100 ~LI of IMX~' Tris buffer injected from three adjacent nozzles at a linear rate of 2 5 210 cm/sec. The lransferred mixture was allowed to drain through the fibrous pad for 12 seconds. 50 ~ul of the AMPPD substrate solution prepared as preYiously described in Example 1 were then dispensed in each det~ction well to saturate the fibrous matrix. The disposable tray was moved on the timing belt to allow subsequent well pairs to be located under the transfer device to affect transfer of the 3 0 reaction mixture. The disposable device then was moved to a deteotion position where a chemiluminescence detector (described previously) was located. After a 4.8 rninute substrate incubation time had elapsed, 85 ~LI of 0.3% alkaline peroxide trigger solution was injected, and the resulting chemiluminescence signal was integrated for eight (~) seconds after an eight (B) seconds delay from the onset of trigger solution injection to give the intensity of the long-lived chemiluminesc~nce signal. The results of ~he assay are shown in Table 2. The cut-ofl of this assay was considered to be 100 ng/ml. The data in Table 2 indicate that all test samples which !SuBsTlTuTE SHEE~

~. ~ : :; ,. .- ;

W~ 92/122~ PCI'/USgl/09714 2 0 ~ ~ S ~ 1 22 ~
contained 100 ng/ml morphine or higher were well-differsntiated from the negative control, which indicated the validity of the assay procedure.
Short -lived signal ~convoluted)= short-lived signal - (lony-lived signal x 0.655 ) Using this deconvolution scheme presented above, the last two columns of Table 2 show that the chemiluminescence signal in th~ hng-lived window is the dose response for morphina in the sample, while the correctRd short-lived signal is independent of morphins concentration. A different ~ime window or a signal deconvoiution scheme can be applied by tho~e skilled in ~he art to improve the dose 1 0 response curve in the first window and aliminate it in the second window. Also, varia~ions to the assay optimization steps such as wash solution volume, wash solution corrlposition, addition of detergents to the transfer buffered solution, pretreatment of tha ~lass fibrous pad by addition of proteins such as fish ~elatin, casein, etc. are variatiorls which can also be applied by those skilled in the art to 1 5 improve the dose response curve and lie within th~ t~achings of this inv~ntion.

ALKA~NE PHOSPHATASE-LABELED MORPHINE CHE~lilLUMlNESCENT IMMUNOASSAY
(CLIA) .
MorphineShort-lived Long-lived % I Corrected ng/ml(0-8 seconds)(8-16 seconds) Short-lived 0 181,152 235,155 0.0 27,125 100 133,473 162,877 30.74 26,788 200 119,695 144,371 38.61 25,132 350 119,431 140,355 40.31 27,355 60Q 116,268 136,237 42.07 27,033 30 1000 118,485 137,646 41.47 28,327 .

!SUBS~ITIJTE SHIE8~

, - . ~ .. . . ~ . .

WO 92/1225~ PCI/US91/09714 2~93~:! 7 . 23 Exampl~L .Sirr~l~n o~ e,~Say For Pheoyl~clidillQ~Q~bi~

Test samples used were phenyleyclidirl0 calibrators from a commercially available fluorescence polarization kit (TDX~ kit, Abbott Laboratories, North Chicago, IL) at 500, 250, 120, 60, 25 and 0 nglml, or TDX~9 opiate calibrators (commercially available from Abbott Laboratories, North Chicago, IL) at 1000, 600, 350, 200, 100 and 0 ng/ml prepared in human urine.

80 ~J of IMX ~9 Tris buffer solution (available ~rom Abbott Laboratories, North Chicago, IL) followad by 80 ~11 of 0.5% Calquat~L-200 solution w0re dispensed on ~lass fiber matrices of the disposabie reaction tray previously described. Solutions were dispensad using two FM!-RH pumps and controlled via a triac board by an Intel 310 Development System (Intel, Inc., Sunnyvale, CA). Thetray was moved on a linear track using a timing belt and stepper motor. The stepper motor was controlled by a board employing eomponents known to those of ordinary skili in the art. After 4.8 minutes, 50 111 of test sampla was pipetted into theshallow reaction wall of thQ disposable reaction tray, usin~ an automated pipettor.
~0 ~1 of the simultaneous assay probe containin~ 118 n0/ml acrtdinium-labeled anti-PCP antibodies and 48.2 ng/ml alkaline phosphatase labeled anti-morphine 2 0 antibody prepared as described in E~ample 1 was dispensed into each incubation well.
This mixture was incubated for 9.6 minutes while the disposable reaction tray was moving on the track in a temperature controlled tunnel at 32C. The timing bel~
steps at the rate of 0.8 inches per minute, tha reaction tray was stationary for 36 seconds aRer each step for a reaction step to take place. ARer 9.6 minutes of incubation on the moving timing belt, 50 1ll of the simultaneous assay capture reagent solution containing PCP-PGA at a concentration of 1.875 llg/ml and morphine-ovalbumin-PGA at a ooncentration of 0.25 ~lg/rrll prapared as in Example 1 was dispensed into the incuba~ion well through a tip centered on the well. Thereaction rnixture was further incubated while the disposable tray continued 3 0 movement along the track. After 4.B minutes, each ~uaternary ammonium polymer treated glass fiber matrix was rinsed with 100 ~LI of IMX~9 Tris buffer dispensed from a tip centered on the detection well. After 4.8 more minutes of incubation, the disposable reaction tray was located under the transfer device described in co-pending U. S. Patent Application Serial No. 425,~43. The 150 ~1 assay mixture then 3 5 was transferred from the shallow incubation well onto the pre-treated glass fib~r matrix in the detection well. Transfer was affected using one 350 ,ul pulse of IM
Tris buffer injected from three adjacent nozzles at a lin~ar rate of 210 cm/sec.After 12 seconds of drain tirne, 50 ~11 of the substrate solution was dispensed in each su~TurE 5HE~:T

;:

WO 92/12255 PCI`/VS91/09714 20~8~7 detection well to saturate the fibrous matrix. The disposable tray was moved on the timing belt to allow subsequent well pairs to be located under the transfer device to eflect transfer of the reaction mixture. Tha dis~sable device then was moved to a detection position where a chemiluminescence detector (described previously) waslocated. After 4.8 minutes of substrate incubation time had elapssd, 85 111 of 0.3%
alkalins peroxide trigger solution was inject~d into the detr~ction well. The rasulting chemiluminescence was integrated for eight (B) seconds immeciiately after the onset of trigger solution injection to giv8 the intensity of the short-lived chemiluminescencs signal. The chemiluminescence si~nal was inte~rated for another eight (8) subsequent seconds to give the iong-lived chemilurninescence signal. The results of the assay are shown in Tables 3 and 4. The cut-off of the PCP assay was considered to be 25 ng/mi. The data in Tables 3 and 4 indicate that all test samples which contained 2~ ng/ml PCP or higher were well-diffsrentiated from the negative control. The cut-off of the morphina assay was considcred to be 1W ng/ml. The data in Tables 3 and 4 indicate that the test samples which contained 100 ng/ml morphine or higher were well-differentiated from the negative control. The data also indicates that the cut-off of either assay was not affected by using the simultanaous assay procedure as described herein. A longer time window or a signal deconvolution scheme are varia~ions which can be applied by those skilled in the art to improve the 2 0 dose response curve in the first window and eliminate il in the second window. Also, variations of assay optimization steps such as wash solution volume, wash solution composition, addi~ion of detQrgents to the transfer buffered solution, pretreatment of the glass fibrous matrix by addition of proteins such as fish gelatin, casein, etc., are variations which can also be applied by those skilled in the art to improve the 2 5 dose response curve, and these variations lie within the teachings of this invention.

$UI~TITV7-E SH~:IE7 .

, . . ~ , . -WO 92/12255 PCr/US91/09714 2a~2~& l~

SIMULTANEOUS PHENYLCYCUDiNE AND M~FlPHiNE C:HEMIWMINESCENCE ASSAY

[PCP] IMorphine~ Short-lived % I Lon~-lived % I
ng/ml n3/mlSignal (0-8 sec.~ Signal (8-16 sec.) .
o o 199,414 0 73,845 0 73,667 63 67.713 8 0 60 0 68,297 66 70,41~ 5 120 0 73,270 63 75,019 0 25~ 0 61,036 69 69,D23 7 500 0 62,521 69 71,760 3 o ~ 229,~36 0 85,552 0 1 5 0 100 211,149 8 72,248 1 6 0 200 195,605 15 ~4,181 37 o 350 199,325 13 54,975 36 ~LE~ : ' SIMULTANEOUS PHENYLCYCLIDINE AND MORPHINE ASSAY

25lPCP]Shorl-lived Signal IMorphine]Lon~-livad Signal ng/mLCounts/first 8 sec. ng/mLCounts/seoond8sec.
:
1~0~28 0 231879 250 9367 : 600 141899 The dose response of each analyte in the simultaneous assay resembles that:in ~he individual assay of the analyte, which indicates the ability to detect either of the two analytes in a ~est sample using the novel assay method of the invention.
... . .
.
SUeSl'lTllTE SH~l WO 92/122S~ PCl/US91/09714 2~3~-~7 2~ - ~

Al~hou~h the present invention has been dflscribed in terms of preferr~d embodiments, i~ is anticipated that various modifications and Improv~man1s will occur to those skilled in the art upon consideration of the prssent invention. Thus, 5 other assay configurations that include more than one captura raa~snt and more than one chemiluminescent probe with varying chemilumincsc~ncs si~nal lifs times is contemplated to lie within the taachings and scope of this invention. Various solid phases such as plastic tubes coated with mixtures of antibodies and dstected in tube luminomaters or plastic beads such 8s 1/4" polystyrene beads coated with mixtures 10 of antibodies and transferred aft~r the completion of ths bindin0 raaction to a test tube and then detected in a iuba luminometer can be us~d as media for sirnultaneous assays based on chemiluminescenca si~nal tima resolution.

Micropar~icles can ba made from any suitabls particulats matarial that are 15 easily recognizable by those skilled in tha art, such as polystyrone, polymethyl acrylate, derivatized cellulose fibers, polyacrylamida, ~nd 2he lika.

The ion captura procedures previously described used poly~lutamic acid as the polyanion, and Celquat~L-200 as the polycation. However, oth~r polyanionic 2 0 materials and other derivatked polycationic material, as well as oSher methods of attachment of these compounds to the assay components or the porous slarnent, iscontemplated as obvious variations of this invention and are considered to lie within its scope.

2 5 Moreover, it is contemplated 1hat th~ assay method of this inv~ntion may be extended to other small molecules, macromolecules, or to nucJeic acir~ probe assays.
Furthermore, although the present invention has been described using acridinium sulfonamide-labeled and alkaline phospha~ase-labaled compounds as tracars, it iscontemplated that other acridinium compounds or their analogs, or other 3 0 chemiluminescent compounds (labels) may be used.

The assay of the present invention may be employed for the detection of viral particles, such as HBsAg or HIV antigens, or specific fragments thereof.
Simultaneous assays for macromolecular diseasa state markers, such as carcino-embryonic antigen (CEA) and alpha-fetoprotein (AFP) may also be performed, as well as nutritional status markers, such as simultaneous determination of Vitamin B12 and foiates or ferritin. Also usefully detected according to the rnethod of the present inYention are hormones such as LH and FSH, bacteria (e.g., streptoco~i~, SVBSTITUTE SHE~

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Claims (21)

WHAT IS CLAIMED IS:

1. A method for the determination of multiple analytes which may be present in a test sample comprising:
(a) incubating the test sample with a mixture of members of analyte-specific binding pairs attached to a solid phase for a time and under conditionssufficient for analyte/anti-analyte specific binding pairs to form;
(b) incubating with the so-formed specific binding pairs a mixture of labeled members of specific binding pairs for each analyte wherein each analyte is bound to a different chemiluminescent label capable of generating a different short-lived or long-lived chemiluminescent signal upon contact with a triggering solution;
(c) triggering the signal with a triggering solution;
(d) measuring the chemiluminescent signal detected; and (e) determining the presence and the amount of each analyte present in the test sample by calculating the difference in time-profile of the signals generated from the chemiluminescent compounds.

2. The method of claim 1 wherein the solid phase is a suspension of microparticles comprising a mixture of groups of particles, each group having attached thereto a member of a specific binding pair for one analyte.

3. The method of claim 1 wherein the solid phase is a tube coated with a mixture of members of specific binding pairs for the analytes.

4. The method of claim 1 wherein the solid phase comprises a suspension of magnetizable particles comprising a mixture of groups of particles wherein each group of particles has attached thereto members of a specific binding pair for an analyte.

5. The method of claim 1 wherein the solid phase comprises a plastic bead coated with a mixture of members of specific binding pairs for the analytes.

6. The method of claim 1 wherein the solid phase is a derivatized membrane having attached thereto by chemical binding members of specific bindingpairs for the analytes which cover all the membrane or discrete regions of the membrane.

nucleic acid species (e.g., DNA or RNA). The present invention also is useful insmall molecular competitive binding assays such as those for T3 and T4 and digoxin and its analogs. Substances of abuse and their metabolites such as cocaine and benzolecogonine in serum, nicotine and cotinine, codeine and nor-codeine, diazepam and nor-diazepam may be detected using the method of the present invention.
Simultaneous assay of therapeutic drugs and simultaneous determination of two steroids also can be performed according to the method of the present invention.Other combinations of analytes can be contemplated and assayed for, by those skilled in the art, using the method of this invention.

Although the examples of this invention were given for simultaneous determination of two analytes, more than two analytes can be determined by choice of trigger conditions and time gating. Those skilled in the art can contemplate reaction conditions, timing schemes and signal deconvolution algorithms to determine morethan two analytes in the same assay procedure upon consideration of the leachingprovided by the present invention. These choices thus are considered within the scope of the present invention.

7. The method of claim 1 further comprising the step of separating the solid phase comprising analyte/anti-analyte specific binding pairs by microparticle separation on a porous element and washing said solid phase.

8. The method of claim 4 further comprising the step of separating said solid phase comprising analyte/anti-analyte specific binding pairs by magnetic separation.

9. A method for performing a simultaneous determination of multiple analytes in a test sample which may contain said analytes using a competitive binding chemiluminescence assay comprising:
(a) incubating the test sample with a known amount of chemiluminescence labeled analytes each capable of generating a short-lived or long-lived chemiluminescent signal and a solid phase which has a mixture of members of specific binding pairs for said analyte attached thereto for a time. and under conditions sufficient for analyte/anti-analyte specific binding pairs to form;
(b) adding a substrate specific for one of the labels and incubating to allow a long-lived chemiluminescence-generating reaction to occur;
(c) triggering the resultant mixture with a triggering solution specific for the other label; and (d) integrating and time-discriminating the short-lived and the long-lived components of the chemiluminescence signal generated.

10. The method of claim 9 wherein said analytes are selected from the group consisting of haptens, macromolecules, metabolites and antibodies.

11. A method for performing a simultaneous chemiluminescence assay for multiple analytes which may be present in a test sample, comprising:
(a) incubating the test sample with a solid phase coated with a mixture of members of specific binding member pairs for the analytes to form analyte/anti-analyte specific binding pairs;
(b) separating the solid phase;
(c) adding a mixture of members of specific binding pairs for the analytes having attached thereto different chemiluminescent labels capable of generating a different short-lived or long-lived chemiluminescent` signal upon contact with atriggering solution and incubating same;
(d) adding a substrate to one of the labels and incubating same to allow a long-lived chemiluminescence generating reaction to proceed;

(e) triggering the resultant mixture with a triggering solution; and (f) integrating the generated chemiluminescence signal and time-discriminating the short-lived and the long-lived components of the signal generated.

12. The method of claim 11 wherein said analytes are selected from the group consisting of infectious disease antigens, hormones, cancer markers and DNA
probe sequences.

13. A method for the determination of multiple analytes in a test sample which may contain any of the analytes, comprising:
(a) incubating the test sample with mixture of members of specific binding pairs of each analyte attached to polymeric ionic molecules;
(b) adding a mixture of chemiluminescent labeled members of specific binding pairs for each analyte wherein each analyte is bound to a different chemiluminescent label capable of generating a different short-lived or long-lived chemiluminescent signal upon contact with a triggering solution and incubating same to form a reaction mixture of analyte/anti-analyte specific binding pairs (c) transferring the reaction mixture to a porous membrane treated with a polymeric cationic compound;
(d) triggering a chemiluminescent signal with a triggering solution;
(e) detecting the chemiluminescent signal generated;
(f) determining the presence and the amount of each analyte from the difference in time-profile of the signals generated from the chemiluminescent compounds.

14. The method of claim 13 wherein the analytes are selected from the group consisting of haptens, macromolecules, metabolites, antibodies, infectiousdisease antigens, hormones, cancer markers and DNA probe sequences.

15. A kit for performing a simultaneous determination of two analytes comprising:
containers containing members of specific binding pairs for each analyte wherein each analyte is bound to a different compound capable of generating a different short-lived or long-lived chemiluminescence signal upon contact with atriggering solution.

16. The kit of claim 15 wherein the compound generating the short-lived chemiluminescence signal is an acridinium sulfonamide compound.

17. The kit of claim 15 wherein the compound generating the long-lived chemiluminescence signal is an alkaline phosphatase substrate.

18. The kit of claim 15 wherein the compound generating the long-lived chemiluminescence signal is a .beta.-galactosidase substrate.

19. A method for the determination of multiple analytes in a test sample which may contain any of the analytes, comprising:
(a) incubating the test sample with microparticles having bound to them a member of specific binding pair for one analyte, and polyionic residues having bound to them member of specific binding pairs for the second analyte;
(b) adding a mixture of chemiluminescent labeled members of specific binding pairs for each analyte wherein each analyte is bound to a different chemiluminescent label capable of generating a different short-lived or long-lived chemiluminescence signal upon contact with a triggering solution and incubating same to form a reaction mixture of analyte/anti-analyte specific binding pairs;
(c) transferring the reaction mixture to a porous membrane treated with a poly-ionic compound of opposite charge;
(d) triggering a chemiluminescence signal with a triggering solution;
(e) detecting the chemiluminescence signal generated;
(f) determining the presence and the amount of each analyte from the difference in time-profile of the signals generated from the chemiluminescent compounds.

20. A method for the determination of multiple macromolecular analytes in a test sample which may contain any of the analytes, comprising:
(a) incubating the test sample with microparticles having bound to them a member of specific binding pair for one analyte, and polyionic residues having bound to them member of specific binding pairs for the second analyte;
(b) transferring the reaction mixture to a porous membrane treated with a poly-ionic compound of opposite charge;
(c) adding a mixture of chemiluminescent labeled members of specific binding pairs for each analyte wherein each analyte is bound to a different chemiluminescent compound capable of generating a different short-lived or long-lived chemiluminescent signal upon contact with a triggering solution and incubating same;
(e) washing excess unbound conjugate;

(f) triggering a chemiluminescent signal with a triggering solution;
(g) detecting the chemiluminescent signal generated;
(h) determining the presence and the amount of each analyte from the difference in time-profile of the signals generated from the chemiluminescent compounds.

21. A method for performing a simultaneous determination of multiple analytes in a test sample which may contain said analytes using a competitive binding chemiluminescence assay comprising;
(a) incubating the test sample with a known amount of chemiluminescent labeled specific binding pair members each member capable of generating either ashort-lived or a long-lived chemiluminescent signal and a solid phase which has a mixture of analyte derivatives attached thereto, for a time and under conditionssufficient for analyte/anti-analyte specific binding pairs to form;
(b) adding a substrate specific for one of the labels and incubating to allow a long-lived chemiluminescence-generating reaction to occur;
(c) triggering the resultant mixture with a triggering solution specific for the other label; and (d) integrating and time-discriminating the short-lived and the long-lived components of the chemiluminescence signal generated.