Simultaneous detection of multiple analytes

Abstract

Methods of detecting one or more analytes in a sample are described herein using different chemiluminescent labels. Typically the methods involve an algorithm that leverage different chemiluminescent kinetics in order to calculate the concentration of specific analytes mixed in a sample. Solid supports, reagents, and compounds for use in these methods are also described. Typically, the methods involve the detection of nearly simultaneous detection of multiple analytes in a sample via conjugation to different acridinium labels.

Description

SIMULTANEOUS DETECTION OF MULTIPLE ANALYTES CROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority to and the benefit of U.S. App. No.63 / 705,294, filed October 9, 2025, which is hereby incorporated by reference in its entirety. FIELD OF DISCLOSURE

[0001] The present disclosure relates to an assay method of detection or measurement of multiple analytes in a sample by measurement of multiple chemiluminescent signals. The signals may have different emission wavelengths and time frames, multiple chemiluminescent signals resulting from multiple acridinium labels, each having distinguishable emission speed (kinetics) from others and labeled to a binder specific to one of the multiple analytes present in the sample. BACKGROUND

[0002] Traditionally, the detection and differentiation of multiple analytes in a test sample has involved performing separate assays for each analyte. However, current trends in medical diagnostics are moving away from relying solely on a single analyte or biomarker for clinical decision-making. Instead, there is a growing recognition of the importance of incorporating multiple analytes and biomarkers, along with comprehensive testing algorithms, to ensure more precise and reliable healthcare outcomes. This broader approach requires the ability to not only simultaneously identify the presence of one or multiple analytes but also to be able to provide individual results for each analyte targeted.

[0003] The ability to provide multiple results holds the potential to enhance diagnostic accuracy and improve patient care. However, this same approach results in laboratories facing the challenge of accommodating an increasing number of tests within tight timeframes while striving to control costs.

[0004] Therefore, it would be beneficial to develop an assay that allows for the simultaneous detection and differentiation of multiple analytes while still enabling individual quality control for each analyte.SUMMARY

[0005] In accordance with the foregoing objectives and others, the present disclosure includes methods for the detection of analytes in a sample through the use of varying chemiluminescent labels. Detection of multiple analytes can be achieved by utilizing separate distinct AE molecules to distinguish between various analytes within the system. Such a method would offer the advantages of efficient and comprehensive analyte detection while maintaining the ability to assess and control the quality of each analyte individually.

[0006] The present disclosure includes method for the detection or quantification of multiple analytes in a sample (e.g., a biological sample such as blood, saliva, serum, a sample derived from a biological sample such as a diluted biological sample) which may comprise: (a) providing a first set of chemiluminescent labels capable of binding to a first analyte and a second set of chemiluminescent labels capable of binding to a second analyte, wherein the first set of chemiluminescent labels and the second set of chemiluminescent labels comprise at least two chemiluminescent labels having different rates of emission; wherein the first analyte is optionally different from the second analyte; (b) mixing the first set and said second set with said sample; (c) preparing the mixture of said first set, second set, and sample to measure chemiluminescence from the first set and the second set of chemiluminescent labels; (d) triggering chemiluminescence from the first set and second set of chemiluminescent labels following preparation of the mixture (e.g., by the addition of one or more triggering compositions which trigger chemiluminescence of the acridinium labels); (e) measuring the chemiluminescence in the time domain (e.g., measuring light intensity as a function of time from the triggering step) at more than one time point; (f) calculating the concentration of the first analyte and the second analyte in the sample using the measured chemiluminescent signal at the more than one time point and a coefficient matrix comprising the slope of the chemiluminescent signal (e.g., with respect to analyte concentration, with respect to a metric such as the total RLUoutput associated which may be associated with analyte concentration)of the first and second analyte individually obtained (e.g., chemiluminescence obtained at a range of concentrations of standards comprising the analyte not in a mixture) at the more than one time point. In particular embodiments, the at least two chemiluminescent labels are selected from the compounds in Table 1 (e.g., wherein the reactive functional group is used to conjugate to the first or second analyte). For example, the at least two chemiluminescent labels may be NSP- DMAE-NHS and TSPAE. The slope of the chemiluminescent signal of each analyte at each timepoint in the coefficient matrix may be based on at least two (e.g., at least three, at least four, two to four) different concentrations or another metric associated with concentration such as total RLU output of each analyte. The coefficient matrix may include columns associated with the analytes, each column including the slopes correlated with the analyte of that column at each timepoint. In various implementations, the coefficient matrix may be based on a polymeric expression (e.g., Taylor series) to correlate the chemiluminescent signal at each timepoint to concentration. In various embodiments, the coefficient matrix may include rows associated with the analytes, each row including the metrics of the curve shape (e.g. slopes correlated with the analyte of that column at each timepoint (and the matrix multiplication described below may not involve any inversion)). In some embodiments, the coefficient matrix comprises a first column comprising the slopes of the first analyte (with respect to analyte concentration) and a second column comprising the slopes of the second analyte signal (e.g., with respect to analyte concentration, with respect to a metric such as the total RLU output associated which may be associated with analyte concentration). For example, the calculating step may be peformed by multiplying the inverse (e.g., inverse, left-inverse, right inverse, Moore-Penrose inverse) of the coefficient matrix with a measured chemiluminescent matrix (e.g., single column matrix) comprising the relative light units (RLUs) at one or more of the more than one time points. In some embodiments, the calculating step is peformed by multiplying the inverse of the coefficient matrix with a measured chemiluminescent matrix (e.g., single column matrix) comprising the relative light units (RLUs) at each of the more than one time points. Typically, the calculating step is performed by multiplying the Moore-Penrose inverse of the coefficient matrix with the measured chemiluminescent matrix.

[0007] In various implementaions, the measuring step may comprise the addition of one or more chemiluminescence triggering reagents to said separated solid support and / or said separated mixture.

[0008] In some embodiments, the solid support comprises at least two molecules (e.g., two, three, four, five, six, seven, eight, nine ten), each capable of forming a binding complex with different analytes and capable of forming a binding complex with at least two different chemiluminescent labels in the first set and / or second set of chemiluminescent labels.

[0009] The first set of chemiluminescent labels and the second set of chemiluminescent labels may independently have the structure of formula (I): (I)wherein A is an analyte or binding partner for an analyte, L is absent (i.e., it is a bond) or a linker optionally comprising a group LCor ZL, and Ψ is a chemiluminescent acridinium comprising the structure: “j” and “k” are independentlyall R3 groups are hydrogen), 1, 2, 3, or 4; R1 is hydrogen, –R, –Xb, –RL–Xb, –LC–R, –LC–Xb(e.g., –L1–Xb), –Z, –RL–Z, –LC–Z (e.g., – L1–Z), or –RL–LC–RL–Z (e.g., –RL–L1–RL–Z); R2and R3are independently selected at each occurrence from hydrogen, –R, an electron donating group, and –Z; wherein two vicinal R2 or R3 groups may together form a fused cyclic group (e.g., 5-7 membered fused aryl or heteroaryl group, 5-7 membered fused heterocyclic group) and wherein R2or R3may comprise a linkage to an imaging agent such as a fluorophore (e.g., rhodamine); LCis a divalent C1-35 alkyl, alkenyl, alkynyl, aryl, or arylalkyl radical, optionally substituted (e.g., with 1 to 20 heteroatoms, with 1-20 substituents);ZLis a zwitterionic linker group having the structure: ; “m” is 0 (i.e. it is a bond) or 1;“n” and “p” are independently at each occurrence an integer from 0 (i.e. it is a bond) to 10; Z is a zwitterionic group independently at each occurrence has the structure: ; “q” and “l” are“r” is independently an integer from 0 to 10 (e.g., from 1 to 10, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10); Xaand Xbare independently at each occurrence an anionic group; L1is independently at each occurrence –O–, –S–, –NH–, –N(RN)–, –(CH2)1-10–, –S(=O)1-2–, –C=C–, –C=C–(CH2)1-3–, –C(O)–, –O–C(O)–, –C(O)–(CH2)1-4–, –(CH2)1-4–C(O)–, –C(O)–O– , –C(O)–N(RN)–, –C(O)–NH–, –N(RN)–C(O)–, –NH–C(O)–, –C(O)–N(RN)–(CH2)1-3–, – (CH2)1-3–C(O)–N(RN)–, –(CH2)1-3–N(RN)–C(O)–, –NH–S(O)1-2–, –N(RN)–S(O)1-2–, –S(O)1-2– N(RN)–, –S(O)1-2–NH–, –(CH2)1-3–NH–S(O)1-2–, –(CH2)1-3–N(RN)–S(O)1-2–, –(CH2)1-3–S(O)1-2–N(RN)–, –(CH2)1-3–S(O)1-2–NH–, –O–(CH2)1-4–, –(CH2)1-4–O–, –S–(CH2)1-4–, –(CH2)1-4–S– , –NH–(CH2)1-4–, –N(RN)–(CH2)1-4–, –(CH2)1-4–N(RN)–, –(OCH2)1-10–, –(CH2O)1-10–, – (OCH2CH2)1-10–, or –(CH2CH2O)1-10–; RLis independently at each occurrence a C1-20 bivalent hydrocarbon radical (e.g., alkyl, alkenyl, aryl, phenyl, mono alkyl substituted phenyl, di alkyl substituted phenyl, alkynyl, arylalkyl), optionally having one or more (e.g., 1-10, 1-5) points of substitution (e.g., with 1-10 heteroatoms, with 1-10 substituents);R is independently at each occurrence hydrogen or C1-35 hydrocarbon (e.g., alkyl, alkenyl, alkynyl, or aralkyl) radical, optionally having one or more (e.g., 1-20, 1-10, 1-5) points of substitution (e.g., with 1-20 heteroatoms, with 1-20 substituents); R’ and R” are independently at each occurrence hydrogen or a C1-10alkyl; RNis independently at each occurrence from hydrogen or C1-5alkyl (e.g., methyl, ethyl, propyl); and R’ is hydrogen or a C1-10alkyl; or a salt thereof (e.g., a halide salt such as a chloride salt, a sulfonate salt such as a halosulfonate salt, a haloalkyl sulfonate salt a fluoroalkyl sulfonate salt, a carboxylate salt such as a haloalkyl carboxylate salt, fluoroalkyl carboxylate salt).

[0010] In some embodiments, the first analyte is HIV-1 and the second analyte is HIV-2. In various aspects, the first set of chemiluminescent labels comprise a monoclonal antibody capable of binding to the first analyte and the second set of chemiluminescent labels comprise a monoclonal antibody capable of binding to the second analyte.

[0011] In some embodiments, the method further comprises providing a third set of chemiluminescent labels capable of binding to a third analyte and the coefficient matrix comprises the slope of the chemiluminescent signal signal (e.g., with respect to analyte concentration, with respect to a metric such as the total RLU output associated which may be associated with analyte concentration) of the first, second, and third analytes individually obtained at a range of concentrations at the more than one time point. For example, the third analyte may be HIV-1 p24 antigen.

[0012] Systems for the detection of multiple analytes in a sample are also provided. The system may comprise: a) light detectors (e.g., photomultiplier tubes), wherein the light detectors measure chemiluminescent signal from the sample in binned time points; b) a computer system in communication with the light detectors; wherein the light detectors communicate the chemiluminescent signal (or relative light units (RLU)) to the computer system at each binned time point;wherein the computer system may further comprise a processor that accesses memory or a storage device comprising data for a coefficient matrix comprising the slope of the chemiluminescent signal (e.g., with respect to analyte concentration, with respect to a metric such as the total RLU output associated which may be associated with analyte concentration) of the two or more of the multiple analytes individually obtained (e.g., chemiluminescence obtained at a range of concentrations of standards comprising the analyte not in a mixture) at the binned time points; and the processor may access instructions to convert the chemiluminescent signal to analyte concentration with the coefficient matrix. In some embodiments, the instructions comprise multiplication of the coefficient matrix (or an inverse thereof) with a single column matrix the measured RLU signal from the light detectors. In some embodiments, the instructions comprise a constant correction to the measured RLU signal (e.g., prior to multiplication with the coefficient matrix).

[0013] In some embodiments, the sample is blood, saliva, urine, cerebrospinal fluid (CSF), or serum. In some embodiments, the sample derived from a biological sample such as a diluted biological sample (e.g., as mixed with saline).

[0014] The analyte may also be bound to the chemiluminescent label (via the analyte and the intermediary conjugate). Accordingly, by providing one or more immunoassay reagents comprising multiple chemiluminescent labels and / or one or more intermediary conjugates, detection of multiple analytes may be achieved through a single binding complex.

[0015] Various assay formats may be employed which afford the ability to measure many analytes nearly simultaneously (e.g., by collection of the chemiluminescence from a single chemiluminescence event such as one initiated by the addition of one or more triggering agents).

[0016] Each set, and each chemiluminescent label within a set, may be mixed with the biological sample in any order. For example, in some embodiments, one or more of the chemiluminescent labels is mixed with the sample individually. In some embodiments, the first set of chemiluminescent labels is mixed with the sample together, such as by the addition of a composition comprising the chemiluminescent labels in the first set. In some embodiments, the second set of chemiluminescent labels is mixed with the sample together,such as by the addition of a composition comprising the chemiluminescent labels in the second set. In some embodiments, the first and second set of chemiluminescent labels is mixed with the sample together, such as by the addition of a composition comprising the chemiluminescent labels in the first set and second set.

[0017] Following addition of the two sets of chemiluminescent labels, the sample is typically prepared to induce chemiluminesence in a manner that the analytes can be measured and / or their concentration quantified. For example, the preparing step may comprise: (c1) providing a solid support having immobilized thereon a molecule capable of forming a binding complex with said at least one analyte and capable of forming a binding complex with a chemiluminescent label in the first set and / or the second set of chemiluminescent labels; and (c2) separating said solid support from said mixture. In some embodiments, the solid support may comprise at least two molecules (e.g., two, three, four, five, six, seven, eight, nine ten), each capable of forming a binding complex with different analytes and capable of forming a binding complex with at least two different chemiluminescent labels in the first set and / or second set of chemiluminescent labels. In some embodiments, the solid support may comprise molecules capable of forming a binding complex with an analyte or binding partner thereof, wherein, collectively, the molecules can bind with each chemiluminescent label in the first and / or second set.

[0018] For certain assays, there may be an advantage to include positive control and / or negative control, or calibrators into the test. Also, for certain assay combination there may be an advantage to measure the analytes in the same reaction vessel. E.g., if an algorithm like a ratio is required, e.g., for the measurement of placental growth factor (PLGF) and soluble FMS- like tyrosine kinase-1 (sFlt-1) to aid in the diagnosis or prognosis of preeclampsia, the variability of the results may be reduced, because the same sample aliquot is used and the processing errors are the same for each analyte. Therefore, it is desirable to develop an assay method where multiple analytes present in a sample can be detected or measured in one test reaction. On the other hand, there are a number of diagnostic assays that are frequently tested as a group, such as a thyroid test panel, a liver fibrosis (e.g., the Enhanced Liver Fibrosis (ELF™)) test panel, a fertility hormone test panel, a cancer marker screen panel, a human T-lymphotropic virus (e.g., HTL-V) test panel, and HIV antibody and antigen panels. The systems and methods described herein may be used on these test panels.

[0019] In another aspect of the invention, a reagent is provided for the detection of multiple analytes comprising a detectable conjugate bound to a chemiluminescent acridinium, wherein different detectable conjugates are bound to different chemiluminescent acridiniums which have chemiluminescence separated in the time domain (e.g., fast reaction kinetics, slow reaction kinetics). The detectable conjugate may comprise one or more (e.g., one, two) zwitterionic functional groups. The reagent may comprise a concentration of each detectable conjugate of from 10 to 30 ng / mL. Reagents of the present disclosure include compositions comprising the indicated components, and optionally an excipient, carrier, or solvent. The reagents of the present disclosure may include a surfactant.

[0020] These and other aspects of the invention will be better understood by reference to the following detailed description including the appended claims. BRIEF DESCRIPTION OF FIGURES

[0021] FIG. 1A provides a set of flash curves for an chemiluminescent immunoassay measuring HIV-1 at low, medium, and high concentrations. The inset provides the slope of the RLU with respect to these concentrations at timepoints T1, T2, and T3.

[0022] FIG. 1B provides a set of flash curves for an chemiluminescent immunoassay measuring HIV-2 at low, medium, and high concentrations. The inset provides the slope of the RLU with respect to these concentrations at timepoints T1, T2, and T3.

[0023] FIG. 1C provides a schematic for construction and calibration of the coefficient matrix.

[0024] FIG.2A provides the RLU measured of a mixture of unknown concentrations of HIV- 1 and HIV-2 (dotted) and the respective RLU contributions of each component (as determined by the coefficient matrix analysis provided herein).

[0025] FIG. 2B provides the algebraic matrix multiplication that may be used to determine the relative concentrations of HIV1 and HIV2 using the measured RLU output (RLUMIX) and the coefficient matrix.DETAILED DESCRIPTION

[0026] For convenience, certain terms employed in the specification, including the examples and appended claims, are collected here. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0027] Unless otherwise explicitly defined, the following terms and phrases are intended to have the following meanings throughout this disclosure:

[0028] All percentages given herein refer to the weight percentages of a particular component relative to the entire composition, including the carrier, unless otherwise indicated. It will be understood that the sum of all weight % of individual components within a composition will not exceed 100%.

[0029] The terms “a” or “an,” as used in herein means one or more. As used herein, the term “consisting essentially of” is intended to limit the invention to the specified materials or steps and those that do not materially affect the basic and novel characteristics of the claimed invention, as understood from a reading of this specification. Recitations of “comprising” include “consisting essentially” and “consisting.”

[0030] The following definitions of various groups or substituents are used, unless otherwise described. Specific and general values listed below for radicals, substituents, and ranges, are for illustration only; they do not exclude other defined values or other values within defined ranges for the radicals and substituents. Unless otherwise indicated, alkyl, alkenyl, alkynyl, alkoxy, and the like denote straight, branched, and cyclic groups, as well as any combination thereof.

[0031] The term hydrocarbon may refer to a radical or group containing carbon and hydrogen atoms which may be bound at an indicated position (e.g., R, R’, R”, RN, Y, Y’, Ω, L1, LC, RL, RC, R1, R2, R2a, R2b, R2c, R3, R4, R5, R6, R7). Examples of hydrocarbon radicals include, without limitation, alkyl, alkenyl, alkynyl, aryl, aryl-alkyl, alkyl-aryl, and any combination thereof (e.g., alkyl-aryl-alkyl). As used herein, unless otherwise indicated, hydrocarbons may be monovalent or multivalent (e.g., divalent, trivalent) hydrocarbon radicals. A radical of the form –(CH2)n–, including a methylene radical, i.e., –CH2–, is regarded as an alkyl radical if it doesnot have unsaturated bonds between carbon atoms. Unless otherwise specified, all hydrocarbon radicals (including substituted and unsubstituted alkyl, alkenyl, alkynyl, aryl, aryl-alkyl, alkyl- aryl) may have from 1-35 carbon atoms. In other embodiments, hydrocarbons will have from 1-20 or from 1-12 or from 1-8 or from 1-6 or from 1-3 carbon atoms, including for example, embodiments having one, two, three, four, five, six, seven, eight, nine, or ten carbon atoms. Hydrocarbons may have from 2 to 70 atoms or from 4 to 40 atoms or from 4 to 20 atoms.

[0032] A substituted hydrocarbon may have as a substituent one or more hydrocarbon radicals, substituted hydrocarbon radicals, or may comprise one or more heteroatoms. Any hydrocarbon substituents disclosed herein (e.g., R, R’, R”, RN, Y, Y’, Ω, L1, LC, RL, RC, R1, R2, R2a, R2b, R2c, R3, R4, R5, R6, R7) may optionally include from 1-20 (e.g., 1-10, 1-5) heteroatoms. Examples of substituted hydrocarbon radicals include, without limitation, heterocycles, such as heteroaryls. Unless otherwise specified, a hydrocarbon substituted with one or more heteroatoms will comprise from 1-20 heteroatoms. In other embodiments, a hydrocarbon substituted with one or more heteroatoms will comprise from 1-12 or from 1-8 or from 1-6 or from 1-4 or from 1-3 or from 1-2 heteroatoms. Examples of heteroatoms include, but are not limited to, oxygen, nitrogen, sulfur, phosphorous, halogen (e.g., F, Cl, Br, I), boron, or silicon. In some embodiments, heteroatoms will be selected from the group consisting of oxygen, nitrogen, sulfur, phosphorous, and halogen (e.g., F, Cl, Br, I). In certain embodiments, the heteroatoms may be selected from O, N, or S. In some embodiments, a heteroatom or group may substitute a carbon. In some embodiments, a heteroatom or group may substitute a hydrogen. In some embodiments, a substituted hydrocarbon may comprise one or more heteroatoms in the backbone or chain of the molecule (e.g., interposed between two carbon atoms, as in “oxa”). In some embodiments, a substituted hydrocarbon may comprise one or more heteroatoms pendant from the backbone or chain of the molecule (e.g., covalently bound to a carbon atom in the chain or backbone, as in “oxo”).

[0033] When an indicated group is substituted with an indicated substituent, the specified group may be substituted with one or more of any or all of the named substituents. For example, where a group, such as an alkyl or heteroaryl group, is substituted with an unsubstituted C1-C20alkyl, or unsubstituted 2 to 20 membered heteroalkyl, the group may contain one or more unsubstituted C1-C20 alkyls, and / or one or more unsubstituted 2 to 20 membered heteroalkyls. Moreover, where a moiety is substituted with an R substituent, the group may be referred to as “R-substituted.” Where a moiety is R-substituted, the moiety is substituted with at least one Rsubstituent and each R substituent is optionally different. If an indicated group is used multiple times in chemical genus (e.g., R groups), it will be understood that each group is independently selected at each occurrence.

[0034] Unless otherwise specified, any compound disclosed herein which has one or more chiral centers may be in the form of a racemic mixture with respect to each chiral center, or may exist as pure or substantially pure (e.g., great than 98% ee) R or S enantiomers with respect to each chiral center, or may exist as mixtures of R and S enantiomers with respect to each chiral center, wherein the mixture comprises an enantiomeric excess of one or the other configurations, for example an enantiomeric excess (of R or S) of more than 60% or more than 70% or more than 80% or more than 90%, or more than 95%, or more than 98%, or more than 99% enantiomeric excess. In some embodiments, any chiral center may be in the “S” or “R” configurations.

[0035] It will be understood that the description of compounds herein is limited by principles of chemical bonding. Accordingly, where a group may be substituted by one or more of a number of substituents, such substitutions are selected so as to comply with principles of chemical bonding such as regard to valencies, and to give compounds which are not inherently unstable. For example, any carbon atom will be bonded to two, three, or four other atoms, consistent with the four valence electrons of carbon.

[0036] Substituent (radical) prefix names may be derived from the parent hydride by either (i) replacing the “ane” or in the parent hydride with the suffixes “yl,” “diyl,” “triyl,” “tetrayl;” or (ii) replacing the “e” in the parent hydride with the suffixes “yl,” “diyl,” “triyl,” “tetrayl,” (here the atom(s) with the free valence, when specified, is (are) given numbers as low as is consistent with any established numbering of the parent hydride). Accepted contracted names, e.g., adamantyl, naphthyl, anthryl, phenanthryl, furyl, pyridyl, isoquinolyl, quinolyl, and piperidyl, and trivial names, e.g., vinyl, allyl, phenyl, and thienyl are also used herein throughout.

[0037] Alkyl groups typically refer to a saturated hydrocarbon chain that may be a straight chain or branched chain, containing the indicated number of carbon atoms. For example, C1- C6alkyl indicates that the group may have from 1 to 6 (inclusive) carbon atoms in it. Any atom can be optionally substituted, e.g., by one or more substituents. Examples of alkyl groups include without limitation methyl, ethyl, n-propyl, isopropyl, and tert-butyl. Any alkyl groupreferenced herein (e.g., R, R’, R”, RN, Y, Y’, Ω, L1, LC, RL, RC, R1, R2, R2a, R2b, R2c, R3, R4, R5, R6, R7) may have from 1-35 carbon atoms. In other embodiments, alkyl groups will have from 1-20 or from 1-12 or from 1-8 or from 1-6 or from 1-3 carbon atoms, including for example, embodiments having one, two, three, four, five, six, seven, eight, nine, or ten carbon atoms. Alkyl groups may be lower alkyl (e.g., C1-C4 alkyl).

[0038] Haloalkyl groups are typically alkyl groups where at least one hydrogen atom is replaced by halo. In some embodiments, more than one hydrogen atom (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14) are replaced by halo. In these embodiments, the hydrogen atoms can each be replaced by the same halogen (e.g., fluoro) or the hydrogen atoms can be replaced by a combination of different halogens (e.g., fluoro and chloro). Haloalkyl may include alkyl moieties in which all hydrogens have been replaced by halo (sometimes referred to herein as perhaloalkyl, e.g., perfluoroalkyl, such as trifluoromethyl). Haloalkyl groups may be optionally substituted.

[0039] Typically, alkoxy groups have the formula –O(alkyl). Alkoxy can be, for example, methoxy (-OCH3), ethoxy, propoxy, isopropoxy, butoxy, iso-butoxy, sec-butoxy, pentoxy, 2- pentoxy, 3-pentoxy, or hexyloxy. Likewise, the term “thioalkoxy” refers to a group of formula –S(alkyl). Finally, the terms “haloalkoxy” and “halothioalkoxy” refer to –O(haloalkyl) and – S(haloalkyl), respectively. The term “sulfhydryl” refers to –SH. As used herein, the term “hydroxyl,” employed alone or in combination with other terms, refers to a group of formula – OH. Any alkoxy, thioalkoxy, or haloalkoxy group referenced herein (e.g., R, R’, R”, RN, Y, Y’, Ω, L1, LC, RL, RC, R1, R2, R2a, R2b, R2c, R3, R4, R5, R6, R7) may have from 1-35 carbon atoms. In other embodiments, alkoxy, thioalkoxy, or haloalkoxy groups will have from 1-20 or from 1-12 or from 1-8 or from 1-6 or from 1-3 carbon atoms, including for example, embodiments having one, two, three, four, five, six, seven, eight, nine, or ten carbon atoms. Alkoxy groups may be lower alkoxy (e.g., C1-C4 alkoxy).

[0040] Aralkyl groups typically refers to groups where an alkyl moiety in which an alkyl hydrogen atom is replaced by an aryl group. One of the carbons of the alkyl moiety serves as the point of attachment of the aralkyl group to another moiety. Any ring or chain atom can be optionally substituted, e.g., by one or more substituents. Non-limiting examples of aralkyl include benzyl, 2-phenylethyl, and 3-phenylpropyl groups.

[0041] The term “alkenyl” may refer to a straight or branched hydrocarbon chain containing the indicated number of carbon atoms and having one or more carbon-carbon double bonds. Any atom can be optionally substituted, e.g., by one or more substituents. Alkenyl groups can include, e.g., vinyl, allyl, 1-butenyl, and 2-hexenyl. One of the double bond carbons can optionally be the point of attachment of the alkenyl substituent. Any alkenyl group referenced herein (e.g., R, R’, R”, RN, Y, Y’, Ω, L1, LC, RL, RC, R1, R2, R2a, R2b, R2c, R3, R4, R5, R6, R7) may have from 1-35 carbon atoms. In other embodiments, alkenyl groups will have from 1-20 or from 1-12 or from 1-8 or from 1-6 or from 1-3 carbon atoms, including for example, embodiments having one, two, three, four, five, six, seven, eight, nine, or ten carbon atoms.

[0042] The term alkynyl may refer to a straight or branched hydrocarbon chain containing the indicated number of carbon atoms and having one or more carbon-carbon triple bonds. Alkynyl groups (e.g., R, R’, R”, RN, Y, Y’, Ω, L1, LC, RL, RC, R1, R2, R2a, R2b, R2c, R3, R4, R5, R6, R7) can be optionally substituted, e.g., by one or more substituents. Alkynyl groups can include, e.g., ethynyl, propargyl, and 3-hexynyl. One of the triple bond carbons can optionally be the point of attachment of the alkynyl substituent.

[0043] The term heterocyclyl typically refers to a fully saturated, partially saturated, or aromatic monocyclic, bicyclic, tricyclic, or other polycyclic ring system having one or more constituent heteroatom ring atoms independently selected from O, N (it is understood that one or two additional groups (e.g., RN) may be present to complete the nitrogen valence and / or form a salt), or S. The heteroatom or ring carbon can be the point of attachment of the heterocyclyl substituent to another moiety. Any atom can be optionally substituted, e.g., with one or more substituents (e.g. heteroatoms or substituent groups X). Heterocyclyl groups can include, e.g., tetrahydrofuryl, tetrahydropyranyl, piperidyl (piperidino), piperazinyl, morpholinyl (morpholino), pyrrolinyl, and pyrrolidinyl. By way of example, the phrase “heterocyclic ring containing from 5-6 ring atoms, wherein from 1-2 of the ring atoms is independently selected from N, NH, N(C1-C6alkyl), NC(O)(C1-C6alkyl), O, and S; and wherein said heterocyclic ring is optionally substituted with from 1-3 independently selected R” would include (but not be limited to) tetrahydrofuryl, tetrahydropyranyl, piperidyl (piperidino), piperazinyl, morpholinyl (morpholino), pyrrolinyl, and pyrrolidinyl.

[0044] The term heterocycloalkenyl typically refers to partially unsaturated monocyclic, bicyclic, tricyclic, or other polycyclic hydrocarbon groups having one or more (e.g., 1-4)heteroatom ring atoms independently selected from O, N (it is understood that one or two additional groups may be present to complete the nitrogen valence and / or form a salt), or S. A ring carbon (e.g., saturated or unsaturated) or heteroatom can be the point of attachment of the heterocycloalkenyl substituent. Any atom can be optionally substituted, e.g., by one or more substituents. Heterocycloalkenyl groups can include, e.g., dihydropyridyl, tetrahydropyridyl, dihydropyranyl, 4,5-dihydrooxazolyl, 4,5-dihydro-1H-imidazolyl, 1,2,5,6-tetrahydro- pyrimidinyl, and 5,6-dihydro-2H-[1,3]oxazinyl.

[0045] Cycloalkyl groups may be fully saturated monocyclic, bicyclic, tricyclic, or other polycyclic hydrocarbon groups. Any atom can be optionally substituted, e.g., by one or more substituents. A ring carbon serves as the point of attachment of a cycloalkyl group to another moiety. Cycloalkyl moieties can include, e.g., cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, adamantyl, and norbornyl (bicycle[2.2.1]heptyl).

[0046] Cycloalkenyl groups may be partially unsaturated monocyclic, bicyclic, tricyclic, or other polycyclic hydrocarbon groups. A ring carbon (e.g., saturated or unsaturated) is the point of attachment of the cycloalkenyl substituent. Any atom can be optionally substituted, e.g., by one or more substituents. Cycloalkenyl moieties can include, e.g., cyclohexenyl, cyclohexadienyl, or norbornenyl.

[0047] Aryl groups are often aromatic monocyclic, bicyclic (2 fused rings), tricyclic (3 fused rings), or polycyclic (> 3 fused rings) hydrocarbon ring system. One or more ring atoms can be optionally substituted, e.g., by one or more substituents. Aryl moieties include, e.g., phenyl and naphthyl.

[0048] Heteroaryl groups typically are aromatic monocyclic, bicyclic (2 fused rings), tricyclic (3 fused rings), or polycyclic (> 3 fused rings) hydrocarbon groups having one or more heteroatom ring atoms independently selected from O, N (it is understood that one or two additional groups may be present to complete the nitrogen valence and / or form a salt), or S in the ring. One or more ring atoms can be optionally substituted, e.g., by one or more substituents. Examples of heteroaryl groups include, but are not limited to, 2H-pyrrolyl, 3H-indolyl, 4H- quinolizinyl, acridinyl, benzo[b]thienyl, benzothiazolyl, β-carbolinyl, carbazolyl, coumarinyl, chromenyl, cinnolinyl, dibenzo[b,d]furanyl, furazanyl, furyl, imidazolyl, imidizolyl, indazolyl, indolyl, isobenzofuranyl, isoindolyl, isoquinolyl, isothiazolyl, isoxazolyl, naphthyridinyl, oxazolyl, perimidinyl, phenanthridinyl, phenanthrolinyl, phenarsazinyl, phenazinyl,phenothiazinyl, phenoxathiinyl, phenoxazinyl, phthalazinyl, pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridyl, pyrimidinyl, pyrrolyl, quinazolinyl, quinolyl, quinoxalinyl, thiadiazolyl, thianthrenyl, thiazolyl, thienyl, triazolyl, and xanthenyl.

[0049] In general, when a definition for a particular variable includes both hydrogen and non-hydrogen (halo, alkyl, aryl) possibilities, the term “substituent(s) other than hydrogen” refers collectively to the non-hydrogen possibilities for that particular variable, unless otherwise specified.

[0050] In general, the limits (end points) of any range recited herein are within the scope of the invention and should be understood to be disclosed embodiments. Additionally, any half- integral value within that range is also contemplated. For example, a range of from 0 to 4 expressly discloses 0, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, and any subset within that range (e.g., from 1 to 2.5).

[0051] The term “substituent” may refer to a group “substituted” on, on a hydrocarbon (e.g., an alkyl, haloalkyl, cycloalkyl, heterocyclyl, heterocycloalkenyl, cycloalkenyl, aryl, heteroaryl) group at any atom of that group, typically replacing one or more hydrogen atoms therein. In one aspect, the substituent(s) on a group (e.g., R, R’, R”, RN, Y, Y’, Ω, L1, LC, RL, RC, R1, R2, R2a, R2b, R2c, R3, R4, R5, R6, R7) are independently any one single, or any combination of two or more of the permissible atoms or groups of atoms delineated for that substituent. In another aspect, a substituent may itself be substituted with any one of the above substituents. In some embodiments, an indicated substituent is not further substituted. Further, as used herein, the phrase “optionally substituted” means unsubstituted (e.g., substituted with an H) or substituted. It is understood that substitution at a given atom is limited by valency. Common substituents include halo (e.g. F), C1-12 straight chain or branched chain alkyl, C2-12 alkenyl, C2-12alkynyl, C3-12cycloalkyl, C6-12aryl, C3-12heteroaryl, C3-12heterocyclyl, C1-12alkylsulfonyl, nitro, cyano, –COOR, –C(O)NRR’, –OR, –SR, –NRR’, and oxo, such as mono- or di- or tri-substitutions with moieties such as trifluoromethoxy, chlorine, bromine, fluorine, methyl, methoxy, pyridyl, furyl, triazyl, piperazinyl, pyrazoyl, imidazoyl, and the like, each optionally containing one or more heteroatoms such as halo, N, O, S, and P. R and R’ are independently hydrogen, C1-12 alkyl, C1-12 haloalkyl, C2-12 alkenyl, C2-12 alkynyl, C3-12 cycloalkyl, C4-24cycloalkylalkyl, C6-12aryl, C7-24aralkyl, C3-12heterocyclyl, C3-24heterocyclylalkyl, C3-12 heteroaryl, or C4-24 heteroarylalkyl. Unless otherwise noted, all groupsdescribed herein optionally contain one or more common substituents, to the extent permitted by valency. The term “substituted” typically means that a hydrogen and / or carbon atom is removed and replaced by a substituent (e.g., a common substituent). The use of a substituent (radical) prefix names such as alkyl without the modifier “optionally substituted” or “substituted” is understood to mean that the particular substituent is unsubstituted. However, the use of “haloalkyl” without the modifier “optionally substituted” or “substituted” is still understood to mean an alkyl group, in which at least one hydrogen atom is replaced by halo and any other associated substitutions as necessary. Any hydrocarbon described herein may be considered optionally substituted.

[0052] When a moiety of the compounds of the present disclosure are described as an analyte or a binding partner thereof, it will be understood that a covalent linkage is formed with an analyte or binding partner thereof (e.g., using the reactive functional groups which form covalent linkages), for example, by replacing a hydrogen on the unconjugated analyte or binding partner thereof with a covalent bond to the indicated moiety. The covalent linkage on the analyte or binding partner thereof may be formed, for example, at a group on the analyte, binding partner thereof, or derivatized version of the analyte containing a group for forming a linkage. The group may be, for example, an amine group, a thiol group, a carboxy group, a maleimidyl group, or a carbohydrate group. For example, if a covalent linkage is formed through a primary amine of the analyte or binding partner thereof, the compound may have the structure: where the unconjugated analyte or binding partner A has the structure A’–NH2.

[0053] The methods and systems described herein primarily use an algorithm which makes use of the differing chemical kinetics of different AE molecules which generate distinct time- varying AE flash curves, where the algorithm is capable of leveraging differences in these flash curves to obtain concentrations of each analyte in a mixture. Typically, the algorithm is premised on the generation of a coefficient matrix that could be used to resolve mixtures of two or more analytes.

[0054] This present disclosure relates to immunoassays and provides an improved assay for the simultaneous detection and differentiation of individual analytes in a test sample from asingle one-time chemiluminescence flash. The technique utilizes a heterogeneous mixture of solid phases to capture all targeted analytes but subsequently employs two distinct acridinium esters (AE) to enable the differentiation of each analyte within the single reaction. With this approach, a mixture of complexes comprising of solid phase and various analytes are generated. These complexes are then incubated and bound with binding partner(s) that are each labeled with distinct AE molecules. The capability to not just detect but also differentiate between the presence of multiple analytes in a single reaction depends on the capacity to separate the individual contributions of each AE molecule within a single standardized flash curve. The output of this design would not only be able to identify the presence or either or both analytes in the test sample but would be able to identify each specific analyte reaction in the single test sample and thus provide two separate results, one for each analyte.

[0055] In the methods described herein, the sample may undergo treatment similar to a traditional immunoassay. A solid phase reagent may be used, which comprises a mixture of particles specific to the targeted analytes. The sample may be incubated with the solid phase reagent to create solid phase / analyte complexes. These formed complexes may then be washed and further incubated with a single detection reagent. The detection reagent contains different binding partners, each specific to one of the targeted analytes and labeled with a different unique AE molecule.

[0056] The presence of one or more analytes in the sample is determined by distinguishing between the signals generated by each individual complex formed with a binding partner labeled with distinct AE molecules. The AE flash curves generated by the mixture of AE- analyte complexes serve as an indication of the presence of one or more analytes in the test sample. The basic AE flash curve is processed with an algorithm to separate and identify the different individual analytes within the sample. As shown herein, the ability to separate and identify specific individual analytes within a single sample can be replicated with a high level of accuracy and specificity. The algorithm typically processes the distinctive flash curves produced by the AE-analyte complexes and uses specific criteria to differentiate and identify each analyte. This enables reliable and precise detection of multiple analytes within the sample, enhancing the overall diagnostic or analytical capabilities of the method.

[0057] The coefficient matrix affords for the conversion of a set of RLU (taken at a set of time points) into the corresponding concentrations of each analyte measured. The flash curvesof two or more of the analytes in the mixture are typically individually obtained (when not in mix) for all required analyte concentration levels (including and between blank to highest concentration of the given analyte wanted to be measured). At each timepoint (T) the RLU vs a metric to be calculated (e.g., concentration, total integrated RLU for each analyte) may be plotted and / or the corresponding slope (ε) may be obtained. This slope gives an indication of the shape of the flash curves at timepoint (T). For example, the timepoints around the peak of the flash curve would have larger slopes than at the timepoints at the end of the flash curve. The slopes obtained at each time point are then populated into the coefficient matrix where, for example, each column of the matrix represents the measured correlation and shape parameters for the individual analyte (e.g., slopes, Taylor coefficients) and each row represents a unique timepoint. For a mixture of 2 analytes, the coefficient matrix would have 2 columns and a number of rows matching the time points measured (e.g., 1280 rows representing each timepoint on the flash curve).

[0058] The coefficient matrix for an analyte mixture may be obtained once and the same matrix can be operated on flash curves of unknown mixtures, to obtain the constituent analytes (e.g., and stored on a computer system for integration in a processor). In some embodiments, the computer system stores the coefficient matrix. In some embodiments, the computer system stores the inverse of the coefficient matrix. In some embodiments, the computer system stores the Moore-Penrose pseudoinverse of the coefficient matrix. In various implementations, the computer system stores a constant matrix which may be used to apply corrections to the measured light outputs at each timepoint (e.g., prior to multiplication with the coefficient matrix).

[0059] Using the coefficient matrix to convert the RLU dataset to metric to be calculated (e.g., concentration, total integrated RLU for each analyte) can be achieved through matrix multiplication. For example, the concentration of n analytes (e.g., n is 2, 3, 4, 5) may be calculated by: ^^ି^ ^^^^^^^ … ^^^^^^^^^^^^^^^ெூ^^^^^^ ^^^^^^^ ^^^^^^^^^^^wherein an as 1, 2, 3, 4, 5, 6) with respect to analyte concentration (e.g., as measured from two or more (e.g., three,four, five) individual analyte concentrations spanning, for example, the range of interest for analyte concentrations) at timepoint Ty, y represents the total number of measured time points (e.g., from 1 to 10000, from 100 to 5000, from 1000 to 3000), RLUMIX(Ty) represents the RLU of the mixture of analytes at timepoint Ty, C(Ty) is a constant associated at each time point, and ConcAn is the measured concentration of analyte An in the mixture as calculated using the coefficient matrix and the measured chemiluminescent data. In some embodiments, the RLU measured from the mixture associated with each analyte may be computed directly using a coefficient matrix based on total RLU output of each individual analyte sample: ^^ି^ ^^^^^^^ … ^^^^^^^^^^^^^^^ெூ^^^^^^ ^^^^^^^ ^^^^^^^^൭^^^^^^^ଶ^ … ^^^^^^^ଶ^^ ^൭ ^^^^^^ெூ^^^^ଶ^^ െ ൭^^^^^ଶ^^^ ൌ ൭… ^ …… …… … ^^^^^^^^wherein ^^^^^^^௬^ represents the slope of individual analyte An (n is an integer such as 1, 2, 3, 4, 5, 6) with respect to RLU output (e.g., as measured from two or more (e.g., three, four, five) individual analyte concentrations, wherein the RLU output may represent, for example, the total integrated signal, or the signal integrated over a portion output curve (e.g., from the peak to steady state and / or 0 chemiluminescence, from 0 s to the peak or some combination thereof). In these embodiments, the concentration of each individual analyte RLU contribution to the measured mixture may then be calculated by comparison to standard curves (e.g., the concentration is calculated first by determining the each RLUA1…RLUAnwith the coefficient matrix and then, optionally, converting those RLUA1…RLUAn to concentrations. The constant matrix: ^^^^^^^ ^may be used to provide a correction light output of the mixture at each timepoint (e.g., RLUMIX(Ty)). For example, each element in the constant matrix may represent the extrapolated value of the shape correlation at no analyte concentration (e.g., y-intercept). For example, for linear extrapolations represented by the function RLUn(Ty) = εn× Concn(Ty) + cn, where RLUn represents the measured RLU output for analyte n at a concentration (Concn) at timepoint Ty and εnis the slope, the constant matrix may be populated by each extrapolated c (or y-intercept). In various implementation, each element in the constant matrix (e.g., C(Ty)) is the sum of the y-intercepts for the analytes measured.

[0060] The inverse of the coefficient matrix may be the Moore-Penrose inverse of the coefficient matrix. The Moore-Penrose inverse is typically determined using the singular value decomposition of the coefficient matrix such that when C=UΣVTwhere C is a m×n coefficient matrix, U may be a m×m unitary matrix, Σ may be an m×n rectangular diagonal matrix, V may be an n×n unitary matrix, and VTis the conjugate transpose of V. The Moore-Penrose inverse may be VΣ-1UT(and Σ-1may be an inverse such as the Moore-Penrose inverse of Σ). The Moore-Penrose inverse may be calculated (or the same result as the calculation performed by) the pinv Matlab function. In some embodiments, the calculation is performed by solving a least squares fit for the function: Conc = C RLUmix + c, where Conc is the vector represented the concentration for each analyte, C is the coefficient matrix, RLUmix is the measured RLU output vector, and c is the extrapolated value vector of the shape correlation at no analyte concentration (e.g., y-intercept). For example, Conc may be calculated (or the same as the result as the calculation performed by) the lsqnonneg Matlab function.

[0061] Typically, the chemiluminescent labels have the structure of formula (I): (I)wherein A is an analyte or binding partner for an analyte, L is absent (i.e., it is a bond) or a linker optionally comprising a group LCor ZL, and Ψ is a chemiluminescent acridinium comprising the structure: “j” and “k” areall R3groups are hydrogen), 4; R1 is hydrogen, –R, –Xb, –RL–Xb, –LC–R, –LC–Xb(e.g., –L1–Xb), –Z, –RL–Z, –LC–Z (e.g., – L1–Z), or –RL–LC–RL–Z (e.g., –RL–L1–RL–Z); R2and R3are independently selected at each occurrence from hydrogen, –R, an electron donating group, and –Z; wherein two vicinal R2 or R3 groups may together form a fusedcyclic group (e.g., 5-7 membered fused aryl or heteroaryl group, 5-7 membered fused heterocyclic group) and wherein R2or R3may comprise a linkage to an imaging agent such as a fluorophore (e.g., rhodamine); LCis a divalent C1-35 alkyl, alkenyl, alkynyl, aryl, or arylalkyl radical, optionally substituted (e.g., with 1 to 20 heteroatoms, with 1-20 substituents); ZLis a zwitterionic linker group having the structure: ; “m” is 0 (i.e. it is a bond) or 1;“n” and “p” are independently at each occurrence an integer from 0 (i.e. it is a bond) to 10; a zwitterionic group independently at each occurrence has the structure: ; “q” and “l” are independently“r” is independently an integer from 0 to 10 (e.g., from 1 to 10, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10); Xaand Xbare independently at each occurrence an anionic group; L1 is independently at each occurrence –O–, –S–, –NH–, –N(RN)–, –(CH2)1-10–, –S(=O)1-2–, –C=C–, –C=C–(CH2)1-3–, –C(O)–, –O–C(O)–, –C(O)–(CH2)1-4–, –(CH2)1-4–C(O)–, –C(O)–O–, –C(O)–N(RN)–, –C(O)–NH–, –N(RN)–C(O)–, –NH–C(O)–, –C(O)–N(RN)–(CH2)1-3–, –(CH2)1-3–C(O)–N(RN)–, –(CH2)1-3–N(RN)–C(O)–, –NH–S(O)1-2–, –N(RN)–S(O)1-2–, –S(O)1-2–N(RN)–, –S(O)1-2–NH–, –(CH2)1-3–NH–S(O)1-2–, –(CH2)1-3–N(RN)–S(O)1-2–, –(CH2)1-3–S(O)1-2–N(RN)–, –(CH2)1-3–S(O)1-2–NH–, –O–(CH2)1-4–, –(CH2)1-4–O–, –S–(CH2)1-4–, –(CH2)1-4–S–, –NH–(CH2)1-4–, –N(RN)–(CH2)1-4–, –(CH2)1-4–N(RN)–, –(OCH2)1-10–, –(CH2O)1-10–, –(OCH2CH2)1-10–, or –(CH2CH2O)1-10–;RLis independently at each occurrence a C1-20 bivalent hydrocarbon radical (e.g., alkyl, alkenyl, aryl, phenyl, mono alkyl substituted phenyl, di alkyl substituted phenyl, alkynyl, arylalkyl), optionally having one or more (e.g., 1-10, 1-5) points of substitution (e.g., with 1-10 heteroatoms, with 1-10 substituents); R is independently at each occurrence hydrogen or C1-35hydrocarbon (e.g., alkyl, alkenyl, alkynyl, or aralkyl) radical, optionally having one or more (e.g., 1-20, 1-10, 1-5) points of substitution (e.g., with 1-20 heteroatoms, with 1-20 substituents); R’ and R” are independently at each occurrence hydrogen or a C1-10alkyl; RNis independently at each occurrence from hydrogen or C1-5alkyl (e.g., methyl, ethyl, propyl); and R’ is hydrogen or a C1-10alkyl; or a salt thereof (e.g., a halide salt such as a chloride salt, a sulfonate salt such as a halosulfonate salt, a haloalkyl sulfonate salt a fluoroalkyl sulfonate salt, a carboxylate salt such as a haloalkyl carboxylate salt, fluoroalkyl carboxylate salt). For example, the chemiluminescent labels may independently have the structure of formula (Ia): wherein Ω is O or N;Y is selected from –R or –RL–Z, or in the case where Ω is O then Y is absent; and Y’ is either absent (i.e. it is a bond), or is selected from –L1–, –RL–, –RL–L1–, –L1–L1–, –L1–RL–, –L1–RL–L1, and –RL–L1–RL–. In some embodiments, the chemiluminescent labels independently have the structure of formula (Ib) or (Ic):(Ib) (Ic) wherein R4-R7are independently hydrogen, an electron donating group, or C1-35alkyl, alkenyl, alkynyl, aryl, alkoxy, alkylthio, or amino; and Y” is either absent (i.e., it is a bond) or–LC–, –L1–, –RL–, or –RL–L1–. In some embodiments, at least one (e.g., one, two, three, four, five, six, each) chemiluminescent label in the first set is a zwitterionic acridinium (e.g., N-sulfopropyl zwitterionic acridinium, a compound having the structure of formula I, Ia, Ib, or Ic wherein R1is selected from –Xb, –RL–Xb, or –LC–Xbsuch as –L1–Xb; R1 is selected from –SO3-, –RL– SO3- such as –(CH2)1-5– SO3-, or –LC– SO3- such as –L1– SO3-). In some embodiments, at least one (e.g., one, two, three, four, five , six, each) chemiluminescent label in the second set is a zwitterionic acridinium (e.g., N-sulfopropyl zwitterionic acridinium, a compound having the structure of formula I, Ia, Ib, or Ic wherein R1is selected from –Xb, –RL–Xb, or –LC–Xbsuch as –L1–Xb, R1 is selected from –SO3-, –RL– SO3- such as –(CH2)1-5– SO3-, or –LC– SO3- such as –L1– SO3-). In some embodiments, at least one (e.g., one, two, three, four, five, six, each) chemiluminescent label in the first and / or second set is an acridinium salt (e.g., acridinium carboxylate salts such as halocarboxylate salts, haloalkyl carboxylate salts, fluoroalkyl carboxylate salts, acridinium sulfonate salts such a halo sulfonatesalts, haloalkyl sulfonate salts, fluoroalkyl sulfonate salts, acridinium halide salts such as acridinium chloride salts, a compound having the structure of formula I, Ia, Ib, or Ic wherein R1 is selected from –R, –LC–R, –Z, –RL–Z, –LC–Z,–L1–Z, –RL–LC–RL–Z, –RL–L1–RL–Z with a negative counterion such as R–COO-, R–SO3-, Cl-, F-).

[0062] Measurably differential chemiluminesence between chemiluminesent labels (e.g., in the wavelength domain and / or in the chemiluminesence rate domain) can be achieved through use of acridinium labels with relevant conjugations to induce such a differentian. For example, in some embodiments, at least one of R4-R7(e.g., R4, R5, R7, R7) are an electron donating group (e.g., and forms a chemiluminescent label with different emission speed as compared to an otherwise identical label without the electron donating group). In some embodiments, one chemiluminescent label in the first set has the structure of formula (IIIa): and another chemiluminescentwherein A1 and A2 areand, optionally, A1is different than A2; Ω is O or N; Y is selected from –R or –RL–Z, or in the case where Ω is O then Y is absent; andY’ is either absent (i.e. it is a bond), or is selected from –L1–, –RL–, –RL–L1–, –L1–L1–, –L1–RL–, –L1–RL–L1, and –RL–L1–RL–; “j” is 1, 2, 3, or 4; “k” is 0 (e.g., all R2groups are hydrogen, all R3groups are hydrogen), 1, 2, 3, or 4; R1 is hydrogen, –R, –Xb, –RL–Xb, –LC–R, –LC–Xb(e.g., –L1–Xb), –Z, –RL–Z, –LC–Z (e.g., – L1–Z), or –RL–LC–RL–Z (e.g., –RL–L1–RL–Z); R2and R3are independently selected at each occurrence from hydrogen, –R, an electron donating group, and –Z; wherein two vicinal R2 or R3 groups may together form a fused cyclic group (e.g., 5-7 membered fused aryl or heteroaryl group, 5-7 membered fused heterocyclic group) and wherein R2 or R3 may comprise a linkage to an imaging agent (IA) such as a fluorophore (e.g., rhodamine); and at least one R2group is not hydrogen (e.g., at least one R2 is an electron donating group such as –OG, at least one R2 group comprises a linkage to an imaging agent); LCis a divalent C1-35alkyl, alkenyl, alkynyl, aryl, or arylalkyl radical, optionally substituted (e.g., with 1 to 20 heteroatoms, with 1-20 substituents); ZLis a zwitterionic linker group having the structure: ; “m” is 0 (i.e. it is a bond) or 1;“n” and “p” are independently at each occurrence an integer from 0 (i.e. it is a bond) to 10; group independently at each occurrence has the structure: ;10, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10);Xaand Xbare independently at each occurrence an anionic group; L1 is independently at each occurrence –O–, –S–, –NH–, –N(RN)–, –(CH2)1-10–, –S(=O)1-2–, –C=C–, –C=C–(CH2)1-3–, –C(O)–, –O–C(O)–, –C(O)–(CH2)1-4–, –(CH2)1-4–C(O)–, –C(O)–O–, –C(O)–N(RN)–, –C(O)–NH–, –N(RN)–C(O)–, –NH–C(O)–, –C(O)–N(RN)–(CH2)1-3–, –(CH2)1-3–C(O)–N(RN)–, –(CH2)1-3–N(RN)–C(O)–, –NH–S(O)1-2–, –N(RN)–S(O)1-2–, –S(O)1-2–N(RN)–, –S(O)1-2–NH–, –(CH2)1-3–NH–S(O)1-2–, –(CH2)1-3–N(RN)–S(O)1-2–, –(CH2)1-3–S(O)1-2–N(RN)–, –(CH2)1-3–S(O)1-2–NH–, –O–(CH2)1-4–, –(CH2)1-4–O–, –S–(CH2)1-4–, –(CH2)1-4–S–, –NH–(CH2)1-4–, –N(RN)–(CH2)1-4–, –(CH2)1-4–N(RN)–, –(OCH2)1-10–, –(CH2O)1-10–, –(OCH2CH2)1-10–, or –(CH2CH2O)1-10–; RLis independently at each occurrence a C1-20 bivalent hydrocarbon radical (e.g., alkyl, alkenyl, aryl, phenyl, mono alkyl substituted phenyl, di alkyl substituted phenyl, alkynyl, arylalkyl), optionally having one or more (e.g., 1-10, 1-5) points of substitution (e.g., with 1-10 heteroatoms, with 1-10 substituents); R is independently at each occurrence hydrogen or C1-35hydrocarbon (e.g., alkyl, alkenyl, alkynyl, or aralkyl) radical, optionally having one or more (e.g., 1-20, 1-10, 1-5) points of substitution (e.g., with 1-20 heteroatoms, with 1-20 substituents); R’ and R” are independently at each occurrence hydrogen or a C1-10alkyl; RNis independently at each occurrence from hydrogen or C1-5alkyl (e.g., methyl, ethyl, propyl); and R’ is hydrogen or a C1-10 alkyl; or a salt thereof (e.g., a halide salt such as a chloride salt, a sulfonate salt such as a halosulfonate salt, a haloalkyl sulfonate salt a fluoroalkyl sulfonate salt, a carboxylate salt such as a haloalkyl carboxylate salt, fluoroalkyl carboxylate salt). For example, the chemiluminescent label of formula (IIIb) may have the structure of formula (IIIb1):(IIIb1) . In some embodiemnts, at least one R3is not hydrogen (e.g., an electron donating group such as alkoxy). In some embodiments, least one R3 and / or at least one R2 group is an electron donating group (e.g., alkoxy). In various implementations, the first set of chemiluminescent labels further comprise a compound having the structure of formula (IIIc): wherein IA is an imaging agentmodified rhodamine); and A3 is a different analyte or binding partner for an analyte than A1 and A2. In some embodiments, the chemiluminescent label of formula (IIIc) has the structure of formula (IIIc1) or (IIIc2):(IIIc1) (IIIc (2I)IIb3 ) . For example, the chemiluminescent label may have the structure: or(IIIb3 ) . In some embodiments, the chemiluminescent label of formula (IIIb) may have the structure of formula (IIIb3): wherein IA is an imagingrhodamine). For example, the chemiluminescent label of formula (IIIb3) may have the structure of formula (IIIb4) or (IIIb5):(IIIb3) (IIIb (4I)IIb3 ) . For example, the chemiluminescent label may have the structure: or(IIIb3 ) .

[0063] In some embodiments, one chemiluminescent label in the first set has the structure of formula (IVa): and another chemiluminescentwherein A1 and A2 areis different than A2; Ω is O or N; Y is selected from –R or –RL–Z, or in the case where Ω is O then Y is absent; andY’ is either absent (i.e. it is a bond), or is selected from –L1–, –RL–, –RL–L1–, –L1–L1–, –L1–RL–, –L1–RL–L1, and –RL–L1–RL–; “k” is 0 (e.g., all R2groups are hydrogen, all R3groups are hydrogen), 1, 2, 3, or 4; is hydrogen, –R, –Xb, –RL–Xb, –LC–R, –LC–Xb(e.g., –L1–Xb), –Z, –RL–Z, –LC–Z (e.g., – L1–Z), or –RL–LC–RL–Z (e.g., –RL–L1–RL–Z); R3 is independently selected at each occurrence from hydrogen, –R, an electron donating group, and –Z; wherein two vicinal R2 or R3 groups may together form a fused cyclic group (e.g., 5- 7 membered fused aryl or heteroaryl group, 5-7 membered fused heterocyclic group) and wherein R2or R3may comprise a linkage to an imaging agent such as a fluorophore (e.g., rhodamine). LCis a divalent C1-35alkyl, alkenyl, alkynyl, aryl, or arylalkyl radical, optionally substituted (e.g., with 1 to 20 heteroatoms, with 1-20 substituents); ZLis a zwitterionic linker group having the structure: ; “m” is 0 (i.e. it is a bond) or 1;“n” and “p” are independently at each occurrence an integer from 0 (i.e. it is a bond) to 10; group independently at each occurrence has the structure: ;10, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10); Xaand Xbare independently at each occurrence an anionic group; L1 is independently at each occurrence –O–, –S–, –NH–, –N(RN)–, –(CH2)1-10–, –S(=O)1-2–, –C=C–, –C=C–(CH2)1-3–, –C(O)–, –O–C(O)–, –C(O)–(CH2)1-4–, –(CH2)1-4–C(O)–,–C(O)–O–, –C(O)–N(RN)–, –C(O)–NH–, –N(RN)–C(O)–, –NH–C(O)–, –C(O)–N(RN)–(CH2)1-3–, –(CH2)1-3–C(O)–N(RN)–, –(CH2)1-3–N(RN)–C(O)–, –NH–S(O)1-2–, –N(RN)–S(O)1-2–, –S(O)1-2–N(RN)–, –S(O)1-2–NH–, –(CH2)1-3–NH–S(O)1-2–, –(CH2)1-3–N(RN)–S(O)1-2–, –(CH2)1-3–S(O)1-2–N(RN)–, –(CH2)1-3–S(O)1-2–NH–, –O–(CH2)1-4–, –(CH2)1-4–O–, –S–(CH2)1-4–, –(CH2)1-4–S–, –NH–(CH2)1-4–, –N(RN)–(CH2)1-4–, –(CH2)1-4–N(RN)–, –(OCH2)1-10–, –(CH2O)1-10–, –(OCH2CH2)1-10–, or –(CH2CH2O)1-10–; RLis independently at each occurrence a C1-20 bivalent hydrocarbon radical (e.g., alkyl, alkenyl, aryl, phenyl, mono alkyl substituted phenyl, di alkyl substituted phenyl, alkynyl, arylalkyl), optionally having one or more (e.g., 1-10, 1-5) points of substitution (e.g., with 1-10 heteroatoms, with 1-10 substituents); R is independently at each occurrence hydrogen or C1-35hydrocarbon (e.g., alkyl, alkenyl, alkynyl, or aralkyl) radical, optionally having one or more (e.g., 1-20, 1-10, 1-5) points of substitution (e.g., with 1-20 heteroatoms, with 1-20 substituents); R’ and R” are independently at each occurrence hydrogen or a C1-10alkyl; RNis independently at each occurrence from hydrogen or C1-5alkyl (e.g., methyl, ethyl, propyl); and R’ is hydrogen or a C1-10 alkyl; or a salt thereof (e.g., a halide salt such as a chloride salt, a sulfonate salt such as a halosulfonate salt, a haloalkyl sulfonate salt a fluoroalkyl sulfonate salt, a carboxylate salt such as a haloalkyl carboxylate salt, fluoroalkyl carboxylate salt).

[0064] In some embodiments, any hydrocarbon or substituted hydrocarbon disclosed herein (e.g., R, R’, R”, RN, Y, Y’, Ω, L1, LC, RL, RC, R1, R2, R2a, R2b, R2c, R3, R4, R5, R6, R7) may be substituted with one or more (e.g., from 1-6 or from 1-4 or from 1-3 or one or two or three) substituents X, where X is independently selected at each occurrence from one or more (e.g., 1-20) heteroatoms or one or more (e.g., 1-10) heteroatom-containing groups, or X is independently selected at each occurrence from –F, –Cl, –Br, –I, –OH, –OR*, –NH2, –NHR*, –N(R*)2, –N(R*)3+, –N(R*)–OH, –N(→O)(R*)2, –O–N(R*)2, –N(R*)–O–R*, –N(R*)– N(R*)2, –C=N–R*, –N=C(R*)2, –C=N–N(R*)2, –C(=NR*)(–N(R*)2), –C(H)(=N–OH), –SH, –SR*, –CN, –NC, –CHF2, –CCl3, –CF2Cl, –CFCl2, –C(=O)–R*, –CHO, –CO2H, –C(O)CH3, – CO2-, –CO2R*, –C(=O)–S–R*, –O–(C=O)–H, –O–(C=O)–R*, –S–C(=O)–R*, –(C=O)–NH2,–C(=O)–N(R*)2, –C(=O)–NHNH2, –O–C(=O)–NHNH2, –C(=S)–NH2, –(C=S)–N(R*)2, – N(R*)–CHO, –N(R*)–C(=O)–R*, –C(=NR)–OR*, –O–C(=NR*)–R*, –SCN, –NCS, –NSO, – SSR*, –N(R*)–C(=O)–N(R*)2, –CH3, –CH2–CH3, –CH2–CH2–CH3, –C(H)(CH2)2, –C(CH3)3, –N(R*)–C(=S)–N(R*)2, –S(=O)1-2–R*, –O–S(=O)2–R*, –S(=O)2–OR*, –N(R*)–S(=O)2–R*, –S(=O)2–N(R*)2, –O–SO3, –O–S(=O)2–OR*, –O–S(=O)–OR*, –O–S(=O)–R*, –S(=O)–OR*, –S(=O)–R*, –NO, –NO2, –NO3, –O–NO, –O–NO2, –N3, –N2–R*, –N(C2H4), –Si(R*)3, –CF3, –O–CF3, –O–CHF2, –O–CH3, –O–(CH2)1-6CH3, –OC(H)(CH2)2 –OC(CH3)3, –PR*2, –O– P(=O)(OR*)2, or –P(=O)(OR*)2; where, independently at each occurrence, R* may be H or a C1-10 or C1-8 or C1-6 or C1-4 hydrocarbon, including without limitation alkyl, alkenyl, alkynyl, aryl (e.g., phenyl), alkyl-aryl (e.g., benzyl), aryl-alkyl (e.g., tolyl) In some embodiments, X may comprise a C1-C8or C1-C6or C2-C4perfluoroalkyl. In some embodiments, X may be a C1-C8 or C2-C6 or C3-C5 heterocycle (e.g., heteroaryl radical). The term “halo” or “halogen” refers to any radical of fluorine, chlorine, bromine or iodine. In some embodiments, X is independently selected at each occurrence from –OH, –SH, –NH2, –N(R*)2, –C(O)OR*, – C(O)NR*R*, –C(O)NR*R*, –C(O)OH, –C(O)NH2, F, or –Cl. In some embodiments, X is F. R and R* may be, independently at each occurrence, saturated or unsaturated alkyl (e.g., C1- C8alkyl). In some embodiments, R and R* are independently selected from hydrogen, methyl, ethyl, propyl, or isopropyl. In some embodiments, R and R* are independently selected from hydrogen, methoxy, ethoxy, propoxy, or isopropoxy. In some embodiments, X is –CF3or –O– CF3.

[0065] LCmay have the structure: –(X1)0-1–(RL)0-5–(X2)0-1– (RL)0-5–(X3)0-1–(RL)0-5–(X4)0-1–(RL)0-5– wherein X1is selected from =N–, –O–, –S–, or –NRN–; X2–X4are independently selected from –O–, –S–, –NRN–, –C(O)–, –NRN–C(O)–, –C(O)– NRN–, –O–C(O)–, or –C(O)–O–, –S–C(O)–, or –C(O)–S–; and RLis independently selected at each occurrence from –CH2–, –(CH2CH2O)–, or –(OCH2CH2)–. In various embodiments, LCcomprises at least one atom (or at least two atoms) in the chain between A and Ψ (or between A and ZL).

[0066] Anionic groups, such as Xaand Xbmay be, for example, independently at each occurrence carboxylate (–C(O)O-), sulfonate (–SOିଷ), sulfate (–OSOିଷ), phosphate (–OP(O)(ORP)O-), or oxide (–O-), and RPis hydrogen or C1-12 hydrocarbon optionally having one or more (e.g., 1-10, 1-5) points of substitution (e.g., with 1-10 heteroatoms, with 1-10 substituents). For example, R1 may comprise (or be) –RL–SOିଷ (e.g., sulfopropyl). In some embodiments, R1 comprises (or is) sulfopropyl. In some embodiments, R1 is –S(O)2–NH–Z or –(CH2)1-3–S(O)2–NH–Z. In various implementations, R2 and R3 are independently at each occurrence hydrogen, alkyl, or alkoxy (e.g., lower alkoxy such as C1-C4alkoxy, methoxy, ethoxy, propoxy, isopropoxy). In some embodiments, R2 and R3 are each hydrogen. In other embodiments, one of R2or R3is hydrogen and the other of R2or R3is alkoxy (e.g., lower alkoxy such as C1-C4 alkoxy, methoxy, ethoxy, propoxy, isopropoxy). In some embodiments, Xais sulfonate (–SOିଷ), m is 1, RLis propyl, and n and p are each 3. For example, ZLmay have the structure: .

[0067] The a material in a sample such as an analyte (e.g., a biomolecule). In some embodiments, the analyte is a thyroid hormone (e.g., a thyroid stimulating hormone and, for example, A is a binding partner therefor such as an anti-thyroid stimulating hormone monoclonal antibody (AntiTSH-mAb)), an androgen, a steroid hormone (e.g., androstenedione, testosterone), a troponin, thyroglobulin, anti-thyroid peroxidase antibody, triiodothyronine (T3) hormone, thyroxine (T4) hormone, thyroxine- binding globulin (TBG), neurofilament light chain (e.g., serum neurofilament light chain), a vitamin (e.g., vitamin-D such as 25-hydroxy-vitamin D), or an antibody for a virus (e.g., hepatitis).

[0068] The substituents on the chemiluminescent acridinium ester may be modified to vary the rate and yield of light emission, to reduce the non-specific binding, increase stability, or increase hydrophilicity. Typically, these modifications will have minimal interference substantially with the binding of the analyte and its binding partner. Examples of substituent variability are disclosed in Natrajan et al. in U.S. Pat No 7,309,615, hereby incorporated byreference herein, which describes high quantum yield acridinium compounds containing electron donating groups such as alkoxy groups (OR*) at, for example, C2 and / or C7, wherein R* is a group comprising a sulfopropyl moiety or ethylene glycol moieties (e.g., – O(CH2CH2O)0-5CH3) or combinations thereof. In some embodiments, R2(e.g., R2a, R2b, R2c) and / or R3 may be independently at each occurrence hydrogen an electron donating group such as an alkoxy groups (e.g., OR such as –O(CH2CH2O)0-5CH3and / or OR*.). Natrajan et al. in International Pub. No. WO2015 / 006174, hereby incorporated by reference in its entirety, also describes hydrophilic high quantum yield, chemiluminescent acridinium esters possessing certain electron-donating functional groups at the C2 and / or C7 positions as well. These electron donating groups (–OG) may have the structure: wherein R9-R14area methyl group or a group – (CH2CH2O)aCH3, where a is an integer from 1 to 5.

[0069] Ψ may comprise two flanking methyl groups on a phenolic ester to stabilize the bond as disclosed in Law et al. Journal of Bioluminescence and Chemiluminescence 4: 88-89 (1989), hereby incorporated by reference in its entirety. The sets of chemiluminescent labels may be modified in a manner to achieve the proper separation of chemiluminescent wavelength or emission. In some embodiments Ψ (in one or more of the sets of chemiluminescent labels) has the structure:, , , ,, , , , ,, , , , or .G may be independently selected at each occurrence from, for example, hydrogen, alkyl (e.g., C1-C4alkyl), –(CH2CH2O)1-10–OCH3such as –(CH2CH2O)2–OCH3or –(CH2CH2O)5–OCH3and branched groups having the structure:wherein R9-R14 area methyl group or a group –(CH2CH2O)aCH3, where a is an integer from 1 to 5.

[0070] In some embodiments, A, L and Ψ are each covalently linked. Portions of the covalent linkage between A and Ψ may be formed from a reactive functional group for forming covalent linkages with a peptide, a protein, or a macromolecule, wherein the functional group comprises an electrophilic group, nucleophilic group, or a photoreactive group. The reactive functional group may an amine-reactive group, a thiol-reactive group, a carboxy-reactive group, a maleimidyl-reactive group, or a carbohydrate-reactive group. In some embodiments, the reactive functional group may react with a functional group of the analyte or binding partner therefore such as a primary amine. The reactive functional group may comprise (or be) an isiothiocyanate, isocyantate, acyl azide, NHS ester, sulfonyl chloride, aldehyde, glyoxal, epoxide, oxirane, carbonate, aryl halide, imidoester, carbodiimide, anhydride, fluorophenyl ester, or combinations thereof. In various implementations, the reactive functional group labels the analyte or binding partner therefor through acylation or alkylation. For example, the linkage may be formed from a reactive group selected from:, , , , , , , , –NCS, –NCO, , , , –SO2Cl, –N3, –N2+Cl-, , , , , , –Cl, –Br, –I, or –COOH. In some embodiments, the compound comprises a linker group having the structure –NH– C(O)– or –C(O)–NH–. In a preferred embodiment, the compound or moiety thereof (e.g., LC, Ψ) comprises at least one –NH–C(O)– or –C(O)–NH– linker group.

[0071] The chemiluminescent labels are typically conjugated to a binding pair of one of the analytes of interest. The chemiluminescent labels may be formed from a chemiluminescent compound or salt having a reactive functional group for conjugation to the binding pair. For example, the chemiluminescent label may be selected from DMAE-Bz, 3-MeO-DMAE-Bz, DIPAE-Bz, ABAC, LEAE-Bz, DIP-LEAE-Bz, 2-MeO-LEAE-Bz, 3-EtO-LEAE-Bz, 3-QAE- LEAE-Bz, 2-QAE-LEAE-NHS, LEAC-Bz, NSP-LEAE-Bz, 2-MeO-NSE-LEAE-NHS, 2-Meo-LEAE-Imidate, 3-Carboxybutadienyl-AE, P-Carboxyethyl-AE, Rhodamine-2-AM- DMAE-Bz, Rhodamine-2-AM-DMAE-CO2H, Texas Red-2-AM-DMAE-CO2H, CNF-2-AM- DMAE-CO2H, Texas Red-3-AM-DMAE-CO2H, Rhodamine-3-AM-DMAE-b-Alanine, Texas Red-3-AM-DMAE-b-Alanine, Texas Red-ED-NCM-DMPAE, Texas Red-ED-NSP- DMPAE, Rhodamine-2-AM-DMAE-HD-Theophylline, Texas Red-3-APO-DMAE-Bz, Texas Red-3-ABO-DMAE-Bz, DMAE-Bz, and 2-MeO-LEAE-Bz.

[0072] The covalent linkage between A and Ψ (e.g., L) or RFG and Ψ (e.g., L) may comprise (or be) a divalent C1-20alkyl, alkenyl, alkynyl, aryl, or arylalkyl radical, optionally substituted with up to 20 heteroatoms (e.g., N, O, S, P, Cl, F, Br). In some embodiments L comprises a zwitterionic linker. L may have the structure –LC–(ZL)z–, wherein z is 0 or 1. LCmay have the structure –(X1)0-1–(RL)0-5–(X2)0-1– (RL)0-5–(X3)0-1–(RL)0-5–(X4)0-1–(RL)0-5– wherein X1 is selected from –O–, –S–, –NRN–, –C(O)–, –NRN–C(O)–, –C(O)–NRN–, –O– C(O)–, or –C(O)–O–, –S–C(O)–, or –C(O)–S–, =N–, –O–, or –S–; X2–X4 are independently selected from –O–, –S–, –NRN–, –C(O)–, –NRN–C(O)–, –C(O)– NRN–, –O–C(O)–, or –C(O)–O–, –S–C(O)–, or –C(O)–S–; and RLis independently selected at each occurrence from –CH2–, –(CH2CH2O)–, or –(OCH2CH2)– ; with, for example, the proviso that LCcomprises at least one atom (or at least two atoms) in the chain between A and Ψ (or between A and ZL).

[0073] In some embodiments, L and / or Ψ comprises –C(O)–NH–. In some embodiments, LChas the structure: , , , , , , ,, , , , , , ,or .

[0074] The detectable label may comprise a dimethyl acridinium ester (DMAE) moiety and a zwitterionic linker comprising a zwitterionic linker or a polyethylene glycol derived linker to improve properties of the compound. Such properties as non-specific binding, hydrophilicity, or compound stability may be improved when Ψ comprises a zwitterionic linker or a polyethylene glycol derived linker or a dimethyl phenyl ester. In some embodiments, ZLhas the structure: . In several embodiments, R’ ismethyl, ethyl, propyl).

[0075] Exemplary compounds for forming the conjugates are disclosed in Table 1. In some embodiments, the detectable conjugate is formed by reacting a compound (e.g., a compound of Formula (V), a compound from Table 1, a compound from Table 1 with a different reactive functional group (RFG) or –L–RFG in place of, for example, the benzyl ester or the N- hydroxysuccinimid (NHS) ester) with an analyte, binding partner thereof, or derivatized version of the foregoing capable of reacting with a reactive functional group). Suitable sets ofacridinium labels may be prepared, for example, by alkylating the 1,3 positions of the acridinium ring system, by attaching a fluorophore to the 2 or 3 position of the acridinium ring system, by installing electron donating groups (e.g., –OG) on the acridinium ring system (e.g., at the 2 and / or 6 positions), by installing electron donating groups (e.g., –OG, –O–, –(CH2) 0-5CH3, –NH–) on the phenyl ester (e.g., at the 2 and / or 6 positions such as with –OG, at the 4 position (e.g., with a divalent electron donating group conjugated to the linker such as –O–, –(CH2) 0-5CH3, –NH–), by converting the acridinium ring system into a four membered acridinium system, or combinations thereof). The compounds may be an acridinium ester (“AE”). The compounds designations may include “Z” which may refer to a zwitterionic linker, “CMO” which may refer to a carboxy methyl oxime linker, “CME” which may refer to a carboxy methyl ether linker, “CETE” which may refer to carboxy ethyl thioether, “ZAE” which may refer to a zwitterionic acrinidium ester (which is typically an N-sulfopropyl (“NSP”) dimethyl acridinium ester in the examples shown (“NSP-DMAE”)), “ISODIZAE” which may refer to an acridinium nucleus with an isopropoxy functional group attached thereto and a full zwitterionic group (comprising both N+and X-) attached to the positive N of the acridinium. Table 1 † Compound λmax Range* (nm) (nm) Structure^Cl- NN O NTexas Red-3-AM- 590- DMAE-CO2H 612 720 Rhodamine-3-AM- 62 590- DMAE-β-Alanine 0 750Texas Red-3-AM- 590- DMAE-β-Alanine 628 740 Texas Red-ED- 590- NCM-DMPAE 626 760Texas Red-3-APO- 590- DMAE-Bz 644 750 Texas Red-3-ABO- 590- DMAE-Bz 634 750It will be understood that in the event of any variation between compound structures between this and what is known, such as in U.S. Pat. No.5,879,894 or 6,165,800, both compounds will be considered explicitly disclosed as suitable acridinium labels for use with the present methods.

[0076] In some embodiments, the detectable conjugate may have the structure of one or more of:wherein z is independently at each occurrence 0 or 1; y is independently at each occurrence 0, 1, 2, 3, 4, or 5; and A’ is the analyte or binding partner thereof conjugated via a primary amine of an unconjugated analyte or binding partner thereof A; wherein X1 is selected from –O–, –S–, –NRN–, –C(O)–, –NRN–C(O)–, –C(O)–NRN–, –O– C(O)–, or –C(O)–O–, –S–C(O)–, or –C(O)–S–, =N–, –O–, or –S–; X2–X4are independently selected from –O–, –S–, –NRN–, –C(O)–, –NRN–C(O)–, –C(O)– NRN–, –O–C(O)–, or –C(O)–O–, –S–C(O)–, or –C(O)–S–; andRLis independently selected at each occurrence from –(CH2)1-5–, –(CH2CH2O)1-5–, or – (OCH2CH2)1-5–.

[0077] The acridinium labels may also be characterized by their wavelengths of chemiluminescent emission. For example, some acridinium labels may have an emission wavelength maxima (λmax) of from 430 nm to 460 nm or from 460 nm to 490 nm or from 490 nm to 520 nm or from 520 nm to 550 nm or from 550 nm to 580 nm or from 580 nm to 610 nm or from 610 nm to 640 nm or from 640 nm to 670 nm or from 670 nm to 700 nm or from 700 nm to 730 nm or from 730 nm to 760 nm or from 760 nm to 790 nm or from 790 nm to 820 nm or from 820 nm to 850 nm. For example, in some embodiments, one compound in a wavelength separated set has a λmax of from 400 nm to 500 nm and another compound in the wavelength separated set has a λmax of from 500-600 nm.

[0078] Chemiluminescence from multiple acridinium labels with different emission wavelengths can be measured by using multiple photomultiplier tubes (PMT), each of the PMT’s equipped with an optical filter that allows the light from an acridinium ester of interest to pass through while blocking the unwanted light from other acridinium labels such as acridinium esters. In some embodiments, a single PMT can be utilized with a filter wheel installed in the front of the PMT’s detection window. The filter wheel may be mounted with multiple filters corresponding to, for example, the number of acridinium labels used for chemiluminescence, wherein each filter may allow the light from an acridinium label of interest to pass through while blocking the unwanted light from other AEs. Yet another alternative detector is a charge-coupled device (CCD), where the pixels can be grouped into several sections of the two dimensional detector, each section corresponding to the number of AEs to be detected. The chemiluminescence may pass through a grating, such that the wavelength separation may occur along the detector (e.g., CCD detector) and the images can be analyzed accordingly. Each grouped section of the pixels can have a specific optical filter that allows the light from an acridinium ester of interest to pass through while blocking the unwanted light from other AEs. Other types of detection systems are available for detection or measurement of lights of AEs with different emission profiles.

[0079] The presence of overlapping signals due to emission wavelengths of acridinium esters can be minimized by proper selection of optical filters, such as long pass and short pass filters for detection of two AEs and bandpass filters for detection of three or more AEs. The residualoverlapping signals after the use of filters can be deconvolved by an algorithm or artificial intelligence (AI) which can measure emission wavelength profiles of individual AEs at multiple different wavelengths. For example, the emission wavelength profile of acridinium labels having similar emission kinetics can be fit to a summation of typical chemiluminescence distributions (e.g., Normal distributions, Gaussian distribution, Poisson distribution, combinations thereof) to identify the peak and width of each individual acridinium label contribution. The signal of an acridinium label may be ascertained by subtracting the overlapping signal due to the stray light from the unwanted signal which could include noise and signal from other acridinium labels present in the system. The differentiation may be further improved by leveraging the different emission kinetics and measuring the emission wavelength profiles of the individual AEs at multiple wavelengths at different points in time. Through algorithms or AI, trained on data sets involving various sets of acridinium labels, the different kinetic changes together with the multiple wavelength detection can further improve the differentiation of overlapping signals. The multiple analyte detection schemes of the present disclosure offer increased sensitivity and a wider variety of multiple analyte measurement. However, as will be understood by the present disclosure, use of the coefficient matrix may not require any use of optical filters, deconvolution, or artificial intelligence.

[0080] One important consideration is for the acridinium labels to typically have emission kinetics of sufficient separation. Measurement of individual emissions can be made at a different time point and duration following chemiluminescent triggering to afford different measurement between kinetic separated sets. This can be done by turning on and off the detector at a particular time, or by collecting light in small intervals (binning) and properly grouping and binning with an appropriate time width to allow precise measurement of light at a particular time frame. For example, for emission separated acridinium label sets where most emission of one acridinium label occurs in one time domain (e.g., fast emission such as more than 90% more than 95%, more than 98%, more than 100% occurs within three seconds of triggering or within two seconds of triggering or with one second of triggering), and most emission of another acridinium label (e.g., slow emission such as more than 90% more than 95%, more than 98%, more than 100% occurs within 120 seconds of triggering or within 60 seconds of triggering or within 25 second of triggering or within 15 second of triggering or within 10 seconds of triggering), the bins may be chosen to collect the light from one label in certain bins and to collect the light of the other label in other bins. Each binned measuredintensity (or appropriately grouped bins) may individually be analyzed for quantification of the corresponding analyte.

[0081] The faster acridinium labels generally complete emission within 5 seconds of chemiluminescent triggering. In some embodiments, the fast acridinium label of the present disclosure may have faster light emission kinetics as compared to other acridinium compounds such as emitting at least 90% of their light, measured over 5 seconds, within 2 seconds. Slower acridinium labels may complete emission within 120 seconds of chemiluminescent triggering (and have minimal light contribution during the triggering of faster labels). For example, slower acridinium labels may complete less than 30% or less than 20% or less than 10% of their emission within 5 seconds or within 2 seconds and emit at least 90% of their light measured over 120 second or over 60 seconds or over 30 seconds.

[0082] For example, the acridinium labels of U.S. Pat. No. 8,119,422, which is hereby incorporated by reference in its entirety, can be used as materials to form the wavelength emission separated sets of acridinium labels.

[0083] Typically, zwitterionic acridinium esters (“ZAE”) comprising a reactive functional group for forming covalent linkages as described in U.S. Pat Nos.6,664,043 to Natrajan et al., 7,309,615 to Natrajan et al., 9,575,062 to Natrajan et al., or 9,487,480 to Natrajan, each hereby incorporated by reference in their entirety and in particular with respect to the zwitterionic acridinium esters described therein and their syntheses, may be used for synthesizing the compounds disclosed herein. For example, the zwitterionic acridinium ester starting materials may comprise an N-sulfopropyl (“NSP”) group in a zwitterionic moiety and / or comprise a charged nitrogen atom connected to the charged acridinium nucleus (“DIZAE”) and / or comprise a sterically stabilized dimethyl acridinium ester (“DMAE”) and / or comprise an isopropoxy functionalized acridinium nucleus (“ISO”) and / or comprise a zwitterionic (“Z”) and / or hexa(ethylene) glycol derived (“HEG”) and / or glutarate derived (e.g., –C(O)–(CH2)3– C(O)–) linking moieties between the acridinium ester and the reactive functional group. The reactive functional group may by NH2 or N-hydroxysuccinimidyl ester (“NHS”). For example, the compound (e.g., a compound for conjugating with an analyte or binding partner of an analyte such as a peptide, a protein, or a macromolecule including an antibody) may have the structure of formula (IV):wherein RFG is a reactive functional group for conjugating to the analyte or binding partner for an analyte, L is absent (i.e., it is a bond) or a linker, and Ψ is a chemiluminescent acridinium. For example, the reactive functional group (RFG) may be selected from: ,or may be synthesized through the use of acridinium sulfonamide reactants. For example, the acridinium sulfonamides disclosed in US Pat No 5,543,524 to Mattingly et al., hereby incorporated byreference in its entirety, are useful starting materials for the preparation of the chemiluminescent compounds disclosed herein.

[0084] The assay may be, for example, a competitive immunoassay which typically involves the detection of a large molecule, also referred to as macromolecular analyte, using binding molecules such as antibodies antigens, or proteins. The binding molecule may be immobilized or attached to a solid phase such as a particle, bead, membrane, microtiter plate, or any other solid surface. The assay (and system) may leverage components such as those in US 2023 / 0064409 which is hereby incorporated by reference and particularly in relation to filter- less optical detection methods or US 2021 / 0293803 which is hereby incorporated by reference in its entirety and particularly in relation to the devices, systems, and methods for determining the concentration of analytes in a sample. Antibodies, when used as binding molecules may include intact monoclonal antibodies and polyclonal antibodies, multi-specific antibodies (e.g., bispecific antibodies), as well as antibody fragments and conjugates thereof that exhibit the desired biological activity of analyte binding (such as, but not limited to, Fab, Fab′, F(ab′)2, Fv, scFv, Fd, diabodies, single-chain antibodies, and other antibody fragments and conjugates thereof that retain at least a portion of the variable region of an intact antibody), antibody substitute proteins or peptides (i.e., engineered binding proteins / peptides), and combinations or derivatives thereof. The antibody can be of any type or class (e.g., IgG, IgE, IgM, IgD, and IgA) or sub-class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2). Antibody fragments for use as the binding molecule may be obtained using conventional recombinant and / or enzymatic techniques and are screened for antigen binding in the same manner as intact antibodies.

[0085] An binding molecule may specifically bind an antigen when it preferentially recognizes the antigen in a complex mixture of proteins and / or macromolecules. In certain embodiments, the binding molecule includes an antibody having an antigen-binding site that specifically binds to a particular epitope. In certain embodiments, the antibody may be capable of binding different antigens so long as the different antigens comprise that particular epitope or closely related epitopes. In certain instances, for example, homologous proteins from different species may comprise the same epitope. In certain embodiments, an antibody may specifically binds to an antigen with a dissociation constant of no greater than 10−6M, 10−7M, 10−8M or 10−8M. When an antibody specifically binds to a receptor or ligand (i.e., counterreceptor), it may substantially inhibit adhesion of the receptor to the ligand. An antibody may substantially inhibit adhesion of a receptor to a ligand when an excess of antibody reducesthe quantity of receptor bound to ligand by at least about 20%, 40%, 60% or 80%, 85%, or 90% (as measured in an in vitro competitive binding assay).

[0086] In an example of a competitive heterogeneous assay, a support having a binding molecule for an analyte (e.g., antibody, antigen, protein) bound thereto is contacted with a medium containing a sample suspected of containing the analyte and the chemiluminescent conjugates (or “labeled analogs”) described herein. Analyte from the sample competes for binding to the analyte antibody with the labeled analog. After separating the support and the medium, the label activity of the support or the medium is determined by conventional techniques and is related to the amount of analyte in the sample. In a variation of the above competitive heterogeneous assay, the support comprises the analyte analog, which competes with analyte of the sample for binding to an antibody reagent in accordance with the principles described herein. The labeled analyte analog may be covalently attached with a chemiluminescent or fluorescent molecule often referred to as a label or tracer.

[0087] When the solid phase with the immobilized antibody is mixed with a sample containing the analyte and the labeled analyte, a binding complex is typically formed between the analyte or the labeled analyte. This type of assay is often called a heterogeneous assay because of the involvement of a solid phase. The chemiluminescent signal associated with the binding complex can then be measured and the presence or absence of the analyte in the sample can be inferred. Usually, the binding complex is separated from the rest of the binding reaction components such as excess, labeled analyte, prior to signal generation. For example, if the binding complex is associated with a magnetic bead, a magnet can be used to separate the binding complex associated with the bead from bulk solution.

[0088] In an example of a sandwich assay format employing two antibodies (or fragments thereof), a solid support with a first immobilized antibody or fragment thereof for an analyte is mixed with a sample containing the analyte and a labelled conjugate comprising a second antibody or fragment thereof. A binding complex is formed between the solid particle and the labelled conjugate via the analyte in the sample. The different chemiluminescent moieties may be conjugated to the same binding partner for an analyte. In some embodiments, the different chemiluminescent moieties are conjugated to different binding partners for an analyte. In some embodiments, the different chemiluminescent moieties are conjugated to different binding partners for different analytes (all of which may be detected on the same solid support). Thesignal associated with the binding complex and can the measured and the presence or absence or amount of analyte can be inferred. Usually, the binding complex is separated from the rest of the binding reaction components such as excess, labeled analyte, prior to signal generation. For example, if the binding complex is associated with a magnetic bead, a magnet can be used to separate the binding complex associated with the bead from bulk solution. In some embodiments, the first immobilized antibody is a biotinylated mouse monoclonal antibody bound to coated (e.g., streptavidin coated) optionally paramagnetic particles. In some embodiments, the second antibody is a mouse monoclonal antibody fragment labelled with acridinium (e.g., acridinium ester).

[0089] By using a series of “standards,” that is, known concentrations of the analyte, a “dose- response” curve can be generated for the known labeled analyte. These dose response curves may be identified individually for any acridinium label or identified based on combinations of the acridinium labels used in the assay. Thus, the dose-response curve correlates a certain amount of measured signal with a specific concentration of analyte. In a competitive assay, as the concentration of the analyte increases, the amount of signal decreases if the chemiluminescence from the binding complex is measured. The concentration of the analyte in an unknown sample can then be calculated by comparing the signal generated by an unknown sample containing the macromolecular analyte, with the dose-response curve.

[0090] The methodology of the attachment of binding molecules such as antibodies to solid phases typically involves a mixing of the requisite components to induce attachment. For example, an antibody can be covalently attached to a particle containing amines on its surface by using a cross-linking molecule such as glutaraldehyde. The attachment may also be non- covalent and may involve simple adsorption of the binding molecule to the surface of the solid phase, such as polystyrene beads and microtiter plate. Labeling of binding molecules such as antibodies and other binding proteins are also well known in the prior art and are commonly called conjugation reactions and the labeled antibody is often called a conjugate. Typically, an amine-reactive moiety on the label reacts with an amine on the antibody to form an amide linkage. Other linkages, such as thioether, ester, carbamate, and the like between the antibody and the label may also be used.

[0091] In another aspect of the invention, a reagent may be provided for the detection of an analyte comprising a chemiluminescent acridinium compound bound the analyte or bindingpartner. The reagent may comprise from 0.1 to 100 ng / mL of the chemiluminescent acridinium compound or from 1 to 50 ng / mL of the chemiluminescent acridinium compound or from 5 to 30 ng / mL of the chemiluminescent acridinium compound. In some embodiments, the compound is provided in a reagent which further comprises a buffer.

[0092] In some embodiments, the sample derived from a mammal (e.g., human). In some embodiments, the sample comprises saliva and / or blood and / or serum. In some embodiments, the sample is saliva and / or blood and / or serum.

[0093] In some assays, the sample to be analyzed is subjected to a pretreatment to release analyte from endogenous binding substances such as, for example, plasma or serum proteins that bind the analyte. The release of the analyte from endogenous binding substances may be carried out, for example, by addition of a digestion agent or a releasing agent or a combination of a digestion agent and a releasing agent used sequentially. The digestion agent is one that breaks down the endogenous binding substances so that they can no longer bind the analyte.

[0094] The conditions for conducting an assay on a portion of a sample in accordance with the principles described herein may include carrying out the assay in an aqueous buffered medium at a moderate pH, generally that which provides optimum assay sensitivity. The aqueous medium may be solely water or may include from 0.1 to 40 % by volume of a cosolvent. The pH for the medium may be in the range of 4 to 11, or 5 to 10, or 6.5 to 9.5, or 7 to 8. Usually, the pH value of the solution will be a compromise between optimum binding of the binding members of any specific binding pairs, the pH optimum for other reagents of the assay such as members of the signal producing system, and so forth. Various buffers may be used to achieve the desired pH and maintain the pH during the assay. Illustrative buffers include borate, phosphate, carbonate, TRIS, barbital, PIPES, HEPES, MES, ACES, MOPS, and BICINE, for example.

[0095] Various ancillary materials may be employed in the assay methods. For example, in addition to buffers, the composition, reagents, or reaction medium may comprise stabilizers for the medium and for the reagents employed. In some embodiments, the medium may comprise proteins (e.g., albumins), organic solvents (e.g., formamide), quaternary ammonium salts, polyanions (e.g., dextran sulfate), binding enhancers (e.g., polyalkylene glycols), polysaccharides (e.g., dextran, trehalose), and combinations thereof.

[0096] Triggering the chemiluminescence of the analogs may be performed by the addition chemiluminescent triggering reagents. The chemiluminescent triggering reagents may be acidic or basic. Multiple chemiluminescent triggering reagents may be added sequentially. For example, an acidic solution may first be added followed by a basic solution. In some embodiments, the chemiluminescent triggering reagents comprise hydrogen peroxide, hydrogen peroxide salts, nitric acid, nitric acid salts, sodium hydroxide, ammonium salts, or combinations thereof.

[0097] Methods described herein can be implemented in a computer system having a processor that executes specific instructions in a computer program. In some embodiments, a computer system may be arranged to output the concentration of one or more analytes and / or output a biomarker score based on receiving a biomarker profile and / or a level of associated with the biomarker concentration in the sample. In specific embodiments, a computer program may include instructions for the system to select appropriate next steps, including additional medication, a treatment, and / or additional testing for a subject.

[0098] In some embodiments, the computer program may be configured such that the computer system can identify a subject for further testing, identify a subject as being at risk or having a disease, and / or identify a subject to receive medication based on received data (e.g., biomarker profile) and use the data to calculate a biomarker score.

[0099] The computer system may include a processor, a memory, a storage device and an input / output device. Each of the components may be interconnected using a system bus. The computer system may be interconnected with analyzing equipment for initiating and / or measuring the chemiluminescence of the sample.

[0100] The processor is typically capable of processing instructions for execution within the computer system. In one embodiment, the processor may be a single-threaded processor or a multi-threaded processor. The processor is typically capable of processing instructions stored in the memory or on the storage device, including for receiving or sending information through the input / output device. The memory may store information within the system. In one embodiment, the memory is a computer-readable medium. In one embodiment, the memory is a volatile memory unit. In another embodiment, the memory is a non-volatile memory unit. The storage device may be capable of providing mass storage for the system. In one embodiment, the storage device is a computer-readable medium. The input / output device mayprovide input / output operations for the system. In some embodiments, the input / output device includes a keyboard and / or pointing device. In one embodiment, the input / output device includes a display unit for displaying graphical user interfaces.

[0101] Additionally, non-transitory computer readable media containing executable instructions that when executed cause a processor to perform operations including a method as provided herein are provided. For example, a non-transitory computer readable medium containing executable instructions that when executed cause a processor to perform operations including a method described above. In various implementations, the processor is stored on a system comprising one or more components associated with measurement of the mixture (e.g., light detectors, tracks for reagent vessels that may expose the reagent vessels to steps associated with triggering chemiluminescence including incubation, aspiration, washing, mixing, and triggering). In certain aspects, the processor may be stored on a device separate from the immunoassay system (e.g., on a server, or on a computer connected to a server), wherein the immunoassay system is configured to the measured data associated with chemiluminescence for subsequent processing at the processor. For example, a server can include one or more computers operating as a web server, database server, or other type of computer server in a manner to fulfill described roles, responsibilities, or functions. One should further appreciate the disclosed computer-based algorithms, processes, methods, or other types of instruction sets can embodied as a computer program product comprising a non-transitory, tangible computer readable media storing the instructions that cause a processor to execute the disclosed steps. The various servers, systems, databases, or interfaces can exchange data using standardized protocols or algorithms, possibly based on HTTP, HTTPS, AES, public-private key exchanges, web service APIs, known financial transaction protocols, or other electronic information exchanging methods. Data exchanges can be conducted over a packet-switched network, the Internet, LAN, WAN, VPN, or other type of packet switched network. EXAMPLES

[0102] The following Examples illustrate the synthesis of a representative number of compounds, characterization of parameters implicated in assay development, and the use of these compounds in the measurement of samples in heterogeneous competitive assay. Accordingly, the Examples are intended to illustrate but not to limit the disclosure. Additionalcompounds not specifically exemplified may be synthesized using conventional methods in combination with the methods described herein.

[0103] Example 1: Detection of HIV-1 and HIV-2

[0104] The coefficient matrix for an assay measuring HIV-1 and HIV-2 may be obtained by individually determining the slopes for HIV-1 and HIV-2. FIG. 1A shows exemplary flash curves for an HIV-1 immunoassay taken of a sample having low, medium, and high concentrations HIV-1. The inset illustrates the chemiluminesence (RLUHIV1) of these curves as a function of concentrations (Conc) at timepoint T1, T2, and T3. As can be seen, each timepoint is associated with a different slope (ε) of the RLU with respect to concentration. In some embodiments, each timepoint is associated with a different slope (ε) of the RLU of the individual components with respect to the total RLU output at a timepoint. FIG.1B provides a similar analysis for the HIV-2 analyte with a different acridinium ester. As can be seen, the HIV-2 analyte provides a different set of slopes at the timepoints due, in part, to the differing emission kinetics.

[0105] FIG.1C provides a schematic for creation of the coefficient matrix using these slopes (ε). The flash curves of each analyte are plotted for all relevant concentration levels and the slopes (ε) are obtained at each time point, separately for HIV-1 and HIV-2. The coefficient matrix is then populated with one column each for HIV-1 and HIV-2 respectively and rows for each timepoints, resulting in a 2x1280 matrix size (where there are 1280 time points measured). With this coefficient matrix, any given mixture of HIV-1 and HIV-2 can be resolved into individual components of HIV-1 and HIV-2.

[0106] FIG.2A provides the PMT output (RLU) as a function of time for a mixed sample of HIV-1 and HIV-2 (dotted line). This output represents the summation of the contributions of chemiluminescent signal from HIV-1 and HIV-2 in the sample. The coefficient matrix can be used to elucidate these relative contributions. FIG.2B provides an exemplary matrix algebraic operations that may be performed to yield the results of the constituent concentrations of HIV- 1 and HIV-2.NON-LIMITING ILLUSTRATIVE EMBODIMENTS

[0107] Non-limiting illustrative embodiments are provided below, each of which should be considered to be part of the disclosure of the present application. These embodiments may apply to any embodiment described herein.

[0108] Illustrative Embodiment 1. A method for the detection or quantification of multiple analytes in a sample (e.g., a biological sample such as blood, saliva, serum, a sample derived from a biological sample such as a diluted biological sample) comprising: (a) providing a first set of chemiluminescent labels capable of binding to a first analyte and a second set of chemiluminescent labels capable of binding to a second analyte, wherein the first set of chemiluminescent labels and the second set of chemiluminescent labels comprise at least two chemiluminescent labels having different rates of emission; wherein the first analyte is different from the second analyte; (b) mixing the first set and said second set with said sample; (c) preparing the mixture of said first set, second set, and sample to measure chemiluminescence from the first set and the second set of chemiluminescent labels; (d) triggering chemiluminescence from the first set and second set of chemiluminescent labels following preparation of the mixture (e.g., by the addition of one or more triggering compositions which trigger chemiluminescence of the acridinium labels); (e) measuring the chemiluminescence in the time domain (e.g., measuring light intensity as a function of time from the triggering step) at more than one time point; (f) calculating the concentration of the first analyte and the second analyte in the sample using the measured chemiluminescent signal at the more than one time point and a coefficient matrix comprising the slope of the chemiluminescent (e.g., with respect to analyte concentration, with respect to a metric such as the total RLU output associated which may be associated with analyte concentration) of the first and second analyte individually obtained (e.g., chemiluminescence obtained at a range ofconcentrations of standards comprising the analyte not in a mixture) at the more than one time point.

[0109] Illustrative Embodiment 2. The method according to Illustrative Embodiment 1, wherein the slope of the chemiluminescent signal of each analyte at each timepoint in the coefficient matrix is based on at least three different concentrations of each analyte.

[0110] Illustrative Embodiment 3. The method according to Illustrative Embodiment 1 or 2, wherein the coefficient matrix comprises a first column comprising the slopes of the first analyte (e.g., with respect to analyte concentration, with respect to a metric such as the total RLU output associated which may be associated with analyte concentration) and a second column comprising the slopes of the second analyte (e.g., with respect to analyte concentration, with respect to a metric such as the total RLU output associated which may be associated with analyte concentration) .

[0111] Illustrative Embodiment 4. The method according to any one of Illustrative Embodiments 1-3, wherein the calculating step is peformed by multiplying the inverse of the coefficient matrix (e.g., Moore-Penrose inverse of the coefficient matrix) with a measured chemiluminescent matrix (e.g., single column matrix) comprising the relative light units (RLUs) at one or more of the more than one time points.

[0112] Illustrative Embodiment 5. The method according to any one of Illustrative Embodiments 1-4, wherein the calculating step is peformed by multiplying the inverse (e.g., Moore-Penrose inverse of the coefficient matrix) of the coefficient matrix with a measured chemiluminescent matrix (e.g., single column matrix) comprising the relative light units (RLUs) at each of the more than one time points.

[0113] Illustrative Embodiment 6. The method according to any one of Illustrative Embodiments 1-5, wherein said preparing step comprises: (c1) providing a solid support having immobilized thereon a molecule capable of forming a binding complex with said at least one analyte and capable of forming a binding complex with a chemiluminescent label in the first set and / or the second set of chemiluminescent labels; and (c2) separating said solid support from said mixture.

[0114] Illustrative Embodiment 7. The method according to Illustrative Embodiments 6, wherein said measuring step comprises the addition of one or more chemiluminescence triggering reagents to said separated solid support and / or said separated mixture.

[0115] Illustrative Embodiment 8. The method according to claim 6 or 7, wherein said solid support comprises at least two molecules (e.g., two, three, four, five, six, seven, eight, nine ten), each capable of forming a binding complex with different analytes and capable of forming a binding complex with at least two different chemiluminescent labels in the first set and / or second set of chemiluminescent labels.

[0116] Illustrative Embodiment 9. The method according to any one of Illustrative Embodiments 1-8, wherein the first sent of chemiluminescent labels and the second set of chemiluminescent labels independently have the structure of formula (I): (I)wherein A is an analyte or binding partner for an analyte, L is absent (i.e., it is a bond) or a linker optionally comprising a group LCor ZL, and Ψ is a chemiluminescent acridinium comprising the structure: “j” and “k” areall R3groups are hydrogen), 2, 3, or 4; R1 is hydrogen, –R, –Xb, –RL–Xb, –LC–R, –LC–Xb(e.g., –L1–Xb), –Z, –RL–Z, –LC–Z (e.g., – L1–Z), or –RL–LC–RL–Z (e.g., –RL–L1–RL–Z); R2and R3are independently selected at each occurrence from hydrogen, –R, an electron donating group, and –Z; wherein two vicinal R2 or R3 groups may together form a fused cyclic group (e.g., 5-7 membered fused aryl or heteroaryl group, 5-7 membered fused heterocyclic group) and wherein R2 or R3 may comprise a linkage to an imaging agent such as a fluorophore (e.g., rhodamine); LCis a divalent C1-35alkyl, alkenyl, alkynyl, aryl, or arylalkyl radical, optionally substituted (e.g., with 1 to 20 heteroatoms, with 1-20 substituents);ZLis a zwitterionic linker group having the structure: ; “m” is 0 (i.e. it is a bond) or 1;“n” and “p” are independently at each occurrence an integer from 0 (i.e. it is a bond) to 10; Z is a zwitterionic group independently at each occurrence has the structure: ;10, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10); Xaand Xbare independently at each occurrence an anionic group; L1 is independently at each occurrence –O–, –S–, –NH–, –N(RN)–, –(CH2)1-10–, –S(=O)1-2–, –C=C–, –C=C–(CH2)1-3–, –C(O)–, –O–C(O)–, –C(O)–(CH2)1-4–, –(CH2)1-4–C(O)–, –C(O)–O–, –C(O)–N(RN)–, –C(O)–NH–, –N(RN)–C(O)–, –NH–C(O)–, –C(O)–N(RN)–(CH2)1-3–, –(CH2)1-3–C(O)–N(RN)–, –(CH2)1-3–N(RN)–C(O)–, –NH–S(O)1-2–, –N(RN)–S(O)1-2–, –S(O)1-2–N(RN)–, –S(O)1-2–NH–, –(CH2)1-3–NH–S(O)1-2–, –(CH2)1-3–N(RN)–S(O)1-2–, –(CH2)1-3–S(O)1-2–N(RN)–, –(CH2)1-3–S(O)1-2–NH–, –O–(CH2)1-4–, –(CH2)1-4–O–, –S–(CH2)1-4–, –(CH2)1-4–S–, –NH–(CH2)1-4–, –N(RN)–(CH2)1-4–, –(CH2)1-4–N(RN)–, –(OCH2)1-10–, –(CH2O)1-10–, –(OCH2CH2)1-10–, or –(CH2CH2O)1-10–; RLis independently at each occurrence a C1-20 bivalent hydrocarbon radical (e.g., alkyl, alkenyl, aryl, phenyl, mono alkyl substituted phenyl, di alkyl substituted phenyl, alkynyl, arylalkyl), optionally having one or more (e.g., 1-10, 1-5) points of substitution (e.g., with 1-10 heteroatoms, with 1-10 substituents);R is independently at each occurrence hydrogen or C1-35 hydrocarbon (e.g., alkyl, alkenyl, alkynyl, or aralkyl) radical, optionally having one or more (e.g., 1-20, 1-10, 1-5) points of substitution (e.g., with 1-20 heteroatoms, with 1-20 substituents); R’ and R” are independently at each occurrence hydrogen or a C1-10 alkyl; RNis independently at each occurrence from hydrogen or C1-5 alkyl (e.g., methyl, ethyl, propyl); and R’ is hydrogen or a C1-10 alkyl; or a salt thereof (e.g., a halide salt such as a chloride salt, a sulfonate salt such as a halosulfonate salt, a haloalkyl sulfonate salt a fluoroalkyl sulfonate salt, a carboxylate salt such as a haloalkyl carboxylate salt, fluoroalkyl carboxylate salt).

[0117] Illustrative Embodiment 10. The method according to any one of Illustrative Embodiments 1-9, wherein the first analyte is HIV-1 and the second analyte is HIV-2.

[0118] Illustrative Embodiment 11. The method according to any one of Illustrative Embodiments 1-9, wherein the first set of chemiluminescent labels comprise a monoclonal antibody capable of binding to the first analyte and the second set of chemiluminescent labels comprise a monoclonal antibody capable of binding to the second analyte.

[0119] Illustrative Embodiment 12. The method according to any one of Illustrative Embodiments 1-11, wherein the method further comprises providing a third set of chemiluminescent labels capable of binding to a third analyte and the coefficient matrix comprises the slope of the chemiluminescent signal (with respect to individual analyte concentration) of the first, second, and third analytes individually obtained at a range of concentrations at the more than one time point.

[0120] Illustrative Embodiment 13. The method according to Illustrative Embodiment 12, wherein the third analyte is HIV-1 p24 antigen.

[0121] Illustrative Embodiment 14. A system for the detection of multiple analytes in a sample comprising: a) light detectors (e.g., photomultiplier tubes), wherein the light detectors measure chemiluminescent signal from the sample in binned time points;b) a computer system in communication with the light detectors; wherein the light detectors communicate the chemiluminescent signal (or relative light units (RLU)) to the computer system at each binned time point; wherein the computer system further comprises a processor that accesses memory or a storage device comprising data for a coefficient matrix comprising the slope of the chemiluminescent signal (e.g., with respect to analyte concentration, with respect to a metric such as the total RLU output associated which may be associated with analyte concentration) of the two or more of the multiple analytes individually obtained (e.g., chemiluminescence obtained at a range of concentrations of standards comprising the analyte not in a mixture) at the binned time points; and the processor accesses instructions to convert the chemiluminescent signal to analyte concentration with the coefficient matrix.

[0122] Illustrative Embodiment 15. The system according to Illustrative Embodiment 14, wherein the instructions comprise multiplication of the coefficient matrix (or an inverse thereof such as the Moore-Penrose inverse of the coefficient matrix) with a single column matrix of the measured RLU signal from the light detectors.

[0123] All references including patent applications and publications cited herein are incorporated herein by reference and for all purposes to the same extent as if each individual publication or patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. Many modifications and variations of this invention can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. The embodiments described herein are offered by way of example only, and the invention is to be limited only by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled.

Claims

CLAIMS 1. A method for the detection or quantification of multiple analytes in a sample (e.g., a biological sample such as blood, saliva, serum, a sample derived from a biological sample such as a diluted biological sample) comprising: (a) providing a first set of chemiluminescent labels capable of binding to a first analyte and a second set of chemiluminescent labels capable of binding to a second analyte, wherein the first set of chemiluminescent labels and the second set of chemiluminescent labels comprise at least two chemiluminescent labels having different rates of emission; wherein the first analyte is different from the second analyte; (b) mixing the first set and said second set with said sample; (c) preparing the mixture of said first set, second set, and sample to measure chemiluminescence from the first set and the second set of chemiluminescent labels; (d) triggering chemiluminescence from the first set and second set of chemiluminescent labels following preparation of the mixture (e.g., by the addition of one or more triggering compositions which trigger chemiluminescence of the acridinium labels); (e) measuring the chemiluminescence in the time domain (e.g., measuring light intensity as a function of time from the triggering step) at more than one time point; (f) calculating the concentration of the first analyte and the second analyte in the sample using the measured chemiluminescent signal at the more than one time point and a coefficient matrix comprising the slope of the chemiluminescent signal (e.g., with respect to analyte concentration, with respect to a metric such as the total RLU output associated which may be associated with analyte concentration) of the first and second analyte individually obtained (e.g., chemiluminescence obtained at a range of concentrations of standards comprising the analyte not in a mixture) at the more than one time point.least two different chemiluminescent labels in the first set and / or second set of chemiluminescent labels.

9. The method according to any one of claims 1-8, wherein the first sent of chemiluminescent labels and the second set of chemiluminescent labels independently have the structure of formula (I): (I)wherein A is an analyte or binding partner for an analyte, L is absent (i.e., it is a bond) or a linker optionally comprising a group LCor ZL, and Ψ is a chemiluminescent acridinium comprising the structure: “j” and “k” are independently 0 (e.g., all R2groups are hydrogen, all R3groups are hydrogen), 1, 2, 3, or 4; R1 is hydrogen, –R, –Xb, –RL–Xb, –LC–R, –LC–Xb(e.g., –L1–Xb), –Z, –RL–Z, –LC–Z (e.g., – L1–Z), or –RL–LC–RL–Z (e.g., –RL–L1–RL–Z); R2and R3are independently selected at each occurrence from hydrogen, –R, an electron donating group, and –Z; wherein two vicinal R2 or R3 groups may together form a fused cyclic group (e.g., 5-7 membered fused aryl or heteroaryl group, 5-7 membered fused heterocyclic group) and wherein R2 or R3 may comprise a linkage to an imaging agent such as a fluorophore (e.g., rhodamine); LCis a divalent C1-35alkyl, alkenyl, alkynyl, aryl, or arylalkyl radical, optionally substituted (e.g., with 1 to 20 heteroatoms, with 1-20 substituents); ZLis a zwitterionic linker group having the structure: ; 78“m” is 0 (i.e. it is a bond) or 1; “n” and “p” are independently at each occurrence an integer from 0 (i.e. it is a bond) to 10; Z is a zwitterionic group independently at each occurrence has the structure: ; “q” and “l” are independently 0 or 1; “r” is independently an integer from 0 to 10 (e.g., from 1 to 10, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10); Xaand Xbare independently at each occurrence an anionic group; L1 is independently at each occurrence –O–, –S–, –NH–, –N(RN)–, –(CH2)1-10–, –S(=O)1-2–, –C=C–, –C=C–(CH2)1-3–, –(CH2)1-4–, –(CH2)1-4–C(O)–, –C(O)–O–, –C(O)–N (RN)–C(O)–, –NH–C(O)–, –C(O)–N(RN)–(CH2)1-3–,–(CH2)1-3–N(RN)–C(O)–, 1-2–, –N(RN)–S(O)1-2–, –S(O)1-2–N(RN)–, –S(O)1-2–NH–, –(CH2)1-3–NH–S(O)1-2–, –(CH2)1-3–N(RN)–S(O)1-2–, –(CH2)1-3–S(O)1-2–N(RN)–, –(CH2)1-3–S(O)1-2–NH–, –O–(CH2)1-4–, –(CH2)1-4–O–, –S–(CH2)1-4–, –(CH2)1-4–S–, –NH–(CH2)1-4–, –N(RN)–(CH2)1-4–, –(CH2)1-4–N(RN)–, –(OCH2)1-10–, –(CH2O)1-10–, –(OCH2CH2)1-10–, or –(CH2CH2O)1-10–; RLis independently at each occurrence a C1-20 bivalent hydrocarbon radical (e.g., alkyl, alkenyl, aryl, phenyl, mono alkyl substituted phenyl, di alkyl substituted phenyl, alkynyl, arylalkyl), optionally having one or more (e.g., 1-10, 1-5) points of substitution (e.g., with 1-10 heteroatoms, with 1-10 substituents); R is independently at each occurrence hydrogen or C1-35 hydrocarbon (e.g., alkyl, alkenyl, alkynyl, or aralkyl) radical, optionally having one or more (e.g., 1-20, 1-10, 1-5) points of substitution (e.g., with 1-20 heteroatoms, with 1-20 substituents); R’ and R” are independently at each occurrence or a C1-10 alkyl; RNis independently at each or C1-5 alkyl (e.g., methyl, ethyl, propyl); and R’ is hydrogen or a C1-10alkyl; ora salt thereof (e.g., a halide salt such as a chloride salt, a sulfonate salt such as a halosulfonate salt, a haloalkyl sulfonate salt a fluoroalkyl sulfonate salt, a carboxylate salt such as a haloalkyl carboxylate salt, fluoroalkyl carboxylate salt).

10. The method according to any one of claims 1-9, wherein the first analyte is HIV-1 and the second analyte is HIV-2.

11. The method 1-9, wherein the first set of chemiluminescent labels capable of binding to the firstanalyte and the second set of a monoclonal antibody capable of binding to the second analyte.

12. The method according to any one of claims 1-11, wherein the method further comprises providing a third set of chemiluminescent labels capable of binding to a third analyte and the coefficient matrix comprises the slope of the chemiluminescent signal signal (e.g., with respect to analyte concentration, with respect to a metric such as the total RLU output associated which may be associated with analyte concentration) of the first, second, and third analytes individually obtained at a range of concentrations at the more than one time point.

13. The method according to claim 12, wherein the third analyte is HIV-1 p24 antigen.

14. A system for the detection of multiple analytes in a sample comprising: a) light detectors (e.g., photomultiplier tubes), wherein the light detectors measure chemiluminescent signal from the sample in binned time points; b) a computer system in communication with the light detectors; wherein the light detectors communicate the chemiluminescent signal (or relative light units (RLU)) to the computer system at each binned time point; wherein the computer system further comprises a processor that accesses memory or a storage device comprising data for a coefficient matrix comprising the slope of the chemiluminescent signal signal (e.g., with respect to analyte concentration, with respect to a metric such as the total RLU output associated which may be associated with analyte concentration) of the two or more of the multiple analytes individually obtained (e.g., chemiluminescence obtained at a range of concentrations of standards comprising the analyte not in a mixture) at the binned time points; and 80the processor accesses instructions to convert the chemiluminescent signal to analyte concentration with the coefficient matrix.

15. The system according to claim 14, wherein the instructions comprise multiplication of the coefficient matrix (or an inverse thereof such as the Moore-Penrose inverse of the coefficient matrix) with a single column matrix of the measured RLU signal from the light detectors. 81the processor accesses instructions to convert the chemiluminescent signal to analyte concentration with the coefficient matrix.

15. The system according to claim 14, wherein the instructions comprise multiplication of the coefficient matrix (or an inverse thereof such as the Moore-Penrose inverse of the coefficient matrix) with a single column matrix of the measured RLU signal from the light detectors.

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