AU9146491A - Simultaneous determination of multiple analytes using a time-resolved heterogeneous chemiluminescence assay - Google Patents

Description

SIMULTANEOUS DETERMINATION OF MULTIPLE ANALYTES USING A TIME-RESOLVED HETEROGENEOUS CHEMILUMINESCENCE ASSAY

This application is a continuation-in-part application of U. S. Serial No.

07/636,038, which enjoys common ownership and is incorporated herein by reference.

Background of the Invention This application relates generally to chemiluminescent labels and more particularly, relates to the simultaneous determination of multiple analytes using a heterogeneous chemiluminescent assay method.

The generation of light as a result 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, βds., Advances in Physical Organic Chemistry

18:187-238, Academic Press, New York (1982). The application of Chemiluminescence generation and its use in immunoassays also is well-known in the art. See, for example, W. Rudolf Seitz "Immunoassay Labels Based on Chemiluminescence and Bioluminescence," Clinical Chemistry 17: 120-126

( 1 984) .

The use of acridinium compounds as labels for immunoassays and the subsequent generation of short-lived chemiluminescent signals from these labels has been described by I. Weeks et al., "Acridinium Esters as High Specific Activity Labels in Immunoassays," Clinical Chemistry 29:1474-1478 (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 compounds 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 I. Bronstein; I. Bronstein et a!., "1 ,2-

Dioxetanes, Novel Chemiluminescent Substrates, Applications to Immunoassays, "In Proceedings of the V th International Conference on Bioluminescence and Chemiluminescence," Florence-Bologna, Italy, September 25-28, 1988 and also in

iTurε SHEET the Journal of Bioluminescence and Chemiluminescence 4:99 (1988), and US patent 4,950,593.

Triggering chemiluminescent reactions in a porous matrix and the subsequent detection of signals resulting from immobilized microparticles is described in co- pending United States Patent Application Serial No. 206,645, which enjoys common ownership and is incorporated herein by reference. The use of an ion capture separation method with chemiluminescent detection in a porous matrix is described in co-pending United States Patent Application Serial No. 425,643, which enjoys common ownership and is incorporated herein by reference.

The ability to perform simultaneous testing for multiple analytes in the same sample would offer several advantages. First, it would allow obtaining multiple assay results for the same sample processing time, thus it would increase the through-put of automated instruments. Second, it would decrease the cost per test, because the same position of a disposable test device would be used for several test results. Third, it would decrease the amount of volume of used disposable 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 antigens, thus offering better screening tests. Finally, it would allow simultaneous assays for different analytes such as drugs or for a drug and its metabolites, thus increasing the predictive value of the assay, which in turn would lead to more accurate diagnosis.

The simultaneous detection of multiple analytes using chemiluminescence could 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 apply to chemiluminescent measurements. Finally, the majority of light-generating chemical reactions are not compatible with each other in terms of reaction conditions.

The present invention overcomes the problems of existing methods by allowing the simultaneous determination of analytes in a chemiluminescent assay by using time resolutions of the light emissions generated under the same reaction conditions. The present invention provides a novel method in which the simultaneous measurement of analytes in a competitive or in a sandwich assay are achieved. This in turn improves the predictive value of the assay and leads to more accurate diagnosis.

Summary of the Invention

The present invention provides 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 specific binding pairs attached to a solid phase for a time and under conditions sufficient 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 compound (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 (labels). The solid phase can include 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, a tube coated with a mixture of members of specific binding pairs for the analytes, 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, a plastic bead coated with a mixture of members of specific binding pairs for the analytes and a derivatized membrane having attached thereto by chemical binding members of specific binding pairs for the analytes which cover all the membrane or discrete regions of the membrane. The method can further comprise a separation step. If the solid phase comprises analyte/anti-analyte specific binding pairs attached to microparticles, microparticle separation on a porous element and 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 using a competitive binding chemiluminescence assay comprising (a) incubating the test sample with a known amount of chemiluminescent 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 said analyte attached thereto for a time and under conditions sufficient for analyte/anti-analyte specific binding members 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 alkaline peroxide; and (d) integrating and time-discriminating the short-lived and the long-lived components of the chemiluminescence signal generated. The analytes include haptens, macromolecu.es, metabolites and antibodies. In yet another format for a simultaneous determination of multiple analytes in a test sample which may contain the analytes using a competitive binding chemiluminescence assay, the assay comprises (a) incubating the test sample with a known amount of chemiluminescent labeled specific binding pair members each member capable of generating either a short-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 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.

The present invention further provides a method for performing 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 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) 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. The analytes which can be tested include infectious disease antigens, 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, 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 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 porous membrane treated with a polymeric cationic compound; (d) triggering a chemiluminescent signal with a triggering solution; (e) detecting the chemiluminescent signal generated; and (f) determining the presence and the amount of each analyte from the difference in time-profile of the signals generated from the chemiluminescent compounds (labels). The analytes include haptens, macromolecules, metabolites, antibodies, 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 containing members of specific binding pairs for each analyte wherein each analyte is bound to a different chemiluminescent compound (label) capable of generating a different 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 an alkaline phosphatase substrate or a β-galactosidase substrate.

Detailed Description of the Invention

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 solution to produce a chemiluminescent signal that maximizes after approximately two (2) seconds. Light emission is completely extinguished after approximately ten (10) seconds. Acridinium sulfonamide labeling chemistry may be employed according 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 entitled "Chemiluminescent Acridinium Salts" filed June 23, 1989, which enjoys common ownership and is incorporated herein by reference.

UBSTI Chemically catalyzed long-lived chemiluminescent 1 ,2-dioxetanes can be generated in a variety of ways. Thus, a siloxy-substituted dioxetane, 4-(6,tert- butyldimethylsiloxy-2-napthyl)-4-methoxyspiro[1 ,2-dioxetane-3,2'adamantane] is triggerable with tetrabutylammonium flori e solution to produce a chemiluminescent signal lasting for 20 minutes. Also, enzymes such as aryl esterase, alkaline phosphatase or β-galactosidase react with aryl dioxetane derivatives stabilized with an adamantane cage to produce similar long-lived chemiluminescent signals. Further, long-lived emissions from alkaline phosphatase catalyzed reactions of 3(2'-spiroadamantane)-4-methoxy-4-(3"- phosphoryloxy)-phenyl-1,2-dioxetane (AMPPD) and of a similar β-galactosidase substrate have been described in WO 881 00694, along with the use of these compounds (labels) in an immunoassay. Alkaline phosphatase and β-galactosidase labeling techniques are known, and catalyzed dioxetane chemiluminescence can be used according to this invention to generate long-lived signals.

According to the invention, a short-lived chemiluminescent label can be any member of the acridinium and/or phenanthridinium 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 generating reaction performed, at a triggering condition of pH and catalysts, in such a way that the reaction is triggered and decays in a few seconds.

A long-lived chemiluminescent compound (label) according to this invention can be one of the class of dioxetane compounds (labels) that can react with enzyme labels such as alkaline phosphatase or β-galactosidase to generate chemiluminescent signals of long durations. It is also contemplated that any compound which can be configured to generate a long-lived signal, such as luminol, or any compound which generates a long-lived signal can be used. Alkaline phosphatase catalyses the decomposition of AMPPD at pH 8-9 to generate a chemiluminescence signal of long duration. Increasing the pH to ca. 12 enhances the generation of the signal, β- gaiactosidase catalysis 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 long-lived chemiluminescence signal is generated.

A trigger solution according to this invention is an alkaline peroxide solution.

This trigger solution can be prepared by the addition of hydrogen peroxide to a sodium hydroxide solution or by dissolving solid urea-hydrogen peroxide adduct in sodium hydroxide. The alkaline peroxide trigger solution, according to this invention, acts on the acridinium label to generate a short-lived chemiluminescence signal due to the decomposition of a high energy intermediate and the generation of an excited acridone derivative. Alkaline peroxide, also according to this invention, enhances the enolization of the enzyme reaction product and hence, the intensity of the chemiluminescence signal. This appears as a pH-jump-like change in the signal. Alkaline peroxide also according to this invention, reacts with the non-luminescent reaction product of β-galactosidase with AMPGD to generate a chemiluminescence 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 include polystyrene, carboxylated polystyrene, polymethylacrylate or similar particles with radius in the range of from about 0.1 to 20 μm. A preferred separation method for these particles is the use of microparticle capture on a glass fiber matrix followed by triggering a chemiluminescent reaction on this matrix.

Another preferred method of separation is that which is described in co- pending U.S. Patent Application Serial No. 150,278, and U. S. Patent Application Serial No. 375,029, 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 chemically attached to polyanionic acids such as polyglutamic acid or potyacrylic 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 between the positively charged pad and the negatively 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 chromium oxide cores and polystyrene, carboxylated polystyrene, polymethylacrylate coating. A preferred separation method for these particles is the use of constant or pulsed magnets, washing said particles, and then suspending the separated particles in a vessel where a chemiluminescent reaction can be generated and detected. Yet other solid supports are known to those in the art and include the walls of wells of a reaction tray, test tubes, polystyrene beads, nitrocellulose strips, membranes, and others.

Polymeric microparticles with chemically or physically bound antigens or antibodies can be used according to the invention as capture phases in a binding reaction to make use of the fast diffusion rates of these particles in solution to yield rapid results. Microparticles that can be used according to this invention include polystyrene, carboxylated polystyrene, polymethylacrylate or similar particles with radius in the range of from about 0.1 to 20 μm.

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

"Analyte," as used herein, is the substance to be detected which may be present in the test sample. The analyte can be any substance for which there exists a naturally occurring specific binding member (such as, an antibody), or for which a specific binding member can be prepared. Thus, an analyte is a substance that can bind to one or more specific binding members in an assay. "Analyte" also includes any antigenic substances including infectious disease antigens such as viral, bacterial, rickettsial antigens and also cancer markers such as CEA, also macromolecules, haptens and/or their metabolites, antibodies, and combinations thereof. As a member of a specific binding pair, the analyte can be detected by means 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 B12, or the use of lectin as a member of a specific binding pair for the determination of a carbohydrate. The analyte can include a protein, a peptide, an amino acid, DNA or RNA probe sequences, a hormone, a steroid, a vitamin, a drug including those administered for therapeutic purposes as well as those administered for illicit purposes, a bacterium, a virus, and metabolites of or antibodies to any of the above substances. The details for the preparation of such antibodies and the suitability for use as specific binding members are well-known to those skilled in the art.

A "capture reagent", 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 binding member, 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 the performance of the assay or during the performance of the assay, thereby enabling the separation of immobilized complexes from the test sample .

The "indicator reagent" comprises a signal generating compound (label) which is capable of generating a measurable signal detectable by external means conjugated (attached) to a specific binding member for the analyte(s). "Specific binding member" as used herein means a member of a specific binding pair. That is, two different molecules where one of the molecules through chemical or physical means specifically binds to the second molecule. In addition to being an antibody member of a specific binding pair for for the analyte, the indicator reagent also can be a member of any specific binding pair, including either hapten-anti-hapten systems such as biotin or anti-biotin, avidin or biotin, a carbohydrate or a lectin, a complementary nucleotide sequence, an effector or a receptor molecule, an enzyme cofactor and an enzyme, an enzyme inhibitor or an enzyme, and the like. An immunoreactive specific binding member can be an antibody, an antigen, or an antibody/antigen complex that is capable of binding either to an analyte as in a sandwich assay, to the capture reagent as in a competitive assay, or to the ancillary specific binding member as in an indirect assay.

The various signal generating compounds (labels) contemplated in the practice of the invention include short-lived chemiluminescence signal generating compounds (labels) such as an acridinium sulfonamide, an acridinium ester or a phenanthrkJine compound (label), and long-lived chemiluminescence signal generating compounds (labels) whose signals result from action of enzymes or nucleophilic agents on dioxetane compounds containing an adamantane structure.

It is contemplated that the reagent(s) employed for the assay can be provided in the form of a kit with one or more containers such as vials or bottles, with each container containing a separate reagent such as a monoclonal antibody, or a cocktail of monoclonal antibodies, employed in the assay as capture phases or as indicator reagents which comprise chemiluminescent compounds (labels) as signal generating compounds.

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 period of time and under conditions sufficient to allow optimal immunochemical binding reactions for the analytes to take place. The 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 enzyme/substrate reaction reaches an end point, the separated reaction mixture is triggered using an alkaline peroxide solution. The signal is collected over a period of about four to ten seconds and is integrated to give the sum of the pH-jump enhanced alkaline phosphatase catalyzed chemiluminescence and the alkaline peroxide triggered acridinium sulfonamide chemiluminescence. The residual signal is collected and integrated for an equal time interval to give the steady state enzyme catalyzed 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.

In an alternative way of performing the assay 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 period of time and under conditions sufficient to allow optimal immunochemical binding reactions for the analytes to take place. The 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 enzyme/substrate reaction reaches an end point, the generated chemiluminescence signal corresponding to the enzyme label is integrated. The separated reaction mixture is then triggered using an alkaline peroxide 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 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 period of time and under conditions sufficient to allow optimal immunochemical binding reactions for the analytes to take place. The capture phase then is separated and washed. A substrate specific to the β-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 triggered using an alkaline peroxide solution. The signal is collected over a period of about one second and is integrated to give the alkaline peroxide triggered acridinium sulfonamide chemiluminescence. The residual signal is collected after a delay of four to five seconds and integrated for an equal time interval to give the steady state enzyme catalyzed product that is rendered chemiluminescent by the 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 multiple analytes is performed according to this invention, as follows: A test 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 interest, and the second group of particles coated with a polyclonal antibody to the second analyte of interest. A conjugate mixture of labeled antibodies then is added to the reaction vessel. One antibody in this mixture is specific for the first analyte and is labeled with (bound to) a short¬ lived chemiluminescence signal generating compound (label) such as an acridinium sulfonamide, an acridinium ester or a phenanthridine compound. The second antibody of the conjugate mixture is labeled with alkaline phosphatase. The mixture is incubated 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 preferred method of separation is one which employs a glass fiber pad, using the fluid removal method described in co-pending U. S. Patent Application Serial No. 07/184,726, which enjoys common ownership and is incorporated herein 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 chemiluminescence signal when reacted with the substrate, then is added to the separated particles and is allowed to react for a time and under conditions sufficient to approach steady state. This generally takes only a few minutes. The microparticles and substrate reaction product then is triggered with an alkaline peroxide solution in an apparatus such as that described in co-pending U. S. Patent Application 07/206,645.

Another method of the invention comprises contacting the test sample which may contain either or both of the 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 group of microparticles is coated with a monoclonal antibody which specifically binds to one epitope of the second analyte. A conjugate mixture of labeled antibodies is added into the reaction vessel. One antibody of the conjugate mixture is specific to the first analyte and is labeled with a short-lived chemiluminescence signal generating label such as an acridinium sulfonamide, an acridinium ester, or a phenanthridine compound (label). The second antibody of the conjugate mixture is specific for the second analyte and Is labeled with alkaline phosphatase. The mixture is incubated for a time and under conditions sufficient to form analyte/anti- analyte/anti-anti-analyte complexes. Then, the microparticles are separated and 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 labeled polyclonal antibodies.

A microparticle-based two-step sandwich immunoassay for two analytes can be performed according to the invention, as follows. The test sample which may contain either or both analytes of interest and a mixture of microparticles comprising two groups of particles, one group of particles 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 sample and microparticles are incubated for a time and under conditions sufficient to allow binding of the analytes to the particles. Then, the microparticles are separated and washed. A preferred method of separation is on a glass fiber pad, using the fluid removal method described in co-pending application Serial No. 184,726 previously incorporated 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 chemiluminescent generating label such as an acridinium sulfonamide, an acridinium ester or a phenanthridine compound (label). The second antibody of the conjugate mixture is specific for the second analyte and is labeled with alkaline phosphatase. This conjugate mixture is allowed to incubate with the separated microparticles for a time and under conditions sufficient for complexes to form. The excess conjugate mixture then is removed by washing. An alkaline 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 substrate reaction product. The amount of time to reach steady state is usually only a few minutes. The microparticle and substrate reaction product then is triggered with an alkaline peroxide solution in a reaction vessel such as that described in co- pending U. S. Patent Application Serial No. 206,645 previously incorporated herein by reference.

in yet another embodiment of the invention, the test sample which may contain any of the analytes of interest and a mixture comprising two groups of microparticles, one group of particles coated with a monoclonal antibody to an epitope of the first analyte and the second group of particles coated with a monoclonal antibody to an epitope of the second analyte, 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 are separated and washed. A conjugate mixture of labeled antibodies then is added to the so-formed complexes. One antibody of the conjugate mixture is specific to the first analyte and is labeled with a short-lived chemiluminescence-generating label such as an acridinium sulfonamide, an acridinium ester or a phenanthridine compound (label). The second antibody of the conjugate mixture is specific for the second analyte and is labeled with alkaline phosphatase. After incubation for a time and under conditions sufficient for the conjugate to bind to either or both analyte/anti-analyte complexes, the excess conjugate mixture is washed off and the chemiluminescence signals are generated and measured.

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

Ion capture procedures for immobilizing an immobilizable reaction complex with a negatively charged polymer, described in co-pending U. S. Patent Application Serial No. 150,278 filed January 29, 1988, which enjoys common ownership and is incorporated herein by reference, can be employed according to the present 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 charged poly-anion/immune complex and the previously treated, positively charged porous matrix and detected by using chemiluminescent signal measurements as described in co-pending U.S. Patent Application Serial No. 425,643, previously incorporated herein by reference. An ion capture-based competitive chemiluminescent immunoassay for two haptens can be performed according to this invention, as follows. The test sample which may contain any of the analytes of interest is contacted with a mixture of labeled antibodies. One antibody in the mixture is specific to the first hapten, and is labeled with an acridinium sulfonamide, an acridinium ester or a phenanthridine compound (label). The second antibody of the mixture is specific for the second hapten and is labeled with alkaline phosphatase. The test sample and mixture of labeled antibodies are incubated for a time and under conditions sufficient for hapten/anti-hapten complexes to form a reaction mixture. Then, a mixture of capture phase is added to the reaction mixture. The capture phase mixture comprises a mixture of two components each of which is a hapten bound to polyglutamic acid residue. The binding can be accomplished either directly or through a carrier.

Another alternative way of performing these assays according to the present invention is to use a combination of microparticle capture and ion capture separation procedures. Thus in a two-step sandwich assay, a sample suspected of containing multiple analytes can be incubated with a mixture of microparticles having bound thereto a member of specific binding pair for one analyte, and polyionic residues having bound thereto members of specific binding pairs for the second analyte. The reaction mixture is then transferred to a porous matrix that has been treated to carry an opposite charge to the polyionic residue. The microparticles captured analyte is retained on the porous matrix by hydrophobic interactions and the poly¬ ionic captured analyte is retained on the oppositely charged porous matrix by ionic interaction. The retained reaction products are washed and a mixture of conjugates is added on the porous matrix. One of these conjugates is labeled 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 to illustrate, but not to limit, the spirit and scope of the invention. EXAMPLES

Example 1. Preparation of Reagents

The phencyclidine (PCP) capture reagent was prepared by coupling the free acid form of polyglutamic acid with phenylcyclidine-4-chloroformate. The free acid form of polyglutamic acid was prepared from the sodium salt of polyglutamic acid according to the following procedure: 1 gm of polyglutamic acid sodium salt (Sigma Chemical Company; St. Louis, MO) was stirred overnight with 7 gms of AG5OW-X8 cation exchange resin (Bio-Rad Laboratories, Richmond, CA) suspended in 20 mL water. The ion exchange resin was previously swelled and washed in distilled water. The supernatant was separated from the resin and lyophilized to give about 0.8 g of the free 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 benzene (130 mole excess). The reaction was allowed to proceed for 2.5 hours at room temperature. Solvents were evaporated under a stream of nitrogen to yield a residue of PCP-4-chloroformate. The residue was dissolved in 0.5 mL tetrahydrofuran and 1.7 mg of the free acid form of polyglutamic acid (MW 40,000) in 0.5 mL of 1-Methyl-2-Pyrrolidinone (Aldrich Chemical Co., Milwaukee, Wl) was added to it. The reaction was carried out overnight at room temperature and then the reaction mixture was evaporated to dryness. The dried product was dissolved in 1.5 mL 0.1 M sodium phosphate buffer, pH 7.0, and was dialyzed against a volume of the same buffer in a 3,500 molecular weight cut-off dialysis bag overnight at room temperature. The precipitate was filtered. The cloudy aqueous filtrate was extracted with methylene chloride (Fisher Scientific, Itasca, IL) until it was clear.

A fluorescence polarization immunoassay confirmed the presence of PCP on the PGA residue (performed on Abbott TD_X®, available from Abbott Laboratories, Abbott Park, IL 60064). The aqueous layer was diluted in a solution containing 1% fish gelatin, 100 mM sodium chloride, 25 mM Tris, 1 mM magnesium chloride, 0.1 mM zinc chloride, 0.1% sodium azide, pH 7.2, to yield 1.875 μgm/mL of PCP-PGA capture reagent.

The moφhine capture reagent was prepared by coupling the isothiocyanate derivative of the free acid form of polyglutamic acid with morphine-ovalbumin. The free acid form of polyglutamic acid was activated by derivatizing it to an isothiocyanate (ITC-PGAFA). The isothiocyanate derivative was reacted with ovalbumin to form ovalbumin-PGA. Finally, morphine was coupled to ovalbumin- PGA using isobutylchloroformate to yield the capture reagent: morphine- ovalbumin-PGA.

The procedure used was as follows. Ten (10) mg of the free acid form of polyglutamic acid was dissolved in 1 ml dimethylformamide (DMF). 0.01 ml of proton adsorbing agent 4-methylmorpholine and 4.8 mg of 1 ,4-phenylene diisothfocyanate (DITC)(available from the Aldrich Chemical Co., Milwaukee, Wl) in 0.2 ml of dimethylformamide were added to this solution (100 mole excess). This reaction mixture was stirred at room temperature overnight and then it was concentrated using a rotary evaporator. Twenty-five (25) ml of methylene chloride was added dropwise to precipitate the ITC-PGAFA. The flocculent precipitate was centrifuged, and methylene chloride and unreacted DITC were decanted. The precipitate then was suspended in 1 ml of dimethylformamide. The precipitation/centrifugation suspension process was repeated until no DITC was detectable in the supernatant using thin-layer chromatography (TLC) on silica slides. The remaining solid was vacuum dried to yield the ITC-PGAFA as a yellow powder.

Ovalbumin-PGA was prepared according to the following procedure. Ten (10) mg ovalbumin (available from Sigma Chemical 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 μm 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 9.0. The pH then was adjusted to 8.5 with 1 N sodium hydroxide. The mixture was incubated overnight at 37°C, and then it was fractionated on an HPLC instrument using a TSK- 3000SWG preparative gel filtration column (available from Beckman Instruments, Arlington Heights, IL) run at 5 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 cut 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 μg/ml ovalbumin were dialyzed against a volume of 0.1 M sodium bicarbonate, pH 8.5, in a 3,500 molecular weight cut-off dialysis tube.

One (1) mg morphine 3-β-D-glucuronide (Sigma Chemical Co., St. Louis, MO) MW 461.5, was activated by reacting it with 100 mole excess isobutylchloroformate (MW 136.15, available from Sigma Chemical Co., St. Louis, MO) and 100 mole excess 4-methylmorpholine in DMF at 0°C for one-half hour. The activated morphine derivative was added to the dialyzed ovalbumin-PGA and kept in an ice bath for one (1) hour at 0-4°C. The reaction mixture then was kept at room temperature overnight. The product solution was dialyzed against 0.1 M sodium phosphate buffer at pH 8.5, in 3,500 molecular weight cut-off dialysis bag to remove the uncoupled morphine. The recovered dialysate was run in the fluorescence polarization immunoassay for morphine, using a commercial analyzer (TDχ®₍ Abbott Laboratories, North Chicago, IL), which gave 3.5 morphine molecules per ovalbumin-PGA residue. The capture reagent (morphine-ovalbumin- PGA) was diluted in a buffer solution containing 1% fish gelatin, 25 mM Tris, 100 mM sodium chloride, 1 mM magnesium chloride, 0.1 mM zinc chloride 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 reagents. Solutions were diluted together to give 1.875 μg/ml of phenylcyclidine-polyglutamic acid and 0.25 μg/ml of morphine- ovalbumin-polyglutamic acid in a final solution which contained 1% fish gelatin, 25 mM Tris, 100 mM sodium chloride, 1 mM magnesium chloride, 0.1 mM zinc chloride and 0.1% sodium azide at a pH of 7.2.

Monoclonal anti-phenylcyclidine antibody was labeled with acridinium sulfonamide using EDAC coupling procedures known in the art. It then was diluted in a buffer which contained 1% fish gelatin, 25 mM Tris, 100 mM sodium chloride, 1 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 anti-phenylcyclidine-acridinium probe. Its final concentration was 48.2 ng/ml.

The simultaneous assay probe consisted of a monoclonal anti-phenylcyclidine antibody conjugated to acridinium (118 ng/ml) 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-l ,2,dioxetane,

SUBST AMPPD (available from Tropix Inc., Bedford, MA) disodium salt, in a solution of 0.05 M sodium bicarbonate buffer containing 1 mM magnesium chloride at pH 9.5.

The disposable reaction tray used for this assay was that described in co- pending U. S. Patent Application Serial No. 425,651 , which enjoys common ownership and which is incorporated herein by reference. The device comprised a funnel-like structure, a porous element, and an absorbant material, which were assembled to provide intimate contact between the porous element and the absorbent material. The porous element was made of fibrous glass material, Product No. 4111 glass fiber filter paper, which had a nominal thickness of 0.05 inches and which is available from Hollingsworth and Voss Co., East Walpole, MA. It was treated with a 0.5% solution of a polymeric quaternary ammonium compound, Ceiquat ™L-200 (National Starch and Chemical Co., Bridgewater, NJ), to give the solid phase material a positive charge. 80 μl of 0.5% Celquat™L-200 in 10 mM sodium chloride was applied to each porous element of the disposable reaction tray.

Example 2. Acridinium-Labeled Phenylcyclidiπe Assay

Test samples used were phenylcyclidiπe calibrators from a commercially available fluorescence polarization kit ( TD_X® kit, Abbott Laboratories, North Chicago, IL) which contained 500, 250, 120, 60, 25 and 0 ng/ml PCP in human urine. 80 μl 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 μl of a 0.5% Celquat™L-200 solution on each pad. Solutions were dispensed using two FMI-RH pumps (Fluid Metering Inc., Oyster Bay,

NY), and controlled via a triac board by an Intel 310 Development System (Intel Inc., Sunnyvale, CA). The tray was moved on a linear track using a timing belt and a stepper motor. The stepper motor was controlled by a board employing components known in the art. After 4.8 minutes, 50 μl of test sample was pipetted into the shallow reaction well of the disposable reaction tray, using an automated pipettor.

50 μl 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 tunnel at 32°C. The timing belt steps at the rate of 0.8 inches per minute, the reaction tray was stationary for 36 seconds after each step for a reaction step to take place. After 9.6 minutes of incubation on the moving timing belt, 50 μl of a solution containing PCP- PGA capture reagent prepared as in Example 1 at a concentration of 118 πg PGA ml was dispensed into the incubation well through a tip centered on the well and connected to an FMI pump through a Teflon® line. The reaction mixture was further incubated while the disposable tray continued movement along the track. After 4.8 minutes, each quaternary ammonium polymer-treated glass fiber matrix was rinsed with 100 μl of IM_X® Tris buffer (available from Abbott Laboratories, North Chicago, IL) dispensed from a tip centered on the fibrous pad of the detection well. After 4.8 more minutes, the disposable reaction tray was located under the transfer device described in the co-pending Patent Application Serial No. 425,643, previously incorporated herein by reference. The 150 μl assay mixture then was transferred from the shallow incubation well onto the pre-treated glass fiber matrix in the detection well. Transfer was effected using one 350 μl pulse of IM_X ®Tris buffer injected from three adjacent nozzles at a linear rate of 210 cm/sec. The transfer fluid was injected by a stepper-motor controlled syringe pump. A valve directed the transfer solution to each side of the disposable reaction tray. The transferred mixture then was allowed to drain through the fibrous pad for 12 seconds. Then, 50 μl of the AMPPD substrate solution was dispensed in each 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 effect transfer of the reaction mixture. The disposable device then was moved to a detection position where a chemiluminescence detector (described in co-pending U. 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 counter/timer board constructed using components and methods known in the art. The two trigger solution injectors associated with each PMT were connected to an FMI pump using a black Teflon® 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 the disposable. The high voltage to the PMT was gated on, the two trigger solution pumps and the counter/timer boards were activated simultaneously by the Intel 310

Development System (previously described). After the substrate was incubated for 4.8 minutes, 85 μl of 0.3% alkaline peroxide trigger solution was injected and the resulting chemiluminescence was measured for eight (8) seconds from the onset of trigger solution injection to give the intensity of the short-lived chemiluminescence signal. The results of the assay are shown in Table 1. The chemiluminescence signal in the short-lived window followed the PCP concentration in the sample, while the long-lived signal had a slowly varying function of PCP concentration. A longer time window or a signal deconvolution scheme can be applied by those skilled in the art to

SUBSTIT improve the dose response curve in the 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 detergents to the transfer buffered solution, pretreatment of the glass fiber pad by addition of proteins such as fish gelatin, casein, etc., can be applied by those skilled in the art to improve the dose response curve, and these variations lie within the teachings of the invention. The cut-off of this assay was considered to be 25 ng/ml. The data from Table 1 indicate that all test samples which contained 25 ng/ml PCP or higher were well- differentiated from the negative control, which indicated the validity of the assay procedure.

TABLE 1

ACRIDINIUM-LABELED PHENYLCYCUDINE CHEMILUMINESCENCE IMMUNOASSAY

(CLIA)

*%l=Percent Inhibition Example 3. Alkaline Phosphatase Labeled Morphine Assay

Test samples used were calibrators from a commercially available opiates fluorescence polarization kit (TD_X® kit, Abbott Laboratories, North Chicago, IL) which contained 1000, 600, 350, 200, 100 and 0 ng/ml morphine in human urine. 80 μl of IMχ® Tris buffer solution (available from Abbott Laboratories, North Chicago, IL), followed by 80 μl of Celquat™L-200 solution were dispensed on the glass fiber matrices of a disposable reaction tray previously described in Example 2. Solutions were dispensed using two FMI-RH pumps and controlled via a triac board by an Intel 310 Development System (Intel Inc., Sunnyvale, CA). The tray was moved on a linear track using a timing belt and a stepper motor. The stepper motor was controlled by a board employing components known to those skilled in the art. After 4.8 minutes, 50 μl of alkaline phosphatase-iabeled anti-morphine antibody was 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 tunnel at 32°C. The timing belt steps at the rate of 0.8 inches per minute, the reaction tray was stationary for 36 seconds after each step for a reaction step to take place. After 9.6 minutes of incubation on the moving timing belt, 50 μl of a solution containing morphine-ovalbumine-PGA capture reagent as prepared in Example 1 at a concentration of 0.25 μg PGA/ml was dispensed into the incubation well through a tip centered on the well. The reaction mixture was further incubated while the disposable tray continued movement along the track. After 4.8 minutes, each quaternary ammonium polymer-treated glass fiber matrix was rinsed with 100 μl of IMχ® Tris buffer injected from three adjacent nozzles at a linear rate of 210 cm/sec. The transferred mixture was allowed to drain through the fibrous pad for 12 seconds. 50 μl of the AMPPD substrate solution prepared as previously described in Example 1 were then dispensed in each 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 affect transfer of the reaction mixture. The disposable device then was moved to a detection position where a chemiluminescence detector (described previously) was located. After a 4.8 minute substrate incubation time had elapsed, 85 μl of 0.3% alkaline peroxide trigger solution was injected, and the resulting chemiluminescence signal was integrated for eight (8) seconds after an eight (8) seconds delay from the onset of trigger solution injection to give the intensity of the long-lived chemiluminescence signal. The results of the assay are shown in Table 2. The cut-off of this assay was considered to be 100 ng/ml. The data in Table 2 indicate that all test samples which contained 100 ng/ml morphine or higher were well-differentiated from the negative control, which indicated the validity of the assay procedure.

Short -lived signal (convoluted)- short-lived signal - (long-lived signal x 0.655 )

Using this deconvolution scheme presented above, the last two columns of

Table 2 show that the chemiluminescence signal in the long-lived window is the dose response for morphine in the sample, while the corrected short-lived signal is independent of morphine concentration. A different time window or a signal deconvolution scheme can be applied by those skilled in the art to improve the dose response curve in the 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 detergents to the transfer buffered solution, pretreatment of the glass fibrous pad 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 dose response curve and lie within the teachings of this invention.

TABLE 2

ALKALINE PHOSPHATASE-LABELED MORPHINE CHEMILUMINESCENT IMMUNOASSAY (CLIA)

Example 4. Simultaneous Assay For Phenvlcvclidine and Morphine

Test samples used were phenylcyclidine calibrators from a commercially available fluorescence polarization kit (TD_X® kit, Abbott Laboratories, North Chicago, IL) at 500, 250, 120, 60, 25 and 0 ng/ml, or TD ® 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 μl of IMχ ® Tris buffer solution (available from Abbott Laboratories, North Chicago, IL) followed by 80 μl of 0.5% Celquat™L-200 solution were dispensed on glass fiber matrices of the disposable reaction tray previously described. Solutions were dispensed using two FMI-RH pumps and controlled via a triac board by an Intel 310 Development System (Intel, Inc., Sunnyvale, CA). The tray was moved on a linear track using a timing belt and stepper motor. The stepper motor was controlled by a board employing components known to those of ordinary skill in the art. After 4.8 minutes, 50 μl of test sample was pipetted into the shallow reaction well of the disposable reaction tray, using an automated pipettor. 50 μl of the simultaneous assay probe containing 118 ng/ml acridinium-labeled anti-PCP antibodies and 48.2 ng ml alkaline phosphatase labeled anti-morphine antibody prepared as described in Example 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 32°C. The timing belt steps at the rate of 0.8 inches per minute, the reaction tray was stationary for 36 seconds after each step for a reaction step to take place. After 9.6 minutes of incubation on the moving timing belt, 50 μl of the simultaneous assay capture reagent solution containing PCP-PGA at a concentration of 1.875 μg/ml and morphine-ovalbumin-PGA at a concentration of 0.25 μg/ml prepared as in Example 1 was dispensed into the incubation well through a tip centered on the well. The reaction mixture was further incubated while the disposable tray continued movement along the track. After 4.8 minutes, each quaternary ammonium polymer- treated glass fiber matrix was rinsed with 100 μl of IMχ® 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. 425,643. The 150 μi assay mixture then was transferred from the shallow incubation well onto the pre-treated glass fiber matrix in the detection well. Transfer was affected using one 350 μl pulse of IM_X® Tris buffer injected from three adjacent nozzles at a linear rate of 210 cm/sec. After 12 seconds of drain time, 50 μl of the substrate solution was dispensed in each 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 effect transfer of the reaction mixture. The disposable device then was moved to a detection position where a chemiluminescence detector (described previously) was located. After 4.8 minutes of substrate incubation time had elapsed, 85 μl of 0.3% alkaline peroxide trigger solution was injected into the detection well. The resulting chemiluminescence was integrated for eight (8) seconds immediately after the onset of trigger solution injection to give the intensity of the short-lived chemiluminescence signal. The chemiluminescence signal was integrated for another eight (8) subsequent seconds to give the long-lived chemiluminescence 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/ml. The data in Tables 3 and 4 indicate that all test samples which contained 25 ng/ml PCP or higher were well-differentiated from the negative control. The cut-off of the morphine assay was considered to be 100 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 simultaneous assay procedure as described herein. A longer time window or a signal deconvolution scheme are variations which can be applied by those skilled in the art to improve the dose response curve in the first window and eliminate it in the second window. Also, variations of assay optimization steps such as wash solution volume, wash solution composition, addition of detergents 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 dose response curve, and these variations lie within the teachings of this invention.

IΔBL 2 SIMULTANEOUS PHENYLCYCUDINE AND MORPHINE CHEMILUMINESCENCE ASSAY

[PCP] [Morphine] Short-lived %l Long-lived %l ng/ml ng/ml Signal (0-8 sec.) Signal (8-16 sec.)

TABLE 4

SIMULTANEOUS PHENYLCYCUDINE AND MORPHINE ASSAY

The dose response of each analyte in the simultaneous assay resembles that in the individual assay of the analyte, which indicates the ability to detect either of the two analytes in a test sample using the novel assay method of the invention. Although the present invention has been described in terms of preferred embodiments, it is anticipated that various modifications and improvements will occur to those skilled in the art upon consideration of the present invention. Thus, other assay configurations that include more than one capture reagent and more than one chemiluminescent probe with varying chemiluminescence signal life times is contemplated to lie within the teachings and scope of this invention. Various solid phases such as plastic tubes coated with mixtures of antibodies and detected in tube luminometers or plastic beads such as 1/4" polystyrene beads coated with mixtures of antibodies and transferred after the completion of the binding reaction to a test tube and then detected in a tube luminometer can be used as media for simultaneous assays based on chemiluminescence signal time resolution.

Microparticles can be made from any suitable paniculate material that are easily recognizable by those skilled in the art, such as polystyrene, polymethyl acrylate, derivatized cellulose fibers, polyacrylamide, and the like.

The ion capture procedures previously described used polyglutamic acid as the polyanion, and Celquat™L-200 as the polycation. However, other polyanionic materials and other derivatized polycationic material, as well as other methods of attachment of these compounds to the assay components or the porous element, is contemplated as obvious variations of this invention and are considered to lie within its scope.

Moreover, it is contemplated that the assay method of this invention may be extended to other small molecules, macromolecules, or to nucleic acid probe assays. Furthermore, although the present invention has been described using acridinium sulfonamide-labeled and alkaline phosphatase-labeled compounds as tracers, it is contemplated that other acridinium compounds or their analogs, or other 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 disease 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 B-|2 and folates or ferritin. Also usefully detected according to the method of the present invention are hormones such as LH and FSH, bacteria (e.g., streptococci), nucleic acid species (e.g., DNA or RNA). The present invention also is useful in small 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-codβine, 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 more than two analytes in the same assay procedure upon consideration of the teachings provided by the present invention. These choices thus are considered within the scope of the present invention.

Claims (21)

WHAT ISCLAtMED 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 conditions sufficient 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 binding pairs for the analytes which cover all the membrane or discrete regions of the membrane.

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.

5 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 0 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 analytes each capable of generating a short-lived or long-lived chemiluminescent signal and a solid phase which has a mixture of members of 5 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 D for the other label; and

( d ) integrating and time-discriminating the short-lived and the long- lived components of the chemiluminescence signal generated.

1 0. The method of claim 9 wherein said analytes are selected from the 5 group consisting of haptens, macromolecules, metabolites and antibodies.

1 1 . 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 0 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 5 different short-lived or long-lived chemiluminescent signal upon contact with a triggering 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, infectious disease 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 a triggering solution.

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

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

1 8. The kit of claim 15 wherein the compound generating the long-lived chemiluminescence signal is a β-galactosidase substrate.

1 9. 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;

SHEET ( 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 a short-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 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 iong- lived components of the chemiluminescence signal generated.