Increasing light stability of immunoassay reagents

Abstract

The present disclosure provides immunoassay reagent compositions having increased stability (when exposed to light). These compositions typically include a dye which prevents labels, such as fluorescent labels, from degradation. Kits comprising, methods of using, and methods of producing these compositions are provided as well.

Description

INCREASING LIGHT STABILITY OF IMMUNOASSAY REAGENTSCROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority to and the benefit of U.S. App. No. 63 / 605,316, filed December 1, 2023, the entire contents of which are hereby incorporated by reference in their entirety.FIELD OF DISCLOSURE

[0002] This disclosure describes immunoassay reagent compositions comprising a label (e.g., a fluorescent label, chemiluminescent label) that are stabilized by the incorporation of a light absorbent material such as a dye.BACKGROUND

[0003] Detection for the presence of an analyte (or characterization of its concentration) is often achieved through the use of various assay formats involving compositions having compounds with fluorescent moieties (e.g., fluorescein). These compositions are often highly sensitive to certain environmental factors such as light which may cause degradation of the fluorescent compounds. The fluorescent compounds, particularly in highly sensitive assays, may absorb light, causing their degradation which results in inconsistent fluorescent compound concentrations. This degradation can have significant implications for the assay measurements. Degraded fluorescent compounds may result in lower or higher light output when the assay is performed, thereby providing inaccurate measurements of analyte concentrations.

[0004] Degradation can occur during transport, and storage. Typically, the compositions are provided to end users in kits which attempt to minimize this degradation. Minimization of degradation typically involves minimization of the exposure to light during manufacture, storage, and transport through the use of light controlled packaging that decreases as much light as possible from interacting with the composition. However, this minimization of degradation is costly and cumbersome. Moreover, it is difficult to control and minimize light entry in certain ubiquitously used packaging which often promotes degradation of any immunoassay compositions contained therein (particularly highly sensitive compositions). Typically, immunoassay reagent compositions have a shelf life of a year (time betweenmanufacture and degradation) sensitive to light storage conditions due to these light induced degradation mechanisms.

[0005] There is a continuing need for the development of immunoassay reagent compositions and kits which are less prone to this degradation.SUMMARY

[0006] In accordance with the foregoing objectives and others, the present disclosure includes immunoassay reagent compositions which do not (or have decreased) degradation over longer periods of time. In some embodiments, the immunoassay reagent compositions include a light absorbent material, such as a dye. In accordance with the foregoing objectives and others, embodiments of the present disclosure relate to a composition including a liquid medium, a fluorochrome labelling reagent (e.g., a compound comprising a fluorescent moiety optionally conjugated to an antigen or binding partner thereof), and a dye soluble in the liquid medium. Without wishing to be bound by theory, the light absorbent material is able to absorb light and therefore prevent light induced degradation of the conjugates containing fluorescent moieties (e.g., fluorescein containing conjugates). Surprisingly, the present disclosure is partially based on the discover that the use of light absorbent material in immunoassay reagent compositions can prevent light induced degradation while having minimal, if any effect on an immunoassay using the reagent composition. In some embodiments, the absorbent material is dissolved in a liquid composition (e.g., the light absorbent material is a soluble dye). In various implementations, the light absorbent material has an absorbance spectra that substantially overlaps with the absorbance spectra of the labelled compounds (e.g., in the visible spectrum or any part thereof such as the violet, indigo, blue, green, yellow, orange, red, or combinations thereof, in the UV spectrum). In various implementations, the absorbance spectra of the light absorbent material substantially overlaps with the fluorescence emission spectra of the fluorescent compounds (e.g., in the visible spectrum or any part thereof such as the violet, indigo, blue, green, yellow, orange, red, or combinations thereof, in the UV spectrum). In various implementations, the absorbance spectra of the light absorbent material does not or has minimal overlap (e.g, less than 10% overlap, less than 5% overlap, less than 1% overlap) with the fluorescence emission spectra of the fluorescent compounds (e.g., in the visible spectrum or any part thereof such as the violet, indigo, blue, green, yellow, orange, red, or combinations thereof, in the UV spectrum).

[0007] In another aspect of the invention, an immunoassay reagent composition is provided for the detection of an analyte, the composition comprising an analyte or binding partner for an analyte (e.g., an antibody, antibody fragment) bound to a fluorescent moiety (e.g., fluorescein such as fluorescein isocyanate) and a optionally, a light absorbent material such as a dye. In some embodiments, the fluorescent moiety is independently fluorescein such as fluorescein isothiocyanate, rhodamine compounds, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde, or fluorescamine. In some embodiments the immunoassay reagent composition comprises more than compound comprising an analyte or binding partner for an analyte conjugated to a fluorescent moiety (e.g., fluorescein such as fluorescein isothiocyanate). In some embodiments, the immunoassay reagent composition comprises a chemiluminescent acridinium conjugated to an analyte or binding partner for an analyte. The reagent composition may comprise a concentration of each conjugate of, for example, less than 1 mg / mL. Reagents of the present disclosure include compositions comprising the indicated components, and optionally an excipient, carrier, or solvent. The reagent compositions of the present disclosure may include a surfactant.

[0008] The compositions of the present disclosure (e.g., Ancillary Reagents) may comprise a liquid medium, a fluorochrome labelling reagent, and a dye soluble in the liquid medium. In most embodiments, the fluorochrome labelling reagent is a fluorochrome moiety conjugated to an analyte or binding partner thereof. For example, the binding partner may be a monoclonal antibody or fragment thereof for the analyte of interest. In some embodiments, the fluorochrome labelling reagent comprises a fluorescein (e.g., fluorescein isothiocyanate (FITC)) moiety as the fluorophore. In some embodiments, the composition is aqueous and optionally comprises one or more buffers, stabilizers, or salts. In various implementations, the composition comprises bovine serum albumin. The dye soluble in the liquid medium may comprise (or consist essentially of (such as more than 90% of the dye is by weight of the dye or more than 95% of the dye is by weight of the dye more than 99% of the dye is by weight of the dye) or is)6-hydroxy-5-[(2-methoxy-5-methyl-4-sulfonatophenyl)diazenyl]naphthalene-2- sulfonate or salts thereof (e.g., disodium 6-hydroxy-5-[(2-methoxy-5-methyl-4- sulfonatophenyl)diazenyl]naphthalene-2-sulfonate, Allura Red AC) or5-hydroxy-l-(4-sulfonatophenyl)-4-[(E)-(4-sulfonatophenyl)diazenyl]-lH-pyrazole-3- carboxylate or salts thereof (e.g., trisodium 5-hydroxy-l-(4-sulfonatophenyl)-4-[(E)- (4-sulfonatophenyl)diazenyl]-lH-pyrazole-3-carboxylate, tartrazine); or combinations thereof.

[0009] The stability described herein may be afforded to fluorochrome containing reagents capable of binding in several ways to the analyte (e.g., such as to different epitopes of the analyte). For example, the composition may comprise more than one (e.g., two, three, four, five, from two to 10, from two to five) fluorochrome labelling reagents. In various implementations, the composition comprises one or more (e.g., one, two, three, four) fluorochrome labelling reagents each labeling reagent comprising an antibody of different epitopes of said analyte or mutants thereof, wherein each fluorochrome labelling reagent is independently conjugated to fluorescein (e.g., fluorescein isothiocyanate, carboxyfluorescein succinimidyl ester fluorescein maleimide). In some embodiments, the composition further comprises a chemiluminescent labelling reagent (e.g., a compound having a chemiluminescent moiety such as an acridinium ester or sulfonamide conjugated to an analyte or binding partner thereof).

[0010] The present disclosure is partially premised on the discovery that incorporation of the light absorbent materials into fluorochrome containing reagents increases the stability of the fluorochrome containing reagents. This stability has profound impact on manufacture, including in the sensitivity required during reagent manufacture and storage. For example, the composition may be characterized as having within 20% of the fluorescent light output after 8 hours of exposure to LED light (e.g., white LED from 23,000-26,000 lux, 24,500 lux) at 2-8°C as compared said composition before said exposure. Without wishing to be bound by theory, the correlation between light absorbance and fluorescence may be implicated in the stability described herein. For example, the absorbance spectrum of said dye and said fluorochrome labelling reagent overlap in the visible region (e.g., said fluorochrome labelling reagent and said dye absorbs light in the 480-560 nm range (e.g., said fluorochrome labelling reagent and said dye independently have a wavelength of maximum absorbance of from 450- 550 nm or from 480 nm-560 nm).

[0011] In some embodiments, the weight ratio of said fluorochrome labelling reagent (or each of said fluorochrome labelling reagents) to said dye is from 20: 1 to 1 :20 (e.g., from 1 : 1 to 1 :20, from 1 : 1 to 1 : 15, from 1 :5 to 1 : 15). In various implementations, the weight ratio ofall fluorochrome labelling reagents to said dye is from 10: 1 to 1 : 10 (e.g., from 5: 1 to 1 :5, from 3: 1 to 1 :3, from 2: 1 to 1 :2). In some embodiments, the composition comprises less than (or from 0.01 g / L to or from 0.1 g / L to or from 0.2 g / L to) 5 g / L said dye (e.g., less than 4 g / L dye, less than 3 g / L dye, less than 2.5 g / L dye, from 0.05 g / L to 2.5 g / L). In certain aspects, the composition comprises more than 0.1 g / L or more than 0.2 g / L of said dye (e.g., from 0.2 g / L to 5 g / L, from 0.2 g / L to 4 g / L, from 0.2 g / L to 3 g / L from 0.2 g / L to 2.5 g / L, from 0.5 g / L to 5 g / L, from 0.5 g / L to 4 g / L, from 0.5 g / L to 3 g / L from 0.5 g / L to 2.5 g / L). In some embodiments, the composition comprises less than (or from 0.01 g / L to or from 0.1 g / L to or from 0.2 g / L to) 5 mg / mL (e.g., less than 2 mg / mL, less than 1 mg / mL from 0.1 mg / L to 1 mg / L) of said fluorochrome labelling reagent (or each of said fluorochrome labelling reagents). In various aspects, the total concentration of said fluorochrome labelling reagents is less than (or from 0.01 g / L to or from 0.1 g / L to or from 0.2 g / L to) 5 mg / mL (e.g., from 1 mg / mL to 4 mg / mL, from 1 mg / mL to 2 mg / mL); wherein said dye absorbs light in the 480-560 nm range (e.g., said dye has a wavelength of maximum absorbance of from 450-550 nm or from 480 nm-560 nm).

[0012] Kits for immunoassays comprising the compositions of the present disclosure are also provided. In some embodiments, the kit comprises an immunoassay composition of the present disclosure, such as an Ancillary Reagent contained in a container. In some embodiments, the container is cardboard. In various embodiments, the composition is contained in a transparent or translucent vial (e.g., plastic vial) in the container.

[0013] Methods for the detection or quantification of an analyte in a sample comprising:(a) providing a composition of the present disclosure and a chemiluminescent label conjugated to a binding partner of the analyte;(b) providing a solid support having immobilized thereon a molecule capable of forming a binding complex with said fluorochrome labelling agent, wherein said fluorochrome labelling agent is conjugated to a binding partner of the analyte and the binding complex is conjugated to the chemiluminescent label through said analyte and said fluorochrome labelling agent;(c) mixing said composition, said chemiluminescent label, said solid support, and said sample to form said binding complex;(d) separating said binding complex from said mixture;(e) triggering chemiluminescence from said chemiluminescent label (e.g., from the separated binding complex);(f) measuring the amount of chemiluminescent light emission with a luminometer; and(g) detecting the presence or calculating the concentration of said analyte by comparing the amount of light emitted with a standard dose response curve which relates the amount of light emitted to a known concentration of the analyte.In some embodiments, the chemiluminescent label is provided in the composition (e.g., in the Ancillary Reagent). In some embodiments, the chemiluminescent label is provided in another composition (e.g., Lite Reagent). In some embodiments, at least two chemiluminescent labels are provided. For example, a first chemiluminescent label may be provided in the composition comprising the light absorbent material (e.g., soluble dye) and fluorochrome labelling reagent such as the Ancillary Reagent, and a second chemiluminescent label may be provided in another immunoassay composition (e.g., Lite Reagent). In various implementations, the composition comprising the fluorochrome reagent (and optional light absorbent material) may be provided in a kit comprising a container which exposes the composition to light during storage (if light is incident upon the container). In some embodiments, fluorescence is not induced and / or measured. Without wishing to be bound by theory, the present invention is partially premised on the discovery that the dye inhibits degradation of the fluorochrome labelling reagent (which may be used for construction of the binding complex) and, when used in the binding complex, the dye does not inhibit its formation. In certain aspects, the dye does not absorb any fluorescence from the fluorochrome. In various embodiments, the measuring step may measure fluorescence from the fluorochrome labelling reagent.

[0014] Methods for increasing the light stability of an immunoassay reagent composition (e.g., a composition comprising a fluorescein labelling agent and a medium) may include adding a dye to the immunoassay reagent composition. The method may comprise dissolving a dye in a medium and adding a fluorochrome labelling reagent to the mixed composition.

[0015] 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

[0016] FIG. 1 is a schematic of an exemplary binding complex of the present disclosure formed from the use of fluorochrome labelling reagents which conjugate to both the solid phase and the analyte of interest.

[0017] FIGS. 2 A and 2B compare the difference in RLU following light exposure after 8 hours to initial, expressed as a percentage of the original RLU with various concentration standards when dye and no dye was added.

[0018] FIG. 3A provides the RLU measurements at each standard concentration in an ADVIA Centaur® XPT system assay comparing immunoassay reagent compositions having no dye initially (dotted diamond), no dye after 8 hours of light exposure (dashed square), dye initially (solid triangle) and after 8 hours of exposure (dotted circle). FIG. 3B provides the RLU measurements at each standard concentration in a Atellica® IM Analyzer assay comparing immunoassay reagent compositions having no dye initially (dotted diamond), no dye after 8 hours of light exposure (dashed square), dye initially (solid triangle) and after 8 hours of exposure (dotted circle). As can be seen, the compositions with dye show nearly identical curves before and after light exposure while the compositions without dye show dramatic changes evidencing the instability of the non-dyed immunoassay reagent compositions.

[0019] FIG. 4 provides the RLU measurements at each standard concentration as measured from in an Atellica® IM Analyzer comparing immunoassay reagent compositions having no dye initially (TSH3UL OR, solid line black circle), no dye after 8 hours of light exposure (TSH3UL 8 hr, solid line with *), dye initially in the ancillary reagent only (Red Dye 40 Anc. Well only solid line, white square), and dye in the ancillary reagent only after 8 hours of light exposure (Red Dye 40 Anc. Well only 8 hr, dashed line, diamond).DETAILED DESCRIPTION

[0020] 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.

[0021] Unless otherwise explicitly defined, the following terms and phrases are intended to have the following meanings throughout this disclosure:

[0022] All percentages for a composition given herein refer to the weight percentages of a particular component relative to the entire composition, including the carrier, unless otherwise indicated. For example, percentage with weight per volume may also be used when indicated-particularly in the Examples. It will be understood that the sum of all weight % of individual components within a composition will not exceed 100%.

[0023] 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. For example, “consisting essentially of’ may include components at their typical purity levels and have other components in normal tolerance levels (such as with impurity levels and / or measurement tolerances). Recitations of “comprising” include “consisting essentially” and “consisting of’

[0024] 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 5) 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.

[0025] 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 halfintegral 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).

[0026] When a moiety of the conjugates 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 forforming 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:H A' — N— L— where the unconjugated analyte or binding partner A has the structure A’-NFh and is the fluorescent or chemiluminescent moiety and L is a linker (which may optionally include a carrier protein).

[0027] 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’, , Li, Lc, RL, Rc, Ri, R2, R2a, R2b, R2C, R3, R4, Rs, Re, 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 does not 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.

[0028] 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’, , Li, Lc, RL, Rc, Ri, R2, R2a, R2b, R2C, R3, R4, Rs, Re, 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”).

[0029] 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-C20 alkyl, 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 R substituent 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.

[0030] 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 5 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.

[0031] 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 notinherently unstable. For example, any carbon atom will be bonded to two, three, or four other atoms, consistent with the four valence electrons of carbon.

[0032] 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.

[0033] 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, Ci- Ce alkyl 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, / / -propyl, isopropyl, and / e / 7-butyl. Any alkyl group referenced herein group (e.g., R, R’, R”, RN, Y, Y’, , Li, Lc, RL, Rc, Ri, R2, R3, R4, Rs, Rio, R11) 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).

[0034] 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.

[0035] 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 usedherein, 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 group (e.g., R, R’, R”, RN, Y, Y’, Q, LI, LC, RL, RC, RI, R2, R3, R4, RS, RIO, RII) 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., Ci-C4alkoxy).

[0036] 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.

[0037] 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 group (e.g., R, R’, R”, RN, Y, Y’, Q, Li, Lc, RL, Rc, Ri, R2, R3, R4, Rs, Rio, R11) 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.

[0038] 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 group (e.g., R, R’, R”, RN, Y, Y’, Q, Li, Lc, RL, Rc, Ri, R2, R3, R4, Rs, Rio, Rn) 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.

[0039] 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., withone 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(Ci-Ce alkyl), NC(O)(Ci-Ce alkyl), 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.

[0040] 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-lH-imidazolyl, 1, 2,5,6- tetrahydro-pyrimidinyl, and 5,6-dihydro-2H-[l,3]oxazinyl.

[0041] 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 moi eties can include, e.g., cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, adamantyl, and norbomyl (bicyclo[2.2.1]heptyl).

[0042] 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 moi eties can include, e.g., cyclohexenyl, cyclohexadienyl, or norbornenyl.

[0043] 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 moi eties include, e.g., phenyl and naphthyl.

[0044] Heteroaryl groups typically are aromatic monocyclic, bicyclic (2 fused rings), tricyclic (3 fused rings), or polycyclic (> 3 fused rings) hydrocarbon groups having one ormore 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, P-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.

[0045] In general, when a definition for a particular variable includes both hydrogen and nonhydrogen (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.

[0046] 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’, , Li, Lc, RL, Rc, Ri, R2, R3, R4, Rs, Rio, R11) 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, C2-12 alkynyl, C3-12 cycloalkyl, Ce-12 aryl, C3-12 heteroaryl, C3-12 heterocyclyl, C1-12 alkylsulfonyl, 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, C3-12cycloalkyl, C4-24 cycloalkylalkyl, Ce-12 aryl, C7-24 aralkyl, C3-12 heterocyclyl, C3-24 heterocyclylalkyl, C3-12 heteroaryl, or C4-24 heteroarylalkyl. Unless otherwise noted, all groups described 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.

[0047] Fluorescent or fluorochrome moieties in conjugates generally refer to a fluorescent moiety incorporated onto or within a chemical structure having desirable properties, such as binding with a target or attaching to a polypeptide of biotinyl moieties that can be detected by avidin (e.g., streptavidin containing a fluorescent label or enzymatic activity that can be detected by fluorescence detection methods). Conjugation of fluorescent or chemiluminescent compounds may occur by replacing a hydrogen on the unconjugated labelling compound with a covalent bond to a linker or the conjugated analyte or binding partner thereof. Conjugates may be formed by conjugated reactive functional groups on fluorescent moiety to portions of the analyte or binding partner thereof (such as via primary amine reactivity). Various methods of fluorescently labeling polypeptides, glycoproteins and other moieties are known in the art and may be used. Examples of labels for polypeptides include, but are not limited to compounds that fluoresce (e.g., fluorescein such as FITC, fluorescein maleimide, fluorescein N-succinimdyl ester, or rhodamine) conjugated to the polypeptide, intrinsically fluorescent proteins, and lanthanide phosphors. In some embodiments, labels are attached by spacer arms of various lengths to, for example, reduce potential steric hindrance. It will be understood that while the fluorescent conjugated may be considered a dye in some context, the compositions of the present disclosure typically include a fluorescent moiety and a dye distinct from the fluorescent conjugate. The dye, for example, may be in its native form, and not conjugated to an analyte or binding partner of an analyte. When used in some immunoassay formats, such as sandwich assays, the fluorescent moiety may be capable of being bound by an antibody or antibody fragment for the fluorescent moiety. For example, when conjugates having fluorescein such as fluorescein isothiocyanate(FITC) conjugated to a binding partner for an analyte are used, sandwich assay formats may include an anti-FITC capture antibody (e.g., monoclonal mouse anti-FITC capture antibody) which may be, for example, conjugated to the surface of a solid phase.

[0048] In some embodiments, the fluorescein in the fluorochrome labelling reagent has the formula:wherein Ri, R2, R3, and R4 are independently selected from the group consisting of hydrogen, fluoro, chloro, bromo, and — O — C1-4 alkyl,Rs is the point of conjugation to the fluorochrome containing moiety such as through an isothiocyanate reactive functional group or succinimidyl ester reactive functional group or maleimide reactive functional group. Examples of fluorescein derivatives include but are not limited to, 2',7'-dichlorofluorescein, 5(6)-carboxyfluorescein, 5(6)-carboxyfluorescein diacetate, 5-carboxyfluorescein, 6-[fluorescein-5(6)-carboxamido]hexanoic acid, 6- carboxyfluorescein, fluorescein diacetate 5-maleimide, fluorescein-O'-acetic acid, or 2', 7'- difluorofluorescein (OREGON GREEN™)

[0049] Antibody is used herein in the broadest sense and refers to, for example, intact monoclonal antibodies and polyclonal antibodies, multi-specific antibodies (e.g., bispecific antibodies), 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., IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2).

[0050] The labels present in the immunoassay reagent compositions may leverage the use of endogenous binding or specific binding substance (conjugated to a labelling moiety such as a fluorescent moiety or chemiluminescent moiety). Endogenous binding substances are thosefrom which analyte may be displaced by a displacing agent. Endogenous binding substances include both endogenous specific binding substances and endogenous non-specific binding substances. Specific binding substances are substances that have an area on a surface or in a cavity, which specifically binds to and is thereby defined as complementary with, a particular spatial and polar organization of the analyte or vice versa. Specific binding is distinguished from non-specific binding because specific binding involves the specific recognition of one of two different molecules for the other compared to substantially less recognition of other molecules. Non-specific binding substances are substances that may bind to, for example, an analyte, in general, by means of non-covalent binding between molecules that is relatively independent of specific surface structures. Endogenous binding substances for an analyte include, but are not limited to, proteins that specifically bind to an analyte such as, for example, anti -analyte antibodies and receptors (e.g., immunoglobulins); and immunophilins that are composed of major (e.g., cyclophilin that binds CsA, FK-binding proteins that bind tacrolimus and sirolimus) and minor categories. The minor category contains two groups: one with peptidylprolyl cis / trans isomerase activity (12, 25 and 56-50 kDa, the other without peptidylprolyl cis / trans isomerase activity (14, 37, and 52 kDa), target of rapamycin (TOR); al acid glycoprotein, lipoproteins, albumin and globulins; for example, or a combination of two or more of the above.

[0051] The assays of the present disclosure are typically methods of determining in a sample one or both of the presence and the amount of an analyte in the sample. The analyte is a substance of interest or the compound or composition to be detected and / or quantitated. Analytes include, for example, drugs, metabolites, pesticides and pollutants. Representative analytes, by way of illustration and not limitation, include alkaloids, steroids, lactams, aminoalkylbenzenes, benzheterocyclics, purines, drugs derived from marijuana, hormones, polypeptides which includes proteins, immunosuppressants, vitamins, prostaglandins, tricyclic antidepressants, anti-neoplastics, nucleosides and nucleotides including polynucleosides and polynucleotides, miscellaneous individual drugs which include methadone, meprobamate, serotonin, meperidine, lidocaine, procainamide, acetylprocainamide, propranolol, griseofulvin, valproic acid, butyrophenones, antihistamines, chloramphenicol, anticholinergic drugs, and metabolites and derivatives of all of the above. Also included are metabolites related to disease states, aminoglycosides, such as gentamicin, kanamicin, tobramycin, and amikacin, and pesticides such as, for example, polyhalogenated biphenyls, phosphate esters, thiophosphates, carbamates and polyhalogenated sulfenamidesand their metabolites and derivatives. The term analyte also includes combinations of two or more of polypeptides and proteins, polysaccharides and nucleic acids. Such combinations include, for example, components of bacteria, viruses, chromosomes, genes, mitochondria, nuclei and cell membranes. Protein analytes include, for example, immunoglobulins, cytokines, enzymes, hormones, cancer antigens, nutritional markers and tissue specific antigens. Such proteins include, by way of illustration and not limitation, protamines, histones, albumins, globulins, scleroproteins, phosphoproteins, mucoproteins, chromoproteins, lipoproteins, nucleoproteins, glycoproteins, T-cell receptors, proteoglycans, HLA, unclassified proteins, e.g., somatotropin, prolactin, insulin, pepsin, proteins found in human plasma, blood clotting factors, protein hormones such as, e.g., follicle-stimulating hormone, luteinizing hormone, luteotropin, prolactin, chorionic gonadotropin, tissue hormones, cytokines, cancer antigens such as, e.g., PSA, CEA, c-fetoprotein, acid phosphatase, CA19.9, CA15.3 and CA125, tissue specific antigens, such as, e.g., alkaline phosphatase, myoglobin, CPK-MB and calcitonin, and peptide hormones. Other polymeric materials of interest are mucopolysaccharides and polysaccharides. As indicated above, the term analyte further includes oligonucleotide and polynucleotide analytes such as m-RNA, r- RNA, t-RNA, DNA and DNA-RNA duplexes, for example.

[0052] Non-limiting examples of bacteria that can be detected in accordance with the present disclosure include Acinetobacter, Actinomyces, Aeromonas, Aggregatibacter, Atopobium, Bacillus, Bacteroides, Bartonella, Bifidobacterium, Boreilia, Brucella, Campylobacter, Chlamydia, Chlamydophila, Clostridium, Corynebacterium, Coxiella, Eikenella, Enterobacter, Enterococcus, Escherichia, Eubacterium, Francisella, Fusobacterium, Gardnerella, Haemophilis, Helicobacter, Klebsiella, Lactobacillus, Listeria, Mobiluncus, Moraxella, Mycobacterium, Mycoplasma, Neisseria, Parviomonas, Pasteurella, Porphyromonas, Prevotella, Propionibacterium, Proteus, Pseudomonas, Rickettsia, Salmonella, Serratia, Shigella, Staphylococcus, Streptococcus, Tannerella, Treponema, Vibrio, and Yersinia species.

[0053] Non-limiting examples of viruses that can be detected in accordance with the present disclosure include adenoviruses, astroviruses, coronaviruses (such as, but not limited to, severe acute respiratory syndrome coronavirus (SARS-CoV, SARS-CoV2) or Middle East respiratory syndrome coronavirus (MERS-CoV)), Coxsackie viruses, cytomegaloviruses (CMV), echoviruses, encephalitis viruses, enteroviruses, Epstein-Barr viruses (EBV), erythroviruses, hantaviruses, hepatitis viruses, herpes viruses, human immunodeficiencyviruses (HIV), influenza viruses, noroviruses, papilloma viruses, parainfluenza viruses, paramyxoviruses, polio viruses, rabies viruses, respiratory syncytial viruses (RSV), rhinoviruses, rotoviruses, rubella viruses, rubeola viruses, Varicella-Zoster viruses, West Nile viruses, and Zika viruses. Non-limiting examples of protozoans that can be detected in accordance with the present disclosure include Ascaris, Babesia, Cryptosporidium, Cyclospora, Entamoeba, Enterobius, Giardia, Hymenolepis, Necator, Plasmodium, Strongyloides, Taenia, Toxoplasma, and Trichomonas species. Non-limiting examples of fungi that can be detected in accordance with the present disclosure include yeasts, molds, and the like, including (but not limited to) Candida, Cryptococcus, Epidermophyton, Malassezia, Microsporum, and Trichophyton species. In a particular (but non-limiting) embodiment, the microorganism detected by the serology assay is severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), HIV, Hepatitis B Core Total, Epstein-Barr virus, Herpes Virus (HSV), CMV, Rubella, H. pylori, or Toxoplasma gondii. The antigen may be any antigen from the microorganism to be detected. For example, if the microorganism is SARS-CoV-2, the antigen may be any SARS-CoV-2 antigen such as the antigen may be from the nucleocapsid (N) protein, the spike (S) protein, the membrane (M) protein, the envelope (E) protein, or the fusion (F) protein. In particular (but non-limiting) embodiments, the antigen may be from the nucleocapsid protein or the spike protein. In some embodiments, the antigen is the receptor binding domain (RBD) of the SI subunit of SARS-CoV-2 spike protein. The RBD SI antigen can be obtained from any source known in the art. For example (but not by way of limitation), this particular antigen is commercially available from GenScript (Piscataway, N.J.); Meridian Life Sciences, Inc. (Memphis, Tenn.); Sino Biological US Inc. (Wayne, Pa.); ACRO Biosystems (Newark, Del.); Biorbyt, LLC (St. Louis, Mo.); Icosagen, AS (San Francisco, Calif.); and Bios Pacific Inc. (Emeryville, Calif.).

[0054] Table 1 provides exemplary fluorochrome labelling conjugates and chemiluminescent analogs suitable for use in the Ancillary and / or Lite Reagents of the present disclosure. These combinations of conjugates, particularly when used in sandwich assays may benefit by the incorporation of soluble dyes as disclosed herein.Table 1

[0055] Anti-human Ig antibodies may be used in the labels of the presently disclosed immunoassay reagent compositions and specifically bind to any portion of any humanimmunoglobulin molecules known in the art or otherwise contemplated herein. For example (but not by way of limitation), the antibodies may be directed to human IgG, IgE, IgM, IgD, and / or IgA, and / or any portion thereof (such as, but not limited to, anti-human gamma chain, anti-human H+L, anti-human light chain, and the like). Anti-human Ig antibodies (including, but not limited to, anti-human IgG, anti-human IgM, and / or anti-human IgA antibodies, as well as antibodies that recognize two or more human immunoglobulin antibodies) are well known in the art, are widely commercially available, and have been vastly studied. For example (but not by way of limitation), a few commercial sources of anti-human IgG monoclonal and / or polyclonal antibodies include Rockland Immunochemicals, Inc. (Pottstown, Pa.); USBiological Life Sciences (Swampscott, Mass.); Santa Cruz Biotechnology, Inc. (Dallas, Tex.); Jackson Immuno Research Labs, Inc. (West Grove, Pa.); Thermo Fisher Scientific (Waltham, Mass.); and Sigma-Aldrich Corp. (St. Louis, Mo.). However, this list is not inclusive, and there are many additional commercial sources of antihuman Ig antibodies that can be utilized in accordance with the present disclosure. Thus, a person having ordinary skill in the art will clearly and unambiguously be able to identify and select a variety of anti-human Ig antibodies that can be utilized in accordance with the present disclosure, and as such, no further description of the anti-human Ig antibodies or the characteristics thereof is deemed necessary.

[0056] Non-limiting examples of light absorbent materials include dyes, inks, pigments, food coloring, turmeric, titanium dioxide, caretinoids (e.g., bixin, P-carotene, apocarotenals, canthaxanthin, saffron, crocin, capsanthin and capsorubin occurring in paprika ole-oresin, lutein, astaxanthin, rubixanthin, violaxanthin, rhodoxanthin, lycopene, and derivatives thereof), and FD&C colorants [e.g., FD&C Blue No. 1 (brilliant blue FCF); FD&C Blue No. 2 (indigotine); FD&C Green No. 3 (fast green FCF); FD&C Red No. 40 (Allura Red AC); FD&C Red No. 3 (erythrosine); FD&C Yellow No. 5 (tartrazine); and FD&C Yellow No. 6 (sunset yellow)]. In some embodiments, the light absorbent material comprises: p-phenylenediamine, l,4-diazabicyclo[2.2.2]octane,6-hydroxy-5-[(2-methoxy-5-methyl-4-sulfonatophenyl)diazenyl]naphthalene-2- sulfonate or salts thereof (e.g., disodium 6-hydroxy-5-[(2-methoxy-5-methyl-4- sulfonatophenyl)diazenyl]naphthalene-2-sulfonate, Allura Red AC),5-hydroxy-l-(4-sulfonatophenyl)-4-[(E)-(4-sulfonatophenyl)diazenyl]-lH-pyrazole- 3-carboxylate or salts thereof (e.g., trisodium 5-hydroxy-l-(4-sulfonatophenyl)-4- [(E)-(4-sulfonatophenyl)diazenyl]- lH-pyrazole-3 -carboxylate, tartrazine), propyl gallate,1,4-piperazinedi ethanesulfonic acid or salts thereof (e.g., disodium 4- piperazinediethanesulfonic acid, PIPES disodium),5-Benzoyl-4-hydroxy-2-methoxybenzene-l -sulfonic acid or salts thereof (e.g., sulisobenzone), [(3Z)-3-[[4-[(Z)-[7,7-Dimethyl-2-oxo-l-(sulfomethyl)-3- bicyclo[2.2.1]heptanylidene]methyl]phenyl]methylidene]-7,7-dimethyl-2-oxo-l- bicyclo[2.2.1]heptanyl]methanesulfonic acid or salts thereof (e.g., ecamsule),2-Phenyl-3H-benzimidazole-5-sulfonic acid or salts thereof (e.g., ensulizole) a metal oxide (e.g., zinc oxide, titanium oxide, combinations thereof), or combinations thereof.

[0057] Typically, the chemiluminescent labels have the structure of formula (I):A - L - (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 is a chemiluminescent acridinium comprising the structure:and ‘ ’ are independently 0 e.g., all R2 groups are hydrogen, all R3 groups are hydrogen), 1, 2, 3, or 4;Ri is hydrogen, -R, -Xb, -RL-Xb, -Lc-R, -Lc-Xb(e.g., -Li-Xb), -Z, -RL-Z, -Lc-Z (e.g., - Li-Z), or -RL-LC-RL-Z (e g., -RL-LI-RL-Z);R2 and R3 are 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 fusedheterocyclic group) and wherein R2 or R3 may comprise a linkage to an imaging agent such as a fluorophore (e.g., rhodamine);Lcis a divalent C1-35 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:“zw” is 0 (i.e. it is a bond) or 1; and “ / ?” 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:“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;Li is independently at each occurrence -O-, -S-, -NH-, -N(RN)-, -(CH2)I-IO-, -S(=O)I-2- - C=C-, -C=C-(CH2)I-3-, -C(O)-, -O-C(O)-, -C(O)-(CH2)I-4-, -(CH2)I-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)I-3-, - (CH2)I-3-C(O)-N(RN)- -(CH2)I-3-N(RN)-C(O)-, -NH-S(O)I-2-, -N(RN)-S(O)I-2-, - S(O)I-2-N(RN)-, -S(O)I-2-NH- -(CH2)I-3-NH-S(O)I-2-, -(CH2)I-3-N(RN)-S(O)I-2-, - (CH2)I-3-S(O)I-2-N(RN)-, -(CH2)I-3-S(O)I-2-NH- -O-(CH2)I-4-, -(CH2)I-4-O-, -S- (CH2)I-4-, -(CH2)I-4-S-, -NH-(CH2)I-4-, -N(RN)-(CH2)I-4-, -(CH2)I-4-N(RN)- -(OCH2)I- 10-, -(CH20)I-IO-, -(OCH2CH2)I.IO- or -(CH2CH20)I-IO-;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); andR’ 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 labels may independently have the structure of formula (la):wherein is O or N;Y is selected from -R or -RL-Z, or in the case where is O then Y is absent; andY’ is either absent (i.e. it is a bond), or is selected from -Li- -RL-, -RL-LI-, -L1-L1-, -Li- RL— , -LI-RL-LI, and -RL-LI-RL- In some embodiments, the chemiluminescent labels independently have the structure of formula (lb) or (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; R1is 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, R1is selected from – SO3-, –RL– SO3- such -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 sulfonate salts, haloalkyl sulfonate salts, fluoroalkyl sulfonate 25 ACTIVE 704156808v1salts, acridinium halide salts such as acridinium chloride salts, a compound having the structure of formula I, Ia, Ib, or Ic wherein R1is 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-). Suitable acridinium labels are provided in in U.S. Pat No 7,309,615, International Pub. No. WO2015 / 006174, in Law et al. Journal of Bioluminescence and Chemiluminescence 4: 88-89 (1989), U.S. Pat. No. 8,119,422, U.S. Pat No. 6,664,043, U.S. Pat. No. 7,309,615, U.S. Pat. No. 9,575,062, U.S. Pat. No. 9,487,480, and U.S. Pat. No. 5,543,524, each of which are hereby incorporated by reference in their entirety and particularly in relation to assay formats and chemiluminescent acridiniums.

[0058] 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. The antibody is immobilized or attached to a solid phase such as a particle, bead, membrane, microtiter plate, or any other solid surface.

[0059] In an example of a competitive heterogeneous assay, a support having an antibody for an analyte (e.g., 3C3, 3H10, 4G8 bovine monoclonal antibodies) bound thereto is contacted with a medium containing a sample suspected of containing the analyte and the chemiluminescent and fluorescent conjugates (or “labeled analogs”) described herein. Analyte from the sample may competes for binding to the analyte antibody with the labeled analog. In some embodiments, a binding complex is formed conjugating the chemiluminescent label to the fluorescent label via the analyte, which is then bound to a solid support by antibody conjugation to the fluorochrome moiety. 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.

[0060] 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 26 ACTIVE 704156808v1reaction 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 supernatant solution.

[0061] In an example of a sandwich assay format employing at least two antibodies (or fragments thereof), a solid support with a first immobilized antibody or fragment thereof for an analyte is mixed with a reagent comprising a binding partner for the first antibody (e.g., a fluorescent moiety such as fluorescein) conjugated to a binding partner for the analyte of interest. This first conjugate may be provided in what is referred to herein as an “Ancillary Reagent” which is an immunoassay composition typically comprising a conjugate having a fluorescent moiety conjugated to a binding partner for an analyte of interest. The mixture also includes a sample containing the analyte and a labelled conjugate comprising a second antibody or fragment thereof that also binds to the analyte. The labelled conjugate may be labelled with a chemiluminescent moiety such as an acridinium and may be provided to what is referred to herein as a “Lite Reagent” which is an immunoassay composition comprising the conjugate detected in the assay (e.g., a conjugate comprising a chemiluminescent acridinium conjugated to a binding partner of the analyte of interest). A binding complex is formed between the solid particle and the labelled conjugate via the conjugate in the Ancillary reagent conjugated to analyte in the sample which is also bound to the detectable conjugate in the Lite Reagent. An example of this binding complex is shown in FIG.1 which provides an exemplary schematic of such a binding complex.

[0062] In FIG. 1 an exemplary binding complex is shown leveraging a fluorescent moiety conjugation scheme. Solid phase 1 comprising paramagnetic particle (PMP) 2 conjugated to a first monoclonal anti-fluorescent antibody (e.g., monoclonal mouse anti-FITC antibody) 4 is bound to a fluorescent conjugate 5 comprising a fluorescent moiety 6 bound to a second antibody 8. Solid phase 1 may be provided in a Solid Phase reagent which may be a composition comprising the solid phase and one or more buffers and / or preservatives and / or stabilizers. Antibody 8 is a binding partner for the analyte of interest 9. In the detectable binding conjugate of FIG.1, analyte of interest 9 is further conjugated to detectable label 10 which comprises a third antibody for the analyte 11 conjugated to a detectable moiety 13 (e.g., a chemiluminescent acridinium moiety).

[0063] Solid phase 1 may be provided in a solid phase immunoassay reagent composition comprising, for example, bovine serum albumin and sodium azide. The fluorescent conjugate 5 may be provided in an Ancillary Reagent which may further comprise bovine 27 ACTIVE 704156808v1serum albumin. In some embodiments, the Ancillary Reagent comprises the dye of the present disclosure as well and optionally, a buffer and / or stabilizer and / or preservative. In some embodiments, the Ancillary Reagent comprises more than one fluorescent conjugate. For example, the Ancillary Reagent may comprise fluorescent conjugates each comprising the same fluorescent moiety conjugated (e.g., fluorescein conjugated via, for example, a thiocarbamate) to antibodies for an analyte of interest (e.g., Hepatitis B Surface Antigen) and mutants of the analyte of interest. In some embodiments, the Ancillary Reagent further comprises a detectable moiety (e.g., acridinium) conjugated to an antibody for the analyte of interest or mutants thereof. Detectable conjugate 10 may be provided in the Lite Reagent or the Ancillary Reagent or both, each of which may further comprise one or more buffers and / or stabilizers and / or preservatives. Conjugates may be provided in at least one of the Ancillary Reagent or the Lite Reagent to optimize performance and shelf-life stability. Formation of the binding complex may be induced by mixing the biological sample with the Lite Reagent, Ancillary Reagent, and Solid Phase Reagent (sequentially, simultaneously, in any order) and optionally incubating between each mixing step.

[0064] The light signal associated with the binding complex can be 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 for a fluorescent moiety bound to coated (e.g., streptavidin coated) optionally paramagnetic particles. In some embodiments, the second antibody is a mouse monoclonal antibody for the analyte of interest conjugated to fluorescent moiety (e.g., fluorescein). In some embodiments, the third antibody is a mouse monoclonal antibody for the analyte of interest conjugate to a chemiluminescent moiety.

[0065] 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 28 ACTIVE 704156808v1in 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.

[0066] 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 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.

[0067] Compositions in accordance with the principles described herein may be employed in assays that utilize paramagnetic particles. One particular example of such an assay is an acridinium ester label immunoassay using paramagnetic particles as a solid phase (“ADVIA” immunoassay). Other immunoassays suitable for use with the immunoassay reagent compositions of the present disclosure include the “CENTAUR” or “ATELLICA” assays available from Siemens which include the ADVIA CENTAUR® XP and ADVIA CENTAUR® XPT systems and ATELLICA® IM Analyzer.

[0068] One general group of immunoassays in which embodiments of the present compositions may be employed to determine the presence and / or amount of and analyte in a sample includes immunoassays using a limited concentration of one of the assay reagents. Another group of immunoassays involves the use of an excess of one or more of the principal reagents. Another group of immunoassays are separation-free homogeneous assays in which the labeled reagents modulate the label signal upon binding of the present composition and an analyte in the sample. Some known assays utilize a signal producing system (sps) that employs first and second sps members. The sps members may be related in that activation of one member of the sps produces a product such as, for example, light or an activated product, which results in activation of another member of the sps. Some known assays utilize a signal producing system that employs first and second sps members. The sps members may be related in that activation of one member of the sps produces a product such as, e.g., light, which results in activation of another member of the sps. In most embodiments, the assay architecture of the present disclosure uses only one sps member labelled with acridinium. 29 ACTIVE 704156808v1

[0069] In a typical competitive heterogeneous assay, an embodiment of the present composition that comprises a specific binding pair (“sbp”) member that binds to an analyte is contacted with a medium containing the sample suspected of containing the analyte and the analyte conjugated to a label that is reactive with the sps member of the present composition or with a product of the activation of the sps member. Activation of the sps member produces a signal from the label if the analyte is present, which is determined by conventional techniques and is related to the amount of the analyte in the sample.

[0070] In a sandwich assay embodiment, the sample suspected of containing the analyte in a suitable medium is contacted with labeled antibody for the analyte and incubated for a period of time. Then, the medium is contacted with the present composition, which comprises a label that is related to the label of the labeled antibody as discussed above. After an incubation period, the support is separated from the medium and washed to remove unbound reagents. The support or the medium is examined for the presence of a signal, which is related to the presence or amount of analyte. In another variation of the above, the sample, the present composition and the labeled antibody are combined in a medium and incubated in a single incubation step. Separation, wash steps and examination for signal are as described above.

[0071] In one example of an ADVIA immunoassay, a detection system is employed that includes a labeled analyte analog (capture moiety), a specific binding pair (“spb”) member for the analyte bound to copolymer-coated paramagnetic iron oxide particles in accordance with the principles described herein as a solid phase (SP), and an acridinium ester labeled antibody specific for the analyte (detection antibody). The small molecule may be, for example, biotin or fluorescein and the respective sbp member for the small molecule may be streptavidin or antibody for fluorescein. Analyte in a patient sample competes with labeled analyte analog of the capture moiety for binding to the acridinium ester labeled and / or fluorescein labelled detection anti-analyte antibody. The assay may be carried out on a CENTAUR®, CENTAUR® XP, CENTAUR® CP, ADVIA CENTAUR® CP system, apparatus (Siemens Healthcare Diagnostics Inc., Tarrytown, NY) in accordance with the manufacturer's directions. After an appropriate incubation period, the paramagnetic particles are separated from the assay medium by application of a magnetic field. The paramagnetic particles are examined for the amount of signal from the acridinium ester labeled anti-analyte antibody by exposing the paramagnetic particles to an activation agent for the acridinium ester such as, for example, one or more of an acid, an oxidizer, a base and a surfactant. 30 ACTIVE 704156808v1

[0072] In a typical non-competitive sandwich assay, an immune sandwich complex is formed in an assay medium. The complex comprises the analyte, a sbp member (first sbp member) and a second sbp member, both of which that bind to the analyte. One of the spb members may comprise a fluorescent moiety (stabilized with a dye of the present disclosure for example, as provided in an immunoassay reagent composition comprising a soluble dye as disclosed herein) which is conjugated to a solid phase via an anti-fluorescent antibody such as anti-FITC. Subsequently, the immune sandwich complex is detected and is related to the amount of analyte in the sample. The immune sandwich complex is detected by virtue of the presence in the complex of one or more of a label of the present composition and a label of the second sbp member.

[0073] In one approach in a sandwich assay, a first incubation of the present composition is contacted with a medium containing a sample suspected of containing the analyte. After a wash and separation step, the support of the present composition is contacted with a medium containing a second sbp member such as, for example, an antibody for the analyte, which contains a label such as an enzyme, for a second incubation period. The labels may be related in that activation of one of the labels activates the other label if the analyte is present in the medium. In some embodiments, the two spb members are not related to one another inasmuch as activation of one spb member (e.g., chemiluminescent label) is not dependent on the other label (e.g., fluorescent label) aside from both being implicated in the formation of the binding complex. The support may again be washed and separated from the medium and either the medium or the support is examined for the presence of a signal. The presence and amount of signal is related to the presence or amount of the analyte.

[0074] In another aspect of the invention, an immunoassay reagent composition (e.g., Ancillary Reagent) may be provided for the detection of an analyte comprising a fluorescent moiety (e.g., fluorescein) bound the analyte or binding partner thereof. The composition may comprise from, for example 0.01 mg / mL to 10 mg / mL of each fluorescent label or from 0.1 to 5 mg / mL of each fluorescent label or from 0.1 to 1 mg / mL of each fluorescent label. In some embodiments, the compound is provided in a reagent composition which further comprises a buffer. In various implementations, the immunoassay reagent composition further comprises a chemiluminescent label (e.g., an acridinium such as an acridinium ester or acridinium sulfonamide conjugated to an analyte or binding partner thereof). The composition may have a concentration of 0.01 mg / mL to 10 mg / mL of chemiluminescent 31 ACTIVE 704156808v1label or from 0.1 to 5 mg / mL of chemiluminescent label or from 0.1 to 1 mg / mL of chemiluminescent label.

[0075] Samples for measure may include any type of biological sample that may be utilized in accordance with the present disclosure. In certain embodiments, the sample may be any fluidic sample and / or sample capable of being fluidic (e.g., a biological sample mixed with a fluidic substrate). Examples of biological samples that may be utilized include, but are not limited to, whole blood or any portion thereof (i.e., plasma or serum), saliva, sputum, cerebrospinal fluid (CSF), surgical drain fluid, skin, intestinal fluid, intraperitoneal fluid, cystic fluid, sweat, interstitial fluid, extracellular fluid, tears, mucus, bladder wash, urine, swabs, semen, fecal, pleural fluid, nasopharyngeal fluid, combinations thereof, and the like. It should be noted that although the present disclosure is directed towards a biological sample, one skilled in the art will appreciate that the concepts disclosed herein may be applied to any sample wherein a concentration of an analyte (such as, but not limited to, a hapten) may be determined, and as such, the scope of the present disclosure is not limited to biological samples. 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.

[0076] 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.

[0077] 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 32 ACTIVE 704156808v1include borate, phosphate, carbonate, TRIS, barbital, PIPES, HEPES, MES, ACES, MOPS, and BICINE, for example.

[0078] 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.

[0079] Triggering the chemiluminescence of the chemiluminescent analogs (when present) may be performed by the addition chemiluminescent triggering reagents (typically added to the binding complex). 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.

[0080] The assay components / reagents of the compositions / kits / methods may be provided in any form that allows them to function in accordance with the present disclosure. For example, but not by way of limitation, each of the reagents may be provided in liquid form and disposed in bulk and / or single aliquot form within the kit. In some embodiments, the kit may comprise a container, composed of a light transparent material such as plastic including high density polyurethane (HDPE), wherein the container comprises an immunoassay composition of the present disclosure. As described herein, the immunoassay compositions comprising soluble dyes and one or more fluorescent conjugates have better shelf lives as compared to compositions with the dyes. Accordingly, the container may include light transparent materials without sacrifice of composition stability.

[0081] In addition to the assay components / reagents described in detail herein above, the kits may further contain other reagent(s) for conducting any of the particular assays described or otherwise contemplated herein. The nature of these additional reagent(s) will depend upon the particular assay format, and identification thereof is well within the skill of one of ordinary skill in the art; therefore, no further description thereof is deemed necessary. Also, the components / reagents present in the kits may each be in separate containers / compartments, or various components / reagents can be combined in one or more containers / compartments, depending on the cross-reactivity and stability of the components / reagents. In addition, the kit 33 ACTIVE 704156808v1may include a microfluidics device in which the components / reagents are disposed. However, due to the increased light stability afforded by the immunoassay reagent compositions described herein, the container of the kit does not necessarily need to prevent or minimize light (e.g., ambient light) from interacting with the compositions contained therein. In some embodiments, the container of the kit is translucent or clear. In some embodiments, the container of the kit is cardboard. In various implementations the kit contains a translucent or clear vial comprising the immunoassay reagent composition.

[0082] The relative amounts of the various components / reagents in the kits can vary widely to provide for concentrations of the components / reagents that substantially optimize the reactions that need to occur during the assay methods and further to optimize substantially the sensitivity of an assay. Under appropriate circumstances, one or more of the components / reagents in the kit can be provided as a dry powder, such as a lyophilized powder, and the kit may further include excipient(s) for dissolution of the dried reagents; in this manner, a reagent solution having the appropriate concentrations for performing a method or assay in accordance with the present disclosure can be obtained from these components. Typically, the kit comprises the immunoassay reagent composition in liquid form. Positive and / or negative controls may also be included with the kit.

[0083] In addition, the kit can further include a set of written instructions explaining how to use the kit to, for example, induce chemiluminescence from a binding complex. A kit of this nature can be used in any of the methods described or otherwise contemplated herein. EXAMPLES

[0084] 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.

[0085] Example 1: Light Stability using Dyed Fluorescein Containing Reagents

[0086] Dyes were added to an immunoassay reagent composition (Ancillary Reagent) comprising three monoclonal antibodies of different epitopes of hepatitis B surface antigen (HBs) each independently conjugated to a fluorescein label (fluorescein isothiocyanate). One of the monoclonal antibodies was for the common form of the hepatitis B surface antigen (M11193) conjugated with fluorescein isothiocyanate. Two of the monoclonal antibodies were for mutants of the hepatits B surface antigen (2C4, MAK04-75 / 01) conjugated to 34 ACTIVE 704156808v1carboxyfluorescein succinimidyl ester. The Ancillary Reagent also comprised a chemiluminescent acridinium label conjugated to a monoclonal antibody for a common form of hepatitis B surface antigen (M12420). The chemiluminescent acridinium label is a dimethyl acridinium ester (N-sulfopropyl acridinium ester) conjugated via a zwitterionic linker, glutaraldehyde linker comprising an N-succinimidyl reactive functional group for conjugating to the antibody (NSP-DMAE-Z-Glu-NHS). The reagent had a total concentration of fluorochrome labelling reagents of 1.43 g / L.

[0087] The Ancillary Reagent was mixed with a solid phase having surface coated anti-FITC antibody, different standards comprising different concentrations of Hepatitis B surface antigen, and a Lite Reagent (comprising an acridinium ester conjugated to an antibody for hepatitis B surface antigen). The acridinium ester conjugated antibodies (from Ancillary Pack and Light Regent Well), detect different varieties of surface antigen. The system measures the RLU output from the chemiluminescence of both acridinum ester labelled antibodies.

[0088] Table 2 provides the measured relative light units (RLU) from the samples without dye and the calculated dose based on those RLUs for different samples of varying concentration. Each immunoassay reagent composition was exposed to 8 hours of LED light having a 23,000-26,000 lux to mimic accelerated exposure of the immunoassay reagent composition when present a plastic container composed of high-density polyethylene typically used in automated immunoassays. “FC” refers to the Full Curve reduction, which indicates that the standard doses were generated off the standards curve. Table 235 ACTIVE 704156808v1

[0089] Ten different light absorbing chemicals, two of which were dyes, were added and solubilized into otherwise identical reagent compositions. Table 3 provides the chemicals added. Table 3Most of these chemicals provided no or very little protection against LED. p- phenylenediamine detrimentally impacted the Ancillary Reagent performance. However, surprisingly, both Allura Red AC (disodium 6-hydroxy-5-[(2-methoxy-5-methyl-4- sulfophenyl)azo]-2-naphthalenesulfonate) and Tartrazine (sodium 5-oxo-1-(4- sulfonatophenyl)-4-((4-sulfonatophenyl)diazenyl)-4,5-dihydro-1H-pyrazole-3-carboxylate) maintained the ability of immunoassay reagent compositions to function in an immunoassay.

[0090] Table 4 provides the measured relative light units (RLU) from the samples without dye and the calculated dose based on those RLUs for different samples of the same varying standard concentration as provided in Table 2. Table 436 ACTIVE 704156808v1

[0091] As can be seen, using soluble dyes, and specifically, food dyes such as Allura Red AC, in immunoassay fluorescent compositions can prevent light degradation of the immunoassay reagent composition. FIG. 2A compares the RLU difference ((RLU8hrs- RLU0hrs) / RLU0hrs) for each composition for immunoassay compositions with and without dye. As can be seen, nearly consistently at each analyte concentration measured (particularly at higher concentrations), addition of the soluble dye reduced signal deterioration upon exposure to light. RLU increases at lower standards is likely due to light degradation of fluorescein containing conjugates resulting in increases in nonspecific binding. Without wishing to be bound by theory, it is believed that the degradation of fluorescein in the mAbs conjugates can lead to aberrant binding of acridinium ester to the solid phase when the analyte is absent or at low concentrations thus resulting in this increase in signal. As can be seen, use of the dye prevents this nonspecific binding.

[0092] The experiment was repeated on two additional samples having an unknown target concentration and target concentration of 5.32 (n=6). Table 5 provides the RLU, FC Dose, and % coefficient of variance (RLU %CV) data for these measurements. Table 5

[0093] Additionally, measurements were performed on immunoassay reagents having 0.1 g / L Allura Red AC. Table 6 provides the RLU, FC Dose, and % coefficient of variance (RLU %CV) data for these measurements. Table 637 ACTIVE 704156808v1

[0094] FIG.2B compares the RLU difference for immunoassay regents having no dye, 1 g / L dye, and 0.1 g / L dye. As can be seen, even 0.1 g / L dye provides increased light stability to immunoassay reagents and this trend should continue to lower dye concentrations as well. The p-value between data sets having no dye and dye (either at 1.0 g / L or 0.1 g / L) indicated that these results were statistically significant with a 95% confidence. Table 7 provides the p- Values for each standard comparing the reagents with dye to reagents without dye. Each p- value represents statistical differences between RLUs generated using reagents with 0.1 g / L and 1.0 g / L of red dye after 8 hours of light exposure. Table 7

[0095] Dose analyses were performed on standards using immunoassay reagent compositions without dye and with Allura Red AC at 1.00 g / L. Runs were performed on the Siemens Centaur XPT system and the Atellica IM system. Table 8 provides the comparison of RLU output for immunoassay reagent compositions with and without dye in each system (two instruments per system). 38 ACTIVE 704156808v1Table 8FIG. 3A compares the Standard RLU as measured in the Centaur XPT for each formulation tested after 0 hours of light exposure and then after 8 hours of exposure. FIG. 3B compares the Standard RLU as measured in the Atellica IM for each formulation tested after 0 hours of light exposure and then after 8 hours of exposure. In these figures, the Red Dye-0 hr and the Reference – 0 hr curves are nearly identical. As can be seen, the addition of dye to immunoassay reagent compositions provides minimal change in the RLU output for a variety of assay formats and measurement protocols.

[0096] Additional quality control measurement were performed using the immunoassay reagent compositions with and without dye including a negative patient panel. Minimal distinctions between immunoassay reagent compositions with and without dye and the immunoassay reagent compositions met the standard specifications required for immunoassay operation.

[0097] Measurements were repeated following exposure of the immunoassay reagent compositions to 8 hours of LED light at 24,500 lux. Table 9 provides the RLU comparison of the tested reagents in each system. As can be seen, despite the dyes having unexpectedly minimal effect on the RLU output and utility in each assay format, the addition of dyes to immunoassay reagent compositions also unexpectedly increased the light output stability despite light exposure which typically degrades the immunoassay reagent compositions. 39 ACTIVE 704156808v1Table 9

[0098] Example 2: Light Stability in Thyroid Stimulating Hormone Assay

[0099] The addition of dye to reagents in a thyroid stimulating hormone 3-ultra (TSH3-UL) assay and the concomitant increases in stability were assessed. The assay used three reagents: a lite reagent (L / R) comprising bovine serum albumin (BSA) conjugated to mouse monoclonal anti-thyroid stimulating hormone (TSH) (0.3 µg / mL) labeled with acridinium ester in HEPES buffered saline; a solid phase reagent comprising mouse monoclonal anti-fluorescein antibody covalently linked to paramagnetic particles (85 µg / mL) in buffer; an ancillary well reagent (Anc. Well Reagent) comprising fluorescein isothiocyanate (FITC) conjugated to mouse monoclonal anti-TSH (3 µg / mL). Red Dye 40 (1 g / L) was added to the ancillary well reagent only or both the ancillary well reagent and the light reagent. The reagents were either exposed to 8 hours of LED light having a 23,000-26,000 lux to mimic accelerated exposure of the immunoassay reagent composition when present a plastic container composed of high-density polyethylene typically used in automated immunoassays or stored in the dark.

[0100] The ancillary reagent, solid phase reagent, and light reagent were mixed with different standards comprising different concentrations of thyroid stimulating hormone (TSH). The system measured the RLU output from the chemiluminescence of the acridinium 40 ACTIVE 704156808v1moiety. Table 10 provides the accelerated light stability RLU percent differences for each experimental setup as measured in an Atellica IM. Table 10

[0101] TSH3UL pack reagents spiked with Red Dye 40 in the light reagent and / or ancillary well reagent had very low RLU % difference when comparing the 8 hour light exposure condition to the 0 hour light exposure condition (average of 3%). In contrast, TSH3UL readypack reagents without dye had an average RLU % difference of -49% when RLUs of the pack with 8 hr of light exposure were compared to the pack with no light exposure.

[0102] Table 11 provides the standard RLUs of Red Dye 40 spiked TSH3UL Ancillary Well Pack Reagents as compared to Reference at 0 and 8 hours of light exposure. FIG. 4 plots the RLU for each standard level. Standard RLUs of the pack with the Red Dye 40 ancillary well reagent at 8 hours are in line with standard RLUs of the pack with the Red Dye 40 Ancillary well reagent with 0 hours of light exposure. Conversely, standard RLUs of the TSH3UL reference pack reagent at 8 hours were much lower than standard RLUs of the TSH3UL reference pack reagent with 0 hours of light exposure. 41 ACTIVE 704156808v1Table 11

[0103] Table 12 provides the multidiluent pack (MDP) RLUS and doses of Red Dye 40 spiked TSH3UL ancillary well pack reagents at 0 hours and 8 hours of light exposure in Atellica IM Light Stability Testing. Table 12Table 13 provides the MDP RLUs and doses of TSH3UL reference at 0 and 8 hour light exposure in Atellica IM Light Stability Testing. The 2 PT stored reduction was created using the average standard RLUS and high calibrator dose from two quality control (QC) runs which were averaged together. Table 1342 ACTIVE 704156808v1

[0104] The formulation that used Red Dye 40 in solely the ancillary well reagent was comparable to the formulation which used Red Dye 40 in both the ancillary well reagent and LR wells. The acridinium labeled antibody in the lite reagent was not affected by light Additionally, for this formulation, all MDPs met specification for the 8 hour exposure condition when a 2 PT stored curve generated of off data from the pack reagent with the same formulation and no light exposure was used (Table 12). However, the reference stressed TSH3UL reagent had multiple out of range values for MDPs (Table 13).

[0105] For TSH3UL reagents, the addition of Red Dye 40 to at least the ancillary reagent containing fluorescein labeled antibody prevents change in RLUs due to LED light exposure. Results show that addition of dyes to fluorophores (and antibodies labeled with fluorophores) minimizes degradation. By minimizing degradation, more accurate measurements were made and erroneously increased or decreased measurements of the analyte due to degradation were prevented. NON-LIMITING ILLUSTRATIVE EMBODIMENTS

[0106] 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.

[0107] Illustrative Embodiment 1. A composition comprising a liquid medium, a fluorochrome labelling reagent, and a dye soluble in the liquid medium.

[0108] Illustrative Embodiment 2. The composition according to Illustrative Embodiment 1, wherein the fluorochrome labelling reagent is a fluorochrome moiety conjugated to an analyte or binding partner thereof.

[0109] Illustrative Embodiment 3. The composition according to Illustrative Embodiment 2, wherein said binding partner is a monoclonal antibody or fragment thereof. 43 ACTIVE 704156808v1

[0110] Illustrative Embodiment 4. The composition according to any one of Illustrative Embodiments 1-3, wherein said fluorochrome labelling reagent comprises a fluorescein (e.g., fluorescein isothiocyanate (FITC)) moiety as the fluorophore.

[0111] Illustrative Embodiment 5. The composition according to any one of Illustrative Embodiments 1-4, wherein said composition is aqueous.

[0112] Illustrative Embodiment 6. The composition according to any one of Illustrative Embodiments 1-5, wherein said dye soluble in the liquid medium comprises (or consists essentially of or is) 6-hydroxy-5-[(2-methoxy-5-methyl-4-sulfonatophenyl)diazenyl]naphthalene-2- sulfonate or salts thereof (e.g., disodium 6-hydroxy-5-[(2-methoxy-5-methyl-4- sulfonatophenyl)diazenyl]naphthalene-2-sulfonate, Allura Red AC) or 5-hydroxy-1-(4-sulfonatophenyl)-4-[(E)-(4-sulfonatophenyl)diazenyl]-1H-pyrazole- 3-carboxylate or salts thereof (e.g., trisodium 5-hydroxy-1-(4-sulfonatophenyl)-4- [(E)-(4-sulfonatophenyl)diazenyl]-1H-pyrazole-3-carboxylate, tartrazine); or combinations thereof.

[0113] Illustrative Embodiment 7. The composition according to any one of Illustrative Embodiments 1-6, wherein said composition comprises more than one (e.g., two, three, four, five, from two to 10, from two to five) fluorochrome labelling reagents.

[0114] Illustrative Embodiment 8. The composition according to any one of Illustrative Embodiments 1-7, wherein said composition comprise one or more (e.g., one, two, three, four) fluorochrome labelling reagents each labeling reagent comprising an antibody of different epitopes of said analyte or mutants thereof, wherein each fluorochrome labelling reagent is independently conjugated to fluorescein (e.g., fluorescein isothiocyanate, carboxyfluorescein succinimidyl ester fluorescein maleimide).

[0115] Illustrative Embodiment 9. The composition according to any one of Illustrative Embodiments 1-8, wherein said composition further comprises a chemiluminescent labelling reagent (e.g., a compound having a chemiluminescent moiety such as an acridinium ester or sulfonamide conjugated to an analyte or binding partner thereof).

[0116] Illustrative Embodiment 10. The composition according to any one of Illustrative Embodiments 1-9, wherein said composition is characterized as having within (or from 0.1% to) 20% (e.g., within 10%, within 5%) of the fluorescent light output after 8 hours 44 ACTIVE 704156808v1of exposure to LED light (e.g., white LED from 23,000-26,000 lux, 24,500 lux) at 2-8°C as compared said composition before said exposure.

[0117] Illustrative Embodiment 11. The composition according to any one of Illustrative Embodiments 1-9, wherein the weight ratio of said fluorochrome labelling reagent (or each of said fluorochrome labelling reagents) to said dye is from 20:1 to 1:20 (e.g., from 1:1 to 1:20, from 1:1 to 1:15, from 1:5 to 1:15).

[0118] Illustrative Embodiment 12. The composition according to any one of Illustrative Embodiments 1-11, wherein the weight ratio of all fluorochrome labelling reagents to said dye is from 10:1 to 1:10 (e.g., from 5:1 to 1:5, from 3:1 to 1:3, from 2:1 to 1:2).

[0119] Illustrative Embodiment 13. The composition according to any one of Illustrative Embodiments 1-12, wherein said composition comprises less than (or from 0.01 g / L to or from 0.1 g / L to or from 0.2 g / L to) 5 g / L said dye (e.g., less than 4 g / L dye, less than 3 g / L dye, less than 2.5 g / L dye, from 0.05 g / L to 2.5 g / L).

[0120] Illustrative Embodiment 14. The composition according to any one of Illustrative Embodiments 1-13, wherein said composition comprises more than 0.1 g / L or more than 0.2 g / L of said dye (e.g., from 0.2 g / L to 5 g / L, from 0.2 g / L to 4 g / L, from 0.2 g / L to 3 g / L from 0.2 g / L to 2.5 g / L, from 0.5 g / L to 5 g / L, from 0.5 g / L to 4 g / L, from 0.5 g / L to 3 g / L from 0.5 g / L to 2.5 g / L).

[0121] Illustrative Embodiment 15. The composition according to any one of Illustrative Embodiments 1-14, wherein said composition comprises less than (or from 0.01 g / L to or from 0.1 g / L to or from 0.2 g / L to) 5 mg / mL (e.g., less than 2 mg / mL, less than 1 mg / mL from 0.1 mg / L to 1 mg / L) of said fluorochrome labelling reagent (or each of said fluorochrome labelling reagents).

[0122] Illustrative Embodiment 16. The composition according to any one of Illustrative Embodiments 1-15, wherein the total concentration of said fluorochrome labelling reagents is less than (or from 0.01 g / L to or from 0.1 g / L to or from 0.2 g / L to) 5 mg / mL (e.g., from 1 mg / mL to 4 mg / mL, from 1 mg / mL to 2 mg / mL); wherein said dye absorbs light in the 480-560 nm range (e.g., said dye has a wavelength of maximum absorbance of from 450-550 nm or from 480 nm-560 nm).

[0123] Illustrative Embodiment 17. The composition according to any one of Illustrative Embodiments 1-16, wherein the absorbance spectrum of said dye and said 45 ACTIVE 704156808v1fluorochrome labelling reagent overlap in the visible region (e.g., said fluorochrome labelling reagent and said dye absorbs light in the 480-560 nm range (e.g., said fluorochrome labelling reagent and said dye independently have a wavelength of maximum absorbance of from 450- 550 nm or from 480 nm-560 nm).

[0124] Illustrative Embodiment 18. A kit comprising a container and a composition comprising a liquid medium, a fluorochrome labelling reagent (e.g., a compound comprising a fluorescent moiety which may label an analyte to be analyzed), and a dye soluble in the liquid medium, wherein the container allows light to be transmitted to the composition contained therein.

[0125] Illustrative Embodiment 19. The kit according to Illustrative Embodiment 18, wherein the fluorochrome labelling reagent is a fluorochrome moiety conjugated to an analyte or binding partner thereof.

[0126] Illustrative Embodiment 20. The kit according to Illustrative Embodiment 19, wherein said binding partner is a monoclonal antibody or fragment thereof.

[0127] Illustrative Embodiment 21. The kit according to any one of Illustrative Embodiments 18-20, wherein said fluorochrome labelling reagent comprises a fluorescein (e.g., fluorescein isothiocyanate (FITC)) moiety as the fluorophore.

[0128] Illustrative Embodiment 22. The kit according to any one of Illustrative Embodiments 18-21, wherein said composition is aqueous.

[0129] Illustrative Embodiment 23. The kit according to any one of Illustrative Embodiments 18-22, wherein said dye soluble in the liquid medium comprises (or consists essentially of or is) 6-hydroxy-5-[(2-methoxy-5-methyl-4-sulfonatophenyl)diazenyl]naphthalene-2- sulfonate or salts thereof (e.g., disodium 6-hydroxy-5-[(2-methoxy-5-methyl-4- sulfonatophenyl)diazenyl]naphthalene-2-sulfonate, Allura Red AC) or 5-hydroxy-1-(4-sulfonatophenyl)-4-[(E)-(4-sulfonatophenyl)diazenyl]-1H-pyrazole- 3-carboxylate or salts thereof (e.g., trisodium 5-hydroxy-1-(4-sulfonatophenyl)-4- [(E)-(4-sulfonatophenyl)diazenyl]-1H-pyrazole-3-carboxylate, tartrazine); or combinations thereof.

[0130] Illustrative Embodiment 24. The kit according to any one of Illustrative Embodiments 18-23, wherein said composition comprises more than one (e.g., two, three, four, five, from two to 10, from two to five) fluorochrome labelling reagents. 46 ACTIVE 704156808v1

[0131] Illustrative Embodiment 25. The kit according to any one of Illustrative Embodiments 18-24, wherein said composition comprises one or more (e.g., one, two, three, four) fluorochrome labelling reagents each labeling reagent comprising an antibody of different epitopes of said analyte or mutants thereof, wherein each fluorochrome labelling reagent is independently conjugated to fluorescein (e.g., fluorescein isothiocyanate, carboxyfluorescein succinimidyl ester fluorescein maleimide).

[0132] Illustrative Embodiment 26. The kit according to any one of Illustrative Embodiments 18-25, wherein said composition further comprises a chemiluminescent labelling reagent (e.g., a compound having a chemiluminescent moiety such as an acridinium ester or sulfonamide conjugated to an analyte or binding partner thereof).

[0133] Illustrative Embodiment 27. The kit according to any one of Illustrative Embodiments 18-26, wherein said composition is characterized as having within 20% of the fluorescent light output after 8 hours of exposure to LED light (e.g., white LED from 23,000- 26,000 lux, 24,500 lux) at 2-8°C as compared said composition before said exposure.

[0134] Illustrative Embodiment 28. The kit according to any one of Illustrative Embodiments 18-27, wherein the weight ratio of said fluorochrome labelling reagent (or each of said fluorochrome labelling reagents) to said dye is from 20:1 to 1:20 (e.g., from 1:1 to 1:20, from 1:1 to 1:15, from 1:5 to 1:15).

[0135] Illustrative Embodiment 29. The kit according to any one of Illustrative Embodiments 18-28, wherein the weight ratio of all fluorochrome labelling reagents to said dye is from 10:1 to 1:10 (e.g., from 5:1 to 1:5, from 3:1 to 1:3, from 2:1 to 1:2).

[0136] Illustrative Embodiment 30. The kit according to any one of Illustrative Embodiments 18-29, wherein said composition comprises less than (or from 0.01 g / L to or from 0.1 g / L to or from 0.2 g / L to) 5 g / L said dye (e.g., less than 4 g / L dye, less than 3 g / L dye, less than 2.5 g / L dye, from 0.05 g / L to 2.5 g / L).

[0137] Illustrative Embodiment 31. The kit according to any one of Illustrative Embodiments 18-30, wherein said composition comprises more than 0.1 g / L or more than 0.2 g / L of said dye (e.g., from 0.2 g / L to 5 g / L, from 0.2 g / L to 4 g / L, from 0.2 g / L to 3 g / L from 0.2 g / L to 2.5 g / L, from 0.5 g / L to 5 g / L, from 0.5 g / L to 4 g / L, from 0.5 g / L to 3 g / L from 0.5 g / L to 2.5 g / L).

[0138] Illustrative Embodiment 32. The kit according to any one of Illustrative Embodiments 18-31, wherein said composition comprises less than (or from 0.01 g / L to or from 0.1 g / L to or from 0.2 g / L to) 5 mg / mL (e.g., less than 2 mg / mL, less than 1 mg / mL 47 ACTIVE 704156808v1from 0.1 mg / L to 1 mg / L) of said fluorochrome labelling reagent (or each of said fluorochrome labelling reagents).

[0139] Illustrative Embodiment 33. The kit according to any one of Illustrative Embodiments 18-32, wherein the total concentration of said fluorochrome labelling reagents is less than (or from 0.01 g / L to or from 0.1 g / L to or from 0.2 g / L to) 5 mg / mL (e.g., from 1 mg / mL to 4 mg / mL, from 1 mg / mL to 2 mg / mL); wherein said dye absorbs light in the 480-560 nm range (e.g., said dye has a wavelength of maximum absorbance of from 450-550 nm or from 480 nm-560 nm).

[0140] Illustrative Embodiment 34. The kit according to any one of Illustrative Embodiments 18-33, wherein the absorbance spectrum of said dye and said fluorochrome labelling reagent overlap in the visible region (e.g., said fluorochrome labelling reagent and said dye absorbs light in the 480-560 nm range (e.g., said fluorochrome labelling reagent and said dye independently have a wavelength of maximum absorbance of from 450-550 nm or from 480 nm-560 nm).

[0141] Illustrative Embodiment 35. The kit according to any one of Illustrative Embodiments 18-34, wherein the fluorochrome labelling reagent is a fluorophore (e.g., fluorescein such as FITC) conjugated to a mouse monoclonal antibody of thyroid stimulating hormone and / or a fluorophore (e.g., fluorescein such as FITC) conjugated to a mouse monoclonal antibody of hepatits B surface antigen.

[0142] Illustrative Embodiment 36. The kit according to any one of Illustrative Embodiments 18-35, wherein the composition comprises more than one fluorochrome labelling reagents, wherein each fluorochrome labelling reagent comprises a fluorophore (e.g., fluorescein such as FITC) conjugated to an antibody of an antigen (e.g., hepatitis B surface antigen) and each fluorochrome labelling reagent binds to a different epitope of the antigen.

[0143] Illustrative Embodiment 37. The kit according to any one of Illustrative Embodiments 18-36, wherein the container is cardboard (e.g., cardboard box).

[0144] Illustrative Embodiment 38. The kit according to any one of Illustrative Embodiments 18-37, wherein the composition is contained in a transparent or translucent vial (e.g., plastic vial) in the container. 48 ACTIVE 704156808v1

[0145] Illustrative Embodiment 39. The kit according to any one of Illustrative Embodiments 18-38, wherein the kit further comprises a lite reagent and / or a solid phase reagent; wherein the lite reagent comprises a chemiluminescent acridinium conjugated to an analyte or binding partner thereof, and the solid phase reagent comprises a solid phase particle covalently linked to an analyte or binding partner thereof.

[0146] Illustrative Embodiment 40. The kit according to Illustrative Embodiment 39, wherein the solid phase particle is covalently linked to an anti-fluorescein antibody and the fluorochrome labeling reagent is a fluorochrome moiety conjugated to an analyte or binding partner for the analyte.

[0147] Illustrative Embodiment 41. A method for the detection or quantification of an analyte in a sample comprising: (a) providing a composition comprising a liquid medium, a fluorochrome labelling reagent (e.g., a compound comprising a fluorescent moiety which may label an analyte to be analyzed), and a dye soluble in the liquid medium and a chemiluminescent label conjugated to a binding partner of the analyte; (b) providing a solid support having immobilized thereon a molecule capable of forming a binding complex with said fluorochrome labelling agent, wherein said fluorochrome labelling agent is conjugated to a binding partner of the analyte and the binding complex is conjugated to the chemiluminescent label through said analyte and said fluorochrome labelling agent; (c) mixing said composition, said chemiluminescent label, said solid support, and said sample to form said binding complex; (d) separating said binding complex from said mixture; (e) triggering chemiluminescence from said chemiluminescent label (e.g., from the separated binding complex); (f) measuring the amount of chemiluminescent light emission with a luminometer; and 49 ACTIVE 704156808v1(g) detecting the presence or calculating the concentration of said analyte by comparing the amount of light emitted with a standard dose response curve which relates the amount of light emitted to a known concentration of the analyte; and optionally, not inducing and / or measuring fluorescence of the fluorochrome labelling reagent (e.g., during the triggering and / or measuring steps).

[0148] Illustrative Embodiment 42. The method according to Illustrative Embodiment 41, wherein the chemiluminescent label is provided in the composition.

[0149] Illustrative Embodiment 43. The method according to Illustrative Embodiment 41 or 42, wherein the composition is provided in a kit comprising a container which exposes the composition to light during storage (if light is incident upon the container).

[0150] Illustrative Embodiment 44. A method for increasing the light stability of an immunoassay reagent composition (e.g., a composition comprising a fluorescein labelling agent and a medium) comprising adding a dye to the immunoassay reagent composition.

[0151] Illustrative Embodiment 45. A method for forming a kit for an immunoassay reagent composition comprising dissolving a dye in a medium, adding a fluorochrome labelling reagent to the mixed composition to form an immunoassay reagent composition, packaging the immunoassay reagent composition in a container to form a kit which exposes the composition to light during storage (if light is incident upon the container).

[0152] Illustrative Embodiment 46. The method according to any one of Illustrative Embodiments 41-45, wherein the fluorochrome labelling reagent is a fluorochrome moiety conjugated to an analyte or binding partner thereof.

[0153] Illustrative Embodiment 47. The method according to Illustrative Embodiment 46, wherein said binding partner is a monoclonal antibody or fragment thereof.

[0154] Illustrative Embodiment 48. The method according to any one of Illustrative Embodiments 41-47, wherein said fluorochrome labelling reagent comprises a fluorescein (e.g., fluorescein isothiocyanate (FITC)) moiety as the fluorophore.

[0155] Illustrative Embodiment 49. The method according to any one of Illustrative Embodiments 41-48, wherein said composition is aqueous. 50 ACTIVE 704156808v1

[0156] Illustrative Embodiment 50. The method according to any one of Illustrative Embodiments 41-49, wherein said dye soluble in the liquid medium comprises (or consists essentially of or is) 6-hydroxy-5-[(2-methoxy-5-methyl-4-sulfonatophenyl)diazenyl]naphthalene-2- sulfonate or salts thereof (e.g., disodium 6-hydroxy-5-[(2-methoxy-5-methyl-4- sulfonatophenyl)diazenyl]naphthalene-2-sulfonate, Allura Red AC) or 5-hydroxy-1-(4-sulfonatophenyl)-4-[(E)-(4-sulfonatophenyl)diazenyl]-1H-pyrazole- 3-carboxylate or salts thereof (e.g., trisodium 5-hydroxy-1-(4-sulfonatophenyl)-4- [(E)-(4-sulfonatophenyl)diazenyl]-1H-pyrazole-3-carboxylate, tartrazine); or combinations thereof.

[0157] Illustrative Embodiment 51. The method according to any one of Illustrative Embodiments 41-50, wherein said composition comprises more than one (e.g., two, three, four, five, from two to 10, from two to five) fluorochrome labelling reagents.

[0158] Illustrative Embodiment 52. The method according to any one of Illustrative Embodiments 41-51, wherein said composition comprises one or more (e.g., one, two, three, four) fluorochrome labelling reagents each labeling reagent comprising an antibody of different epitopes of said analyte or mutants thereof, wherein each fluorochrome labelling reagent is independently conjugated to fluorescein (e.g., fluorescein isothiocyanate, carboxyfluorescein succinimidyl ester fluorescein maleimide).

[0159] Illustrative Embodiment 53. The method according to any one of Illustrative Embodiments 41-52, wherein said composition further comprises a chemiluminescent labelling reagent (e.g., a compound having a chemiluminescent moiety such as an acridinium ester or sulfonamide conjugated to an analyte or binding partner thereof).

[0160] Illustrative Embodiment 54. The method according to any one of Illustrative Embodiments 41-53, wherein said composition is characterized as having within 20% of the fluorescent light output after 8 hours of exposure to LED light (e.g., white LED from 23,000- 26,000 lux, 24,500 lux) at 2-8°C as compared said composition before said exposure.

[0161] Illustrative Embodiment 55. The method according to any one of Illustrative Embodiments 41-54, wherein the weight ratio of said fluorochrome labelling reagent (or each of said fluorochrome labelling reagents) to said dye is from 20:1 to 1:20 (e.g., from 1:1 to 1:20, from 1:1 to 1:15, from 1:5 to 1:15). 51 ACTIVE 704156808v1

[0162] Illustrative Embodiment 56. The method according to any one of Illustrative Embodiments 41-55, wherein the weight ratio of all fluorochrome labelling reagents to said dye is from 10:1 to 1:10 (e.g., from 5:1 to 1:5, from 3:1 to 1:3, from 2:1 to 1:2).

[0163] Illustrative Embodiment 57. The method according to any one of Illustrative Embodiments 41-56, wherein said composition comprises less than (or from 0.01 g / L to or from 0.1 g / L to or from 0.2 g / L to) 5 g / L said dye (e.g., less than 4 g / L dye, less than 3 g / L dye, less than 2.5 g / L dye, from 0.05 g / L to 2.5 g / L).

[0164] Illustrative Embodiment 58. The method according to any one of Illustrative Embodiments 41-57, wherein said composition comprises more than 0.1 g / L or more than 0.2 g / L of said dye (e.g., from 0.2 g / L to 5 g / L, from 0.2 g / L to 4 g / L, from 0.2 g / L to 3 g / L from 0.2 g / L to 2.5 g / L, from 0.5 g / L to 5 g / L, from 0.5 g / L to 4 g / L, from 0.5 g / L to 3 g / L from 0.5 g / L to 2.5 g / L).

[0165] Illustrative Embodiment 59. The method according to any one of Illustrative Embodiments 41-59, wherein said composition comprises less than (or from 0.01 g / L to or from 0.1 g / L to or from 0.2 g / L to) 5 mg / mL (e.g., less than 2 mg / mL, less than 1 mg / mL from 0.1 mg / L to 1 mg / L) of said fluorochrome labelling reagent (or each of said fluorochrome labelling reagents).

[0166] Illustrative Embodiment 60. The method according to any one of Illustrative Embodiments 41-59, wherein the total concentration of said fluorochrome labelling reagents is less than (or from 0.01 g / L to or from 0.1 g / L to or from 0.2 g / L to) 5 mg / mL (e.g., from 1 mg / mL to 4 mg / mL, from 1 mg / mL to 2 mg / mL); wherein said dye absorbs light in the 480-560 nm range (e.g., said dye has a wavelength of maximum absorbance of from 450-550 nm or from 480 nm-560 nm).

[0167] Illustrative Embodiment 61. The method according to any one of Illustrative Embodiments 41-60, wherein the absorbance spectrum of said dye and said fluorochrome labelling reagent overlap in the visible region (e.g., said fluorochrome labelling reagent and said dye absorbs light in the 480-560 nm range (e.g., said fluorochrome labelling reagent and said dye independently have a wavelength of maximum absorbance of from 450-550 nm or from 480 nm-560 nm).

[0168] Illustrative Embodiment 62. The method according to any one of Illustrative Embodiments 41-61, wherein the fluorochrome labelling reagent is a fluorophore (e.g., fluorescein such as FITC) conjugated to a mouse monoclonal antibody of thyroid stimulating 52 ACTIVE 704156808v1hormone and / or a fluorophore (e.g., fluorescein such as FITC) conjugated to a mouse monoclonal antibody of hepatitis B surface antigen.

[0169] Illustrative Embodiment 63. The method according to any one of Illustrative Embodiments 41-62, wherein the composition comprises more than one fluorochrome labelling reagents, wherein each fluorochrome labelling reagent comprises a fluorophore (e.g., fluorescein such as FITC) conjugated to an antibody of an antigen (e.g., hepatitis B surface antigen) and each fluorochrome labelling reagent binds to a different epitope of the antigen.

[0170] 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. 53 ACTIVE 704156808v1

Claims

CLAIMS1. A kit comprising a container and a composition comprising a liquid medium, a fluorochrome labelling reagent (e.g., a compound comprising a fluorescent moiety which may label an analyte to be analyzed), and a dye soluble in the liquid medium, wherein the container allows light to be transmitted to the composition contained therein.

2. The kit according to claim 1, wherein the fluorochrome labelling reagent is a fluorochrome moiety conjugated to an analyte or binding partner thereof.

3. The kit according to claim 2, wherein said binding partner is a monoclonal antibody or fragment thereof.

4. The kit according to any one of claims 1-3, wherein said dye soluble in the liquid medium comprises (or consists essentially of or is)6-hydroxy-5-[(2-methoxy-5-methyl-4-sulfonatophenyl)diazenyl]naphthalene-2- sulfonate or salts thereof (e.g., disodium 6-hydroxy-5-[(2-methoxy-5-methyl-4- sulfonatophenyl)diazenyl]naphthalene-2-sulfonate, Allura Red AC) or5-hydroxy-l-(4-sulfonatophenyl)-4-[(E)-(4-sulfonatophenyl)diazenyl]-lH-pyrazole-3- carboxylate or salts thereof (e.g., trisodium 5-hydroxy-l-(4-sulfonatophenyl)-4-[(E)- (4-sulfonatophenyl)diazenyl]-lH-pyrazole-3-carboxylate, tartrazine); or combinations thereof.

5. The kit according to any one of claims 1-4, wherein said composition comprises more than one (e.g., two, three, four, five, from two to 10, from two to five) fluorochrome labelling reagents.

6. The kit according to any one of claims 1-5, wherein said composition comprises one or more (e.g., one, two, three, four) fluorochrome labelling reagents each labeling reagent comprising an antibody of different epitopes of said analyte or mutants thereof, wherein each fluorochrome labelling reagent is independently conjugated to fluorescein (e.g., fluorescein isothiocyanate, carboxyfluorescein succinimidyl ester fluorescein maleimide).

7. The kit according to any one of claims 1-6, wherein said composition further comprises a chemiluminescent labelling reagent (e.g., a compound having a chemiluminescent moiety such as an acridinium ester or sulfonamide conjugated to an analyte or binding partner thereof).

8. The kit according to any one of claims 1-7, wherein said composition is characterized as having within 20% of the fluorescent light output after 8 hours of exposure to LED light (e.g., white LED from 23,000-26,000 lux, 24,500 lux) at 2-8°C as compared said composition before said exposure.

9. The kit according to any one of claims 1-8, wherein the weight ratio of said fluorochrome labelling reagent (or each of said fluorochrome labelling reagents) to said dye is from 20: 1 to 1 :20 (e.g., from 1 : 1 to 1 :20, from 1 : 1 to 1 : 15, from 1 :5 to 1 :15).

10. The kit according to any one of claims 1-9, wherein said composition comprises less than (or from 0.01 g / L to or from 0.1 g / L to or from 0.2 g / L to) 5 g / L said dye (e.g., less than 4 g / L dye, less than 3 g / L dye, less than 2.5 g / L dye, from 0.05 g / L to 2.5 g / L).

11. The kit according to any one of claims 1-10, wherein the fluorochrome labelling reagent is a fluorophore (e.g., fluorescein such as FITC) conjugated to a mouse monoclonal antibody of thyroid stimulating hormone and / or a fluorophore (e.g., fluorescein such as FITC) conjugated to a mouse monoclonal antibody of hepatits B surface antigen.

12. The kit according to any one of claims 1-11, wherein the composition comprises more than one fluorochrome labelling reagents, wherein each fluorochrome labelling reagent comprises a fluorophore (e.g., fluorescein such as FITC) conjugated to an antibody of an antigen (e.g., hepatitis B surface antigen) and each fluorochrome labelling reagent binds to a different epitope of the antigen.

13. The kit according to any one of claims 1-12, wherein the container is cardboard (e.g., cardboard box).

14. The kit according to any one of claims 1-13, wherein the composition is contained in a transparent or translucent vial (e.g., plastic vial) in the container.

15. The kit according to any one of claims 1-14, wherein the kit further comprises a lite reagent and / or a solid phase reagent; wherein the lite reagent comprises a chemiluminescent acridinium conjugated to an analyte or binding partner thereof, and the solid phase reagent comprises a solid phase particle covalently linked to an analyte or binding partner thereof.

16. A method for the detection or quantification of an analyte in a sample comprising:(a) providing a composition comprising a liquid medium, a fluorochrome labelling reagent (e.g., a compound comprising a fluorescent moiety which may label an analyte to be analyzed), and a dye soluble in the liquid medium and a chemiluminescent label conjugated to a binding partner of the analyte;(b) providing a solid support having immobilized thereon a molecule capable of forming a binding complex with said fluorochrome labelling agent, wherein said fluorochrome labelling agent is conjugated to a binding partner of the analyte and the binding complex is conjugated to the chemiluminescent label through said analyte and said fluorochrome labelling agent;(c) mixing said composition, said chemiluminescent label, said solid support, and said sample to form said binding complex;(d) separating said binding complex from said mixture;(e) triggering chemiluminescence from said chemiluminescent label (e.g., from the separated binding complex);(f) measuring the amount of chemiluminescent light emission with a luminometer; and(g) detecting the presence or calculating the concentration of said analyte by comparing the amount of light emitted with a standard dose response curve which relates the amount of light emitted to a known concentration of the analyte.

17. The method according to claim 16, wherein the method does not comprise inducing fluorescence of the fluorochrome labelling reagent.

18. A method for increasing the light stability of an immunoassay reagent composition (e.g., a composition comprising a fluorescein labelling agent and a medium) comprising adding a dye to the immunoassay reagent composition.

19. A method for forming a kit for an immunoassay reagent composition comprising dissolving a dye in a medium, adding a fluorochrome labelling reagent to the mixed composition to form an immunoassay reagent composition,packaging the immunoassay reagent composition in a container to form a kit which exposes the composition to light during storage (if light is incident upon the container).

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