阅读说明：本技术 测定 (Measurement of ) 是由 格雷戈里·W·西顿 于 2019-07-10 设计创作，主要内容包括：本发明公开了一种测定样品中的分析物的方法以及用于执行所述测定的试剂盒。(The present invention discloses a method of determining an analyte in a sample and a kit for performing the determination.)

1. A method of determining an analyte in a sample, the method comprising

Forming a reaction mixture, and

mixing a trigger solution with the reaction mixture; wherein

The analyte to be determined may be present in the sample,

the reaction mixture is an aqueous solution comprising

A chemiluminescent-labeled specific binding partner comprising a chemiluminescent label irreversibly bound to the first specific binding partner, the chemiluminescent-labeled specific binding partner capable of binding to the analyte to form an analyte-bound chemiluminescent-labeled specific binding complex,

an activator-labeled specific binding partner comprising an activator label irreversibly bound to a second specific binding partner, and

a selective signal inhibitor; and is

The trigger solution is capable of generating a background signal from unbound chemiluminescent substrate, and

in the presence of an analyte, the trigger solution is capable of generating a detectable analyte signal that is correlated with the amount of the analyte in the sample; it is characterized in that

The reaction mixture also contains unbound chemiluminescent substrate.

2. The method of claim 1, wherein the trigger solution further comprises an enhancer.

3. According to any of the preceding claimsThe method of one item, wherein the enhancer is selected from the group consisting of phenolic compounds, aromatic amines, benzolsOxazoles, hydroxybenzothiazoles, arylboronic acids, and mixtures of any of the foregoing.

4. The method of any one of the preceding claims, wherein the trigger solution comprises an oxidizing agent or a reducing agent.

5. The method of any preceding claim, wherein the trigger solution comprises a peroxide.

6. The method of any one of the preceding claims, wherein the selective signal inhibitor is selected from ascorbic acid, erythorbic acid, 6-hydroxy-2, 5,7, 8-tetramethylchroman-2-carboxylic acid, ascorbic acid 6-palmitate, 5, 6-isopropylidene-ascorbic acid, butylated hydroxytoluene, glutathione, uric acid, one or more tocopherols, and catechins.

7. The method of any one of the preceding claims, wherein the activator label is a peroxidase.

8. The method of any one of the preceding claims, wherein the chemiluminescent-labeled specific binding partner comprises a chemiluminescent label directly or indirectly linked to a specific binding partner, wherein the chemiluminescent label has the formula

Wherein

The wavy line represents the site of attachment to the specific binding partner or to a linker linking the compound to the specific binding partner;

R¹、R²and R³Independently selected from the group consisting of substituted alkyl, unsubstituted alkyl, substituted alkenyl, unsubstituted alkenyl, substituted alkynyl, unsubstituted alkynyl, substituted aralkyl and unsubstituted aralkyl groups of 1 to 20 carbon atoms, wherein when R is¹Or R²When substituted, it is substituted with 1 to 3 substituents, each substituent independently selected from carbonyl, carboxyl, tri (C1-C8) alkylsilyl, -SO₃ ^-、-OSO₃ ^2-Sugar radical, -PO₃ ^-、-OPO₃ ^2-Halogen, hydroxy, mercapto, amino, C (O) NHNH₂Quaternary ammonium and quaternary phosphonium salts

9. The method of any one of the preceding claims, wherein the chemiluminescent-labeled specific binding partner and the activator-labeled specific binding partner each bind to the analyte in the sample.

10. The method of any one of the preceding claims, wherein the chemiluminescent-labeled specific binding partner comprises a chemiluminescent-labeled compound linked to an analog of the analyte, and further wherein the analyte and the chemiluminescent-labeled specific binding partner compete for binding to the activator-labeled specific binding partner.

11. The method according to any one of the preceding claims, wherein the unbound chemiluminescent substrate is selected from luminol, isoluminol, lofenadine, acridine esters, such as lucigenin, and 9, 10-dihydroacridine, phthalylhydrazides, such as 2, 3-dihydro-1, 4-diketophthalazine, fluorescein, and 1, 2-dioxetane-containing compounds, such as diketones, 3- (2' -spiroadamantane) -4-methoxy-4- (3 "-phosphoryloxy) phenyl-1, 2-dioxetane (AMPPD), and 3- (2' -spiroadamantane) -4-methoxy-4- (3" -beta-D ' -galactopyranosyloxy) phenyl-1, 2-dioxetane (AMPGD), disodium 3- (4-methoxyspiro {1, 2-dioxetane-3, 2'- (5' -chloro) tricyclo [3.3.1.13,7] decane } -4-yl) phenyl phosphate and 1, 2-dioxetanedione, adamantyl-1, 2-dioxetane, in particular luminol or isoluminol, and more in particular luminol.

12. The method of any one of the preceding claims, wherein the method further comprises detecting an analyte signal and a background signal.

13. The method of claim 12, wherein detecting an analyte signal comprises detecting at least one standard analyte signal from at least one sample that is a standard sample having a known analyte concentration, and further comprising detecting an assay analyte signal from at least one test sample having an unknown analyte concentration.

14. The method of claim 12 or 13, wherein detecting a background signal comprises detecting at least one standard background signal from at least one sample that is a standard sample having a known analyte concentration, and further comprising detecting a test background signal from at least one test sample having an unknown analyte concentration.

15. A kit for the determination of an analyte, said kit comprising

A chemiluminescent-labeled specific binding partner comprising a chemiluminescent label irreversibly bound to the first specific binding partner, the chemiluminescent-labeled specific binding partner capable of binding to the analyte to form an analyte-bound chemiluminescent-labeled specific binding complex,

an activator-labeled specific binding partner comprising an activator label irreversibly bound to a second specific binding partner, and

a selective signal inhibitor; and wherein

The trigger solution is capable of generating a background signal from unbound chemiluminescent substrate; wherein

In the presence of an analyte, the trigger solution is capable of generating a detectable analyte signal that correlates to the amount of the analyte in the sample;

characterized in that the reaction mixture further comprises an unbound chemiluminescent substrate.

16. The kit of claim 14, wherein the trigger solution further comprises an enhancer selected from the group consisting of phenolic compounds, aromatic amines, benzolsOxazoles, hydroxybenzothiazoles, arylboronic acids, and mixtures of any of the foregoing.

17. The kit according to any one of claims 14 to 15, wherein the unbound chemiluminescent substrate is selected from luminol, isoluminol, lofenadine, acridinium esters, such as lucigenin, and 9, 10-dihydroacridine, phthalylhydrazide, such as 2, 3-dihydro-1, 4-diketophthalazine, fluorescein, and 1, 2-dioxetane-containing compounds, such as diketones, 3- (2' -spiroadamantane) -4-methoxy-4- (3 "-phosphoryloxy) phenyl-1, 2-dioxetane (AMPPD), and 3- (2' -spiroadamantane) -4-methoxy-4- (3" -beta-D ' -galactopyranosyloxy) phenyl-1, 2-dioxetane (AMPGD), disodium 3- (4-methoxyspiro {1, 2-dioxetane-3, 2'- (5' -chloro) tricyclo [3.3.1.13,7] decane } -4-yl) phenyl phosphate and 1, 2-dioxetanedione, adamantyl-1, 2-dioxetane, in particular luminol or isoluminol, and more in particular luminol.

18. A solution phase assay method for an analyte in a sample, the solution phase assay method comprising:

c) forming an aqueous reaction mixture comprising the following components by addition to an aqueous solution in any order or simultaneously:

the samples were taken from the test piece and,

a chemiluminescent-tagged specific binding partner comprising a chemiluminescent-tagged compound directly or indirectly linked to a first specific binding partner, wherein the chemiluminescent-tagged compound is a compound of the formula

Wherein

The wavy line represents the site of attachment to the specific binding partner or to a linker linking the compound to the specific binding partner;

R¹、R²and R³Independently selected from the group consisting of substituted alkyl, unsubstituted alkyl, substituted alkenyl, unsubstituted alkenyl, substituted alkynyl, unsubstituted alkynyl, substituted aralkyl and unsubstituted aralkyl groups of 1 to 20 carbon atoms, wherein when R is¹Or R²When substituted, it is substituted with 1 to 3 substituents, each substituent independently selected from carbonyl, carboxyl, tri (C1-C8) alkylsilyl, -SO₃ ^-、-OSO₃ ^2-Sugar radical, -PO₃ ^-、-OPO₃ ^2-Halogen, hydroxy, mercapto, amino, C (O) NHNH₂Quaternary ammonium and quaternary phosphonium salts

An activator-labeled specific binding partner comprising an activator-labeled compound having peroxidase activity directly or indirectly linked to a second specific binding partner, and

selective signal inhibitors selected fromFrom L-ascorbic acid, 6-hydroxy-2, 5,7, 8-tetramethylchroman-2-carboxylic acid, 2-aminophenol, 3-amino-L-tyrosine, 4-chlorocatechol, thiophenOxazine, 2-bromobenzo-1, 4-diol, 5, 6-isopropylidene ascorbic acid and ascorbic acid 6-palmitate,

wherein all of said components are soluble in said aqueous solution and none of said components are immobilized to a solid support, and

wherein the chemiluminescent-labeled specific binding partner and the activator-labeled specific binding partner bind to the analyte present in the sample to form a bound complex in the aqueous solution; and

d) adding a trigger solution comprising a peroxide compound to the aqueous reaction mixture, wherein the trigger solution releases a detectable chemiluminescent signal in the presence of the selective signal inhibitor that correlates to the amount of the analyte-bound chemiluminescent-labeled specific binding partner and the analyte-bound activator-labeled specific binding partner in the aqueous reaction mixture, and wherein the selective signal inhibitor causes the ratio of the signals produced by the reaction between the chemiluminescent labeling compound and the activator labeling compound to exceed the ratio of the signals from the reaction between the chemiluminescent labeling compound and the activator labeling compound when not in such combination;

it is characterized in that

The reaction mixture further comprises an unbound chemiluminescent substrate that produces a detectable background signal that is not related to the concentration of the analyte in the sample.

19. A kit for detecting an analyte in a sample, the kit comprising:

a first specific binding partner for the analyte;

a chemiluminescent compound conjugated to the first specific binding partner;

a second specific binding partner for the analyte;

an activator compound conjugated to the second specific binding partner;

a selective signal inhibitor; and

the trigger solution is applied to the surface of the substrate,

wherein all of the aforementioned components of the kit are soluble in an aqueous solution,

wherein the chemiluminescent compound is a 9, 10-dihydroacrylketene dithioacetal compound having the formula

Wherein R is₁And R₂Each of which is selected from H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, aralkyl or substituted aralkyl when R is₁And R₂When substituted, it is most commonly substituted with 1 to 3 groups selected from carbonyl, carboxyl, tri (alkyl) silyl, glycosyl, -SO₃ ^-、-OSO₃ ^-、-PO₃ ^-、-OPO₃ ^-Halogen, hydroxy, mercapto, amino, quaternary ammonium and quaternary phosphonium salts

R₃Selected from H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, aralkyl or substituted aralkyl,

R₄to R₁₁Each of which is independently H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, aralkyl, substituted aralkyl, alkoxy, aryloxy, haloAn element, amino, substituted amine, carboxyl, alkoxycarbonyl, carboxamide, cyano or sulfonic group, wherein pairs of adjacent R₄To R₁₁The moieties may be covalently bonded to form a five to seven membered carbocyclic or heterocyclic ring,

wherein the selective signal inhibitor is selected from glutathione, ascorbic acid, especially L-ascorbic acid, ascorbate, especially L-ascorbate, uric acid, L-ascorbic acid-6-palmitate, tocopherol, 5, 6-isopropylidene-L-ascorbic acid, erythorbic acid, including D-erythorbic acid, L-erythorbic acid, or both, sodium sulfite, diethylhydroxylamine, BHT,

and combinations of the foregoing.

20. The kit of claim 19, wherein the chemiluminescent labeling compound is a compound of the formula

Wherein

The wavy line represents the site of attachment to the specific binding partner or to a linker linking the compound to the specific binding partner;

R¹、R²and R³Independently selected from the group consisting of substituted alkyl, unsubstituted alkyl, substituted alkenyl, unsubstituted alkenyl, substituted alkynyl, unsubstituted alkynyl, substituted aralkyl and unsubstituted aralkyl groups of 1 to 20 carbon atoms, wherein when R is¹Or R²When substituted, it is substituted with 1 to 3 substituents, each substituent independently selected from carbonyl, carboxyl, tri (C1-C8) alkylsilyl, -SO₃ ^-、-OSO₃ ^2-Sugar radical, -PO₃ ^-、-OPO₃ ^2-Halogen, hydroxy, mercapto, amino, C (O) NHNH₂Quaternary ammonium and quaternary phosphonium salts

Background

Solution phase luminescence assays are known, for example in WO 2010099486. Known assays are based on the specific recognition of specific binding partners and binding together to detect the presence or amount of a substance. For example, in an immunoassay, an antibody binds to a particular binding partner. As another example, in a nucleic acid binding assay, a strand of nucleic acid (e.g., a nucleic acid ligand) is bound to a specific binding partner. Some assays use chemiluminescence to generate a signal and correlate the signal with the amount of analyte.

Disclosure of Invention

A method of determining an analyte in a sample can include forming a reaction mixture and mixing a trigger solution with the reaction mixture. The analyte to be determined may be present in the sample, but the determination may also show the absence of the analyte. The reaction mixture is an aqueous solution comprising: a chemiluminescent-labeled specific binding partner comprising a chemiluminescent label irreversibly bound to a first specific binding partner capable of binding to the analyte to form an analyte-bound chemiluminescent-labeled specific binding complex; an activator-labeled specific binding partner comprising an activator label irreversibly bound to a second specific binding partner; and selective signal inhibitors.

The reaction mixture also contains unbound chemiluminescent substrate.

The trigger solution is capable of generating a background signal from the unbound chemiluminescent substrate and, in the presence of an analyte, the trigger solution is capable of generating a detectable analyte signal that is related to the amount of the analyte in the sample.

The kit may comprise components other than the sample necessary to perform the above assay.

Detailed Description

Throughout this disclosure, the singular forms such as "a," "an," and "the/the" are often used for convenience; however, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. When referred to in the singular, the term "only one" is often used.

Terms such as "common," "commonly," "common," and "generally" are used to refer to features that are common, typical, or common to the manner in which the invention described herein is made or practiced. These terms should not be construed to imply that such features are present in the prior art to the extent they are common, typical, or common in the art.

Some terms in this disclosure are defined below. Other terms will be familiar to those skilled in the art and should be given their meanings to those of ordinary skill in the art.

An analyte: a substance to be detected or quantified in an assay. One or more substances having specific binding affinity for the analyte (and in particular one or more specific binding partners) are typically used in the assay to facilitate detection of the analyte. The analyte can be a protein, peptide, nucleotide, nucleoside, antibody, hapten, small molecule (i.e., non-polymeric molecule), or the like to which a specific binding partner can bind. Exemplary analytes include not only drugs, such as steroids, hormones, proteins, glycoproteins, mucins, nucleoproteins, phosphoproteins, opioids, vitamins, antimicrobials, antifungals, antivirals, purines, antineoplastics, amphetamines, azepinoids, prostaglandins, and drug metabolites, but also nucleosides, organic nucleosides, nucleotides, organic nucleotides, ribonucleosides, DNA segments, RNA segments, PDNA segments, nucleic acid ligands, toxins (e.g., erythrotoxins, phototoxins, neurotoxins, cyanotoxins, finotoxins, necrotoxins, myotoxins), mycotoxins (e.g., T-2 mycotoxins, aflatoxins, botulinum toxins, ricins, bee toxins), and other environmental or biological toxins. The analyte may also be a cell, virus, bacterium or fungus.

Activating agent: a compound that causes activation of the chemiluminescent compound such that the chemiluminescent compound emits light in the presence of a trigger.

Activator-labeled specific binding partner: a reactant comprising a specific binding member for an analyte and an activator that binds directly or indirectly (e.g., via a linker) to a specific binding partner.

Chemiluminescent compounds: for example, a compound in which a reaction leading to light emission occurs by conversion into another compound formed in an electronically excited state or by conversion into an electronically excited state and then relaxation to a ground state. The excited state may be a singlet excited state or a triplet excited state. The excited state may emit light directly upon relaxation to the ground state, or may first transfer energy to an energy acceptor that in turn emits light, e.g., by a Forester or Dexter mechanism.

Specific binding partners for chemiluminescent labels: a reactant comprising a specific binding member for an analyte and a chemiluminescent compound which binds directly or indirectly (e.g., via a linking group) to a specific binding partner.

Analyte signal: a signal related to the amount of analyte present in the sample, e.g., a chemiluminescent output from the assay.

Background signal: a signal that is not related to the amount of analyte present in the sample, such as a chemiluminescent output.

Irreversible bond: a bond that associates two moieties (typically a specific binding partner and a chemiluminescent label or a specific binding partner and an activator label) that does not break when performing the assays described herein. The irreversible bond can be cleaved by some other means, such as using a compound or under physical conditions such as temperature, which is not exposed to during the assays described herein. Typical bonds that may be irreversible include covalent bonds, ionic bonds, and the like. Two moieties linked by an irreversible bond are said to "irreversibly associate". An irreversible bond is distinguished from a reversible binding interaction, e.g., reversible binding of a specific binding partner or analyte to a specific binding partner.

Assays based on prior art chemiluminescence binding assays (e.g., immunoassays described in US 20100267071) as well as other assays (e.g., those based on nucleic acid ligand binding, etc.) the chemiluminescent signal of a test sample with an unknown analyte concentration is compared to a standard curve generated by using multiple samples with known analyte concentrations.

In a specific chemiluminescent assay, as described in, for example, US20100267071, a test sample comprising an analyte at an unknown concentration is mixed with an assay solution comprising a chemiluminescent-labeled specific binding partner, an activator-labeled specific binding partner, and a selective signal inhibitor to form a reaction mixture. The trigger solution is then mixed with the reaction mixture. The trigger solution contains an oxidizing or reducing agent, typically a peroxide, and in many cases a reinforcing agent.

The assay is performed in one of two formats. In a "competitive assay" format, an activator-labeled specific binding partner is bound to a chemiluminescent-labeled specific binding partner in a complex that can be preformed or can be formed in situ. When the analyte is present, the analyte and the chemiluminescent-labeled specific binding partner compete to bind the analyte. The chemiluminescent label on the chemiluminescent-labeled specific binding partner that is bound to the activator-labeled specific binding partner is operably proximate to the activator-labeled specific binding partner and is thereby activated, resulting in luminescence, in the presence of the trigger solution. The chemiluminescent-labeled specific binding partner bound to the analyte is not in operable proximity to the activator-labeled specific binding partner, and therefore its chemiluminescent label is not activated and does not emit light. In this format, the analyte signal is the luminescence intensity, and it is inversely related to the analyte concentration.

An alternative format is a "sandwich assay" format whereby both the activator-labelled specific binding partner and the chemiluminescent-labelled specific binding partner are bound to the analyte, typically on different parts of the analyte, such that the chemiluminescent label is in operable proximity to the activator-labelled binding partner. The chemiluminescent label of the chemiluminescent-labeled specific binding partner in the resulting "sandwich" complex is in operative proximity to the activator-labeled specific binding partner and therefore emits light. In the absence of analyte, the activator-labeled binding partner is not operably proximate to the activator-labeled specific binding partner and therefore does not emit light. In this format, the analyte signal is the luminescence intensity and it is positively correlated with the analyte concentration.

In either case, the intensity of the analyte signal in the test sample is measured in a photometer and compared to a standard curve of luminescence intensity versus concentration constructed by performing the same assay with a standard sample having a known analyte concentration. The analyte signal is then correlated with a standard curve to provide the concentration of the analyte.

While this is acceptable when the sample being analyzed contains only analyte and water, most commercial applications of chemiluminescence will also contain substances other than analyte. For example, when a test sample from a food substance is analyzed for the presence of a toxin, then in addition to the toxin, there will typically be a number of compounds present, such as residual compounds from the food substance. These other compounds can interfere with the generation of analyte signals, typically by interfering with oxidation or reduction reactions that produce chemiluminescent species. In this case, the correlation of the analyte signal to the standard curve will not provide an accurate concentration of analyte in the test sample, as the standard curve will be generated from a solution that does not contain these other interfering compounds. This can lead to correlation errors where the comparison of the analyte signal to the standard curve does not accurately provide the concentration of analyte in the test sample. For example, one selective signal inhibitor that may be used is ascorbic acid, which is also present in many food substances either naturally or as an added antioxidant. If a significant amount of ascorbic acid is present in the test sample, it may further inhibit the generation of analyte signal from the test sample, and the concentration obtained by correlation of the analyte signal with the standard curve may be too low. This can be a significant problem, especially when determining harmful contaminants in food, as the determination may incorrectly indicate that the food has a safe level of contaminants when in fact it has an unsafe level of contaminants.

More generally, analyte concentration errors that may be caused by interference of other compounds in the sample being analyzed may be significant, in some cases up to 40% or even greater. In examples where food samples are analyzed for toxins, some toxins are unsafe for animal consumption at levels as low as 5ppb or even lower. Thus, these errors may be distinguished by either discarding food samples that are in fact safe to eat, on the one hand, or providing a false belief that toxins are present at safe levels, on the other hand, while toxins are in fact present at unsafe levels that may cause the animal that eats them to become sick or dead.

Prior art solutions to this problem have not been acceptable. Removal of compounds other than analytes from test samples to prevent interfering binding is extremely difficult, if not impossible, and is impractical, if not impossible, to achieve in commercial environments where thousands, tens of thousands, or even more unknown classes of different compounds may be present in the test sample. Another unacceptable prior art solution is to significantly dilute the test sample to a point where interference from compounds other than the analyte can be ignored. This solution is unacceptable because the analyte concentration is also diluted and therefore the minimum detection level ("LLD") and the minimum quantification level (LLQ) of the analyte is reduced. As mentioned above, the concentration of many analytes of interest needs to be determined at ppb levels, and therefore the reduction in LLQ and LLD makes solutions involving extensive dilution of test samples unacceptable.

Not only should an acceptable solution avoid the above disadvantages, that is, should be feasible for use in a commercial environment and not require significant dilution of the test sample, but it should also be usable with a variety of adhesives, meaning that the technique should not rely on the nature of the binding partner (e.g., antibody, nucleic acid ligand, etc.) so that it can be applied to a variety of different types of binding assays.

The present disclosure provides such a technical solution. Briefly, the technical solution described in more detail below involves the use of chemiluminescent substrates as background agents. The background agent provides a luminescent background signal (by the same series of oxidation or reduction reactions that result in analyte signal from the chemiluminescent label) in the presence of the trigger solution, but is independent of the binding of the analyte to the specific binding partner of the chemiluminescent label. That is, the background agent need not bind to any binding partner in order to generate a background signal. The background agent is present in the same known amount in the standard used to construct the standard curve. It is also present in the assay solution at the same concentration together with the binding partner of the chemiluminescent label. Thus, in the absence of a compound interfering with the oxidation or reduction reaction of the analyte signal and the background signal (e.g., the analyte is the only dissolved compound in the test sample), the background signal in the test sample will be the same as the background signal in the standard sample. However, when there is interference with the compound, then the background signal from the test sample will be different from the background signal from the standard sample. The ratio of background signal from the standard sample to the background signal from the test sample is taken to provide a scaling factor. The scaling factor multiplied by the observed analyte signal provides a "corrected analyte signal" that has been corrected for the presence of interfering compounds. Correlating the corrected analyte signal, rather than the observed analyte signal, with the standard curve provides an accurate indication of the analyte concentration in the test sample.

Specific binding partners

Two labeled specific binding partners were used: one is activator-labeled and the other is chemiluminescent-labeled. Each of the specific binding partners is a molecule, typically a biomolecule, having a specific affinity for another substance. Examples include DNA, RNA, oligonucleotides, nucleic acid ligands, antigens, antibodies, antibody-DNA chimeras, haptens, proteins, peptides, lectins, avidin, streptavidin, and biotin.

Each specific binding partner is typically different from the other partners in that the two specific binding partners do not compete for the same or overlapping binding sites on the analyte. For the typical case where the specific binding partner moieties of both the activator-labeled specific binding partner and the chemiluminescent-labeled specific binding partner are antibodies, each of the antibodies has a different, non-competitive epitope on the analyte.

Examples of specific binding partners that can be used in combination (i.e., one can have a chemiluminescent label and the other can have an activator label) include complementary oligonucleotides or polynucleotides (e.g., DNA, RNA, nucleic acid ligands, etc.), avidin-biotin, streptavidin-biotin, hormone-receptors, lectin-carbohydrates, IG protein a-binding protein receptor, nucleic acid-nucleic acid binding protein, nucleic acid ligand-nucleic acid ligand, and nucleic acid-anti-nucleic acid antibody. Specific binding partners as discussed in US20100267071 are suitable.

Any of these methods may be suitable for either competitive assay formats or sandwich assay formats. The type of specific binding partner determines the format of the assay, in particular whether the assay is a sandwich assay or a competitive assay. In either case, the specific binding partner for the chemiluminescent label specific binding partner is selected to specifically bind the analyte. Antibodies are most commonly used, but any of the specific binding partners discussed above may also be employed. To accommodate competitive assay formats, activator-labeled specific binding partners are designed as activator-analyte analog conjugates. In this case, the activating agent is conjugated to an analogue of the analyte, either directly or via a linker comprising an auxiliary substance as discussed herein, which analogue may be the analyte itself or a compound having sufficient structural similarity to the analyte such that the compound also binds to a chemiluminescent-labelled specific binding partner in substantially the same way as the analyte.

To accommodate the sandwich assay format, both the chemiluminescent-labeled specific binding partner and the activator-labeled specific binding partner specifically bind to the analyte. In this case, the specific binding partner for the activator-labelled specific binding partner is typically an antibody, but it may also be a nucleic acid ligand or any of the above-mentioned specific binding partners. In this case, both the chemiluminescent-labeled specific binding partner and the activator-labeled specific binding partner bind to the analyte, typically to different binding sites on the analyte.

Both competitive assays and sandwich assays are known in the art, and methods for accomplishing these adaptations are known. Examples and additional details regarding assay formats and specific binding partners can be found in US 20100267071.

Chemiluminescent-labeled specific binding partners

The specific binding partner of the chemiluminescent label is generally less than 10^-4M, in particular less than 10^-6M, and most specifically 10^-11M to 10^-7The concentration of M is present in the reaction mixture.

Chemiluminescent-labeled specific binding partners include specific binding partners labeled with a chemiluminescent label, typically through an irreversible bond. Typically, each molecule of the specific binding partner has irreversibly bound thereto at least one chemiluminescent label. In some cases, there may be up to 10 binding to each specific binding partner²One or even more chemiluminescent labels. It is not necessary that each specific binding partner molecule has the same number of chemiluminescent labels.

The one or more chemiluminescent labels may be any suitable chemiluminescent moiety that may be bound to a specific binding partner, typically by an irreversible bond. The linkage (typically an irreversible linkage) may be a direct linkage or an indirect linkage. In direct linkage, the one or more chemiluminescent labels are directly linked to the specific binding partner without the use of a linking group or auxiliary substance between the one or more chemiluminescent labels and the specific binding partner. Direct attachment is typically through irreversible bonds, such as ionic bonds, covalent bonds, hydrophobic interactions, hydrogen bonds, and the like, and is most commonly a covalent bond.

When indirect attachment is employed, a linker (sometimes referred to in the art as an auxiliary substance) is used to link the one or more chemiluminescent labels and the specific binding partner. Any suitable linker may be used; suitable linkers will not prevent the one or more chemiluminescent labels from emitting light and will not generally render the specific binding partner of the chemiluminescent label insoluble in aqueous media. Exemplary linkers include proteins such as streptavidin, avidin, neutravidin, biotin, cationized BSA, fos, jun, keyhole limpet hemocyanin, immunoglobulins (including fragments or portions thereof), liposomes, micelles, synthetic dendrimers such as AMAM, synthetic polymers such as polyacrylic acid, natural polymers such as polysaccharides (e.g., functionalized dextrans, polynucleotides, nucleic acid ligands, and oligonucleotides, and the like). Polysaccharides, especially aminodextran or carboxydextran, and self-assembling proteins are most commonly used.

The chemiluminescent label is formed by reacting a compound of the general formula CL-L-RG with a specific binding partner, wherein CL represents a chemiluminescent moiety, L represents a linker or covalent bond, and RG represents a reactive group. Once the reaction is complete, the chemiluminescent moiety becomes a chemiluminescent label on the specific binding partner of the chemiluminescent label. The chemiluminescent label reacts with the oxidizing or reducing agent in the trigger solution or with the activator label in the activator-labeled specific binding partner to form an activated chemiluminescent compound, which is typically the excited state of the chemiluminescent label. The excited state may be a singlet excited state or a triplet excited state. The excited state can either relax with the emission or undergo energy transfer to an emission energy acceptor that in turn emits light. In certain embodiments, luminescence occurs very rapidly upon addition of the trigger solution, more specifically reaching peak intensity within 2 seconds after addition of the trigger solution. However, this is not necessary as slower reactions may also give accurate results as long as the analyte signal is measured over a sufficient period of time.

A variety of chemiluminescent compounds suitable for binding to a specific binding partner (e.g., an antibody or antibody fragment) are known in the art. Any of these chemiluminescent compounds can be used in the assays described herein.

Exemplary chemiluminescent moieties and chemiluminescent labels include aromatic cyclic diacylhydrazines (e.g., luminol, isoluminol, aminobutylethylisoluminol, aminohexylethylisoluminol, 7-dimethylaminonaphthalene-1, 2-dicarboxylic acid hydrazide, ring-substituted aminophthalic hydrazide, anthracene-2, 3-dicarboxylic acid hydrazide, phenanthrene-1, 2-dicarboxylic acid hydrazide, pyrenedicarboxylic acid hydrazide, 5-hydroxyphthalic hydrazide, 6-hydroxyphthalic hydrazide), xanthene dyes (e.g., fluorescein, eosin, rhodamine dyes, rhodol dyes), chemiluminescent aromatic or heterocyclic amines, MCLA, indoleacetic acid, isobutyraldehyde, trihydroxyaromatic compounds (e.g., pyrogallol, phloroglucinol and erythrophenol), and the diketophthalazine compounds disclosed in US 2025475 and US5324835, 9, 10-dihydroacrylketene dithioacetal compounds and combinations of the foregoing. Although any of these markers may or may not be used with an emissive energy receptor, isobutyraldehyde is most commonly used with an emissive energy receptor.

Some chemiluminescent labels may be of formula I:

in the marker of formula I, R₁And R₂Each of which is independently H or an organic moiety containing from 1 to 50 atoms selected from C, N, O, S, P, Si and halogen plus sufficient hydrogen atoms to satisfy a valence other than hydrogen atoms. Most commonly, R₁And R₂Each of which is independently a linker of a specific binding partner, H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, aralkyl, or substituted aralkyl. When R is₁And R₂When substituted, it is most often substituted with 1 to 3 groups selected from carbonyl, carboxyl, tri (alkyl) silyl, glycosyl, -SO₃ ^-、-OSO₃ ^-、-PO₃ ^-、-OPO₃ ^-Halogen, hydroxy, mercapto, amino, quaternary ammonium and quaternary phosphonium salts

In the marker of formula I, R₃Is H or contains 1 to 50 atoms (most commonly 1 to 20 atoms) selected from C, N, O, S, P, Si and halogen plus sufficient to satisfy noneAn organic moiety of a hydrogen atom of hydrogen atom valency. Most commonly, R₃A linker which is a specific binding partner, H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, aralkyl or substituted aralkyl. In this case, R₃Most often 1 to 20 carbon atoms. In many cases, R₃Is alkyl, phenyl, benzyl, substituted benzyl, alkoxyalkyl, carboxyalkyl or alkylsulfonic acid having 1 to 4 carbon atoms. It is possible that R₃(especially when it is alkyl, substituted alkyl, alkenyl or substituted alkenyl, but also in other cases) is covalently bonded to R₇Or R₈To form a ring, typically a five-membered ring or a six-membered ring. When one or more of the above moieties are substituted, it is most often substituted with 1 to 3 groups selected from carbonyl, carboxyl, tri (alkyl) silyl, glycosyl, -SO₃ ^-、-OSO₃ ^-、-PO₃ ^-、-OPO₃ ^-Halogen, hydroxy, mercapto, amino, quaternary ammonium and quaternary phosphonium salts

In the marker of formula I, R₄To R₁₁Each of which is independently H or an organic moiety containing from 1 to 50 atoms selected from C, N, O, S, P, Si and halogen plus sufficient hydrogen atoms to satisfy a valence other than hydrogen atoms. Most commonly, R₁And R₂Each of which is independently a linker group, H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, aralkyl, substituted aralkyl, alkoxy, aryloxy, halogen, amino, substituted amine, carboxyl, alkoxycarbonyl, carboxamide, cyano, or sulfonic group of a specific binding partner. Paired adjacent R₄To R₁₁Moieties, e.g. R₄And R₅、R₈And R₉Etc., may be covalently bonded to form a ring. In this case, the ring is typically a five-to seven-membered ring, and most typically five-membered ringA membered ring or a six membered ring. The ring may be carbocyclic or heterocyclic and in the latter case may contain heteroatoms such as N, O or S, and may be unsubstituted or substituted on one or more carbon atoms or one or more heteroatoms. When one or more of the above moieties are substituted, it is most often substituted with 1 to 3 groups selected from carbonyl, carboxyl, tri (alkyl) silyl, glycosyl, -SO₃ ^-、-OSO₃ ^-、-PO₃ ^-、-OPO₃ ^-Halogen, hydroxy, mercapto, amino, quaternary ammonium and quaternary phosphonium salts

Most commonly, in formula I, R₄To R₁₁Each of which is H.

Most commonly, the label of formula I employed is a label of formula II.

Wherein the wavy line indicates the point of attachment to the specific binding partner. In such cases, R₃Typically H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, aralkyl or substituted aralkyl. In such cases, R₃Alkyl, phenyl, benzyl, substituted benzyl, alkoxyalkyl, carboxyalkyl or alkylsulfonic acids having most often from 1 to 20 carbon atoms, in particular from 1 to 4 carbon atoms. When one or more of the above moieties are substituted, it is most often substituted with 1 to 3 groups selected from carbonyl, carboxyl, tri (alkyl) silyl, glycosyl, -SO₃ ^-、-OSO₃ ^-、-PO₃ ^-、-OPO₃ ^-Halogen, hydroxy, mercapto, amino, quaternary ammonium and quaternary phosphonium salts

In particular, in the label of formula II, the wavy line indicates the site of attachment to the specific binding partner or to a linker linking the compound to the specific binding partner, R¹、R²And R³Independently selected from the group consisting of substituted alkyl, unsubstituted alkyl, substituted alkenyl, unsubstituted alkenyl, substituted alkynyl, unsubstituted alkynyl, substituted aralkyl, and unsubstituted aralkyl, wherein when R is¹Or R²When substituted, it is substituted with 1 to 3 substituents, each substituent independently selected from carbonyl, carboxyl, trialkylsilyl, -SO₃Sugar radical, -PO₃Halogen, hydroxy, mercapto, amino, C (O) NHNH₂Quaternary ammonium and quaternary phosphonium saltsAnd R is³Selected from the group consisting of substituted alkyl, unsubstituted alkyl, substituted alkenyl, unsubstituted alkenyl, substituted alkynyl, unsubstituted alkynyl, substituted aralkyl, and unsubstituted aralkyl, wherein when R is¹Or R²When substituted, it is substituted with 1 to 3 substituents, each substituent independently selected from carbonyl, carboxyl, trialkylsilyl, -SO₃Sugar radical, -PO₃Halogen, hydroxy, mercapto, amino, C (O) NHNH₂Quaternary ammonium and quaternary phosphonium salts

The label of formula II is bound to the specific binding partner by reaction of the specific binding partner with the compound of formula III.

Wherein L and RG are as described above.

Examples of compounds of formula III that may be employed are provided in table 8 of US 20100267071. In general, the compounds of formula I and in particular the compounds of formula III may be prepared according to the methods disclosed in US 20070172878. Example (b)For example, commercially available 9, 10-dihydroacridine or N-substituted 9, 10-dihydroacridine can be treated with a strong base followed by treatment with carbon disulfide to form a 9, 10-dihydroacridinium dithiocarboxylate, which is then esterified or partially esterified by conventional esterification methods to install substituent R₁。R₂Can be added by deprotonation of the remaining thiol with a strong base such as butyllithium or sodium hydride, followed by treatment with an appropriate electrophile to attach R₂. Substituent R₁And R₂Further reactions may take place to manipulate functional groups thereon in order to obtain the desired compound of formula III.

The chemiluminescent label may also be selected from the group consisting of aromatic cyclic dialdehyde hydrazides, trihydroxyaromatic compounds, 9, 10-dihydroacridine ketene dithioacetal compounds, 9, 10-dihydroacridinium esters, 9, 10-dihydroacridinium thioesters, 9, 10-dihydroacridinenol, and compounds having the formula

Wherein

R¹Selected from alkyl, alkenyl, alkynyl, aryl and aralkyl groups of 1 to 20 carbons or independently selected from carbonyl, trialkylsilyl, SO₃-、-OSO₃Sugar radical, PO₃-、-OPO₃Halogen, hydroxy, mercapto, amino, quaternary ammonium or quaternary phosphoniumAny of the foregoing groups partially substituted with 1 to 3 groups, X is selected from C1-C8 alkyl, aryl, aralkyl, alkyl or alkylcarbonyl having 1 to 20 carbon atoms, trialkylsilyl, SO₃-, a glycosyl group and PO (OR ') (OR'), wherein R 'and R' are independently selected from the group consisting of C1-C8 alkyl, cyanoalkyl, cyanoaryl, cyanoaralkyl, trialkylsilyl, alkali metal cation, alkaline earth metal cation, ammonium cation and trialkyiCation, Z¹And Z²Independently selected from O and S atoms, and R²And R³Independently selected from H and C1-C8 alkyl.

Further chemiluminescent labels are disclosed in US 5497072, US523212, US5593845, US5922588, US60130803, US6696569, US6891057 and US 20100267071. Any of these or other chemiluminescent labels may be employed. Particularly suitable chemiluminescent labels and specific binding partners for the chemiluminescent label include those disclosed in US 20100267071.

Activator-labeled specific binding partners

Activator-labeled specific binding partners are generally less than 10^-4M, in particular less than 10^-6M, and most specifically 10^-11M to 10^-7The concentration of M is present in the reaction mixture.

Activator-labeled specific binding partners comprise an activator label that generally irreversibly binds to the specific binding partner. Any suitable activator label may be used. When the compound meets both requirements, it may be suitable as an activator label. First, it is capable of accepting or donating an electron, or in some rare cases multiple electrons, from an oxidizing or reducing agent to form a group, an ionic group, or in unusual cases an ion. Such groups, ionic groups or ions are sometimes referred to as activated activator labels, and the formation of activated activator labels is sometimes referred to as activated activator labels. Second, once the activated activator label is formed, it should be capable of activating the chemiluminescent label on the specific binding partner of the chemiluminescent label and, if applicable, causing the chemiluminescent label to luminesce and, if applicable, the unbound chemiluminescent substrate to luminesce.

Typical activator labels are peroxidases or compounds having peroxidase-like activity. Examples include lactoperoxidase, microperoxidase, myeloperoxidase, haloperoxidase, vanadium bromoperoxidase, horseradish peroxidase, fungal peroxidase, lignin peroxidase, Mn-dependent peroxidase, soybean peroxidase, and peroxidase mimic compounds which are not enzymes but have peroxidase-like activity, such as Mn-TPPS 4.

Activator-labeled specific binding partners may include conjugates or complexes of peroxidase enzymes or compounds having peroxidase-like activity with biomolecules. In such cases, typical biomolecules that may be used include DNA, RNA, nucleic acid ligands, antibodies, antibody fragments, antibody-DNA chimeras, antigens, haptens, proteins, peptides, lectins (lechtins), avidin, streptavidin, and biotin.

One or more activator labels are typically bound to a specific binding partner via an irreversible bond. The linkage (typically an irreversible linkage) may be a direct linkage or an indirect linkage. In direct linkage, the one or more chemiluminescent labels are directly linked to the specific binding partner without the use of a linking group or auxiliary substance between the one or more chemiluminescent labels and the specific binding partner. Direct attachment is typically through irreversible bonds, such as ionic bonds, covalent bonds, hydrophobic interactions, hydrogen bonds, and the like, and is most commonly a covalent bond.

When indirect attachment is employed, a linker (sometimes referred to in the art as an auxiliary substance) is used to link the one or more chemiluminescent labels and the specific binding partner. Any suitable linker may be used; suitable linkers will not prevent the one or more chemiluminescent labels from emitting light and will not generally render the specific binding partner of the chemiluminescent label insoluble in aqueous media. Exemplary linkers include proteins such as streptavidin, avidin, neutravidin, biotin, cationized BSA, fos, jun, keyhole limpet hemocyanin, immunoglobulins (including fragments or portions thereof), liposomes, micelles, synthetic dendrimers such as AMAM, synthetic polymers such as polyacrylic acid, natural polymers such as polysaccharides (e.g., functionalized dextrans, polynucleotides, nucleic acid ligands, and oligonucleotides, and the like). Polysaccharides, especially aminodextran or carboxydextran, and self-assembling proteins are most commonly used.

Suitable activator-labelled specific binding partners include those disclosed in US 20100267071.

Selective signal inhibitors

Selective signal inhibitors reduce the noise signal caused by excess chemiluminescent labeled specific binding partner present in the reaction mixture but not participating in the assay described herein. Their function is described in more detail in US 20100267071.

Typically, the one or more selective signal inhibitors are present at 10^-6M to 10^-1M, most commonly 10^-5M to 10^-4The concentration of M is present in the reaction mixture. Specific concentrations include 5X 10^-6M to 5X 10^-4M, and more specifically 5 × 10^-5M to 5X 10^-4M。

Compounds suitable for use as selective signal inhibitors include antioxidants, particularly sacrificial antioxidants, and other molecules that are reactive with groups, ionic groups, or in some cases ions (or in some cases oxidizing or reducing agents) formed by the interaction of an oxidizing or reducing agent with an activator label on an activator-labeled selective binding partner. Any antioxidant may be employed, as the various options for selective signaling inhibitors function in the same manner for the purposes of this disclosure. Specifically, it is reacted with an oxidizing or reducing agent (typically a peroxide) or with an activated activator label on an activator-labeled specific binding partner to quench the peroxide group or activated activator label.

Some specific antioxidants that can be used as selective signal inhibitors are described in US 20100267071. Examples include glutathione, ascorbic acid, especially L-ascorbic acid, ascorbate, especially L-ascorbate, uric acid, L-ascorbic acid-6-palmitate, tocopherol, 5, 6-isopropylidene-L-ascorbic acid, isoascorbic acid, including D-isoascorbic acid, L-isoascorbic acid orTwo, sodium sulfite, diethylhydroxylamine, BHT, and combinations of the foregoing. Most commonly, tocopherol or ascorbic acid, especially ascorbic acid, is used.

In use, the one or more selective signal inhibitors may be provided in any suitable manner. For example, they may be provided as components of a trigger solution, in which case a reaction mixture is formed when the trigger solution is added. They may also have one or both of a chemiluminescent-labeled specific binding partner or an activator-labeled specific binding partner, or they may be added separately as a solid or as a solution of appropriate concentration. Most commonly, the one or more selective signal inhibitors are provided in the working solution at a concentration ten times higher, or in some cases even more than ten times higher, than that provided in the reaction mixture, especially after addition of the trigger solution. The working solution is typically aqueous and in many cases may be water, such as a buffered water, but in some cases it may also contain surfactants, alcohols, glycols, etc. in order to provide a sufficiently high concentration of the one or more selective signal inhibitors. In any case, they are added in appropriate amounts to obtain the appropriate concentration in the reaction mixture.

Trigger solution

The trigger solution provides the specific binding partner labeled by chemiluminescence and, in some cases, one or more oxidizing or reducing agents required to cause luminescence from the background reagent. The one or more oxidizing or reducing agents may perform this function by reacting directly with the chemiluminescent label and, if applicable, the background agent, but more typically the one or more oxidizing or reducing agents react with the activator label on the specific binding partner of the activator label to facilitate the action of the activator label with the chemiluminescent label.

The one or more oxidizing or reducing agents can be any compound that activates an activator label on an activator-labeled specific binding partner. Most commonly, the one or more oxidizing or reducing agents are one or more peroxides that interact with the activator label, typically a peroxidase or a compound having peroxidase-like activity, to activate the activator label. Although any peroxide that reacts with a peroxidase or a compound having peroxidase-like activity may be used, commonly used peroxides include alkyl peroxides (especially where the alkyl group is ethyl or methyl), alkyl hydroperoxides (especially where the alkyl group is ethyl or methyl), aromatic peroxides (especially benzyl peroxides), lipid hydroperoxides (especially hydroperoxides of eicosapentaenoic acid, docosahexaenoic acid or linoleic acid), peroxy acids (e.g. m-chloroperoxybenzoic acid), hydrogen peroxide, urea peroxide, peroxycarbamates and perborates. The concentration of peroxide can vary, but is typically 10^-8M to 3M, and most commonly 10^-3M to 10^-1M。

Although not required, enhancers are often used as components of the trigger solution. The enhancing agent may be any compound that promotes the reactivity of the activator label (typically a peroxidase), reduces the noise signal in the assay, or both. Typical enhancers include phenolic compounds, aromatic amines, thiophenesMixtures of oxazines or phenothiazines with indophenols or indoanilines, substituted hydroxybenzenesOxazoles, substituted or unsubstituted arylboronic acids, and their esters and anhydrides, and the like. Some suitable enhancers are disclosed in US20100267071, US5171668, US5206149 and US 5512451. When employed, the reinforcing agents are usually in the form of10^-5M to 10^-1The concentration of M is present.

As discussed above, the one or more selective signal inhibitors may also be present in the trigger solution in addition to the oxidizing or reducing agent and, when employed, the enhancing agent.

The trigger solution typically includes various solutes described in an aqueous solvent. The aqueous solvent is typically buffered water. Any buffer that can be used in a biological system can be used so long as it does not interfere with the chemiluminescent label or the luminescence of the assay to the extent that it does not produce a sufficient analyte signal. The most useful buffer maintains the pH at 5 to 10.5. Particularly useful buffers maintain a pH of 6.0 to 9.0, for example 6.5 to 8.5, and most particularly 7.0 to 8.0

Exemplary buffers that can be used are disclosed in US 20100267071. Most commonly, the buffer is selected from the group consisting of phosphate, borate, acetate, tris (hydroxy methyl amine) methane, glycine, tricin, 2-amino-2-methyl-1-propanol, diethanolamine, MOPS, HEPES, MES, and the like.

One or more detergents or polymeric surfactants may be used to enhance luminescence or reduce noise signals. Examples include polyoxyethylated alkylphenols, polyoxyethylated alcohols, polyoxyethylated ethers, polyoxyethylated sorbitol esters, quaternary ammonium salts (e.g. CTAB), and quaternary phosphonium saltsAnd (3) salt. Particularly useful examples are polymeric cationic surfactants, most particularly quaternary ammonium salts and quaternary phosphonium saltsAnd (3) salt.

As discussed above, the one or more selective signal inhibitors may optionally be present in the trigger solution.

For example, the trigger solution may comprise an aqueous buffer at a concentration of 10^-5Peroxide of M to 1M and concentration of 10^-5M to 10^-1An enhancer of M. As another example, the trigger solution may comprise an aqueous buffer at a concentration of 10^-5M to 1M peroxide, concentration 10^-5M to10^-1M, and a concentration such that the one or more selective signal inhibitors have a 10 in the reaction mixture^{- 6}M to 10^-1M, concentration of the one or more selective signal inhibitors.

Unbound chemiluminescent substrate

The unbound chemiluminescent substrate may be any chemiluminescent molecule that is not conjugated or otherwise irreversibly bound to the analyte-labeled selective binding partner or the chemiluminescent-labeled specific binding partner. In most cases, unbound chemiluminescent substrate is capable of being dissolved in the reaction mixture. For measuring the luminescence intensity of the specific binding partner of the chemiluminescent label and the unbound chemiluminescent substrate, respectively, the unbound chemiluminescent substrate may conveniently be selected having a maximum luminescence wavelength different from that of the chemiluminescent label, but this is not essential.

During the assay, the unbound chemiluminescent substrate may react with the oxidizing or reducing agent, most commonly indirectly through an activator label on the activator-labeled specific binding partner. The reaction brings the chemiluminescent substrate into an electronically excited state from which the chemiluminescent substrate can emit light to produce a background signal. Alternatively, but less typically, the excited state of the unbound chemiluminescent substrate may undergo further reaction with an emission energy receptor that can then luminesce to produce a background signal.

Suitable unbound chemiluminescent substrates include cyclic hydrazides such as luminol and isoluminol, imidazole compounds such as lofenadine, acridine esters such as lucigenin, and 9, 10-dihydroacridine, phthalylhydrazides such as 2, 3-dihydro-1, 4-diketophthalazine, fluorescein, and 1, 2-dioxetane-containing compounds such as diketones, 3- (2' -spiroadamantane) -4-methoxy-4- (3 "-phosphoryloxy) phenyl-1, 2-dioxetane (AMPPD) and 3- (2' -spiroadamantane) -4-methoxy-4- (3" -beta-D ' -galactopyranosyloxy) phenyl-1, 2-dioxetane (AMPGD), 3- (4-Methoxysilospiro {1, 2-dioxetane-3, 2'- (5' -chloro) tricyclo [3.3.1.13,7] decane } -4-yl) phenyl disodium phosphate and 1, 2-dioxetanedione, adamantyl-1, 2-dioxetane). The most common are luminol and isoluminol, and in particular luminol.

In an assay, an unbound chemiluminescent substrate is converted to an excited state that produces a detectable background signal, usually in the form of luminescence, by the same photochemical mechanism as the chemiluminescent label, i.e., by reaction with an oxidizing or reducing agent, usually an activator label. The detectable background signal is not related to the concentration of the analyte. Thus, non-analyte components in the sample that may interfere with analyte signal generation will similarly interfere with background signal generation.

It is possible to use this to obtain a correction factor that corrects for the interference of non-analyte compounds with the assay. To this end, a set of standard samples, each containing a different known amount of analyte, is prepared, and the other components of the reaction mixture (i.e., the chemiluminescent-labeled specific binding partner, the activator-labeled specific binding partner, and the unbound chemiluminescent substrate) are exposed to a trigger. Each of the reaction mixtures used with the standard sample has the same amount of chemiluminescent-labeled specific binding partner, activator-labeled specific binding partner, and unbound chemiluminescent substrate. The trigger solution is added and both the analyte signal as well as the background signal at different analyte concentrations in the standard sample are measured, which is usually the same for all standard samples, since the concentration of unbound chemiluminescent substrate is the same in all standard samples. If the background signal is not the same for all test samples, this indicates that there is an error in the portion of the assay or that the analyte interacts with the unbound chemiluminescent substrate. In the latter case, different unbound chemiluminescent substrates may be employed.

Analyte signals at different analyte concentrations can be used to develop a correlation curve of analyte signal to analyte concentration.

Background signals from the test samples can be used to make the following scaling factors. The test sample is assayed and both analyte and background signals are determined. The scaling factor is calculated as the ratio of the background signal generated by the standard sample to the background signal generated by the test sample. The scaling factor takes into account the effect of non-analyte compounds in the test sample, and is 1 if non-analyte compounds that interfere with the assay are not present in the test sample.

To determine the analyte concentration in the test sample, the analyte signal of the test sample is multiplied by a scaling factor to obtain a corrected analyte signal. The corrected analyte signal is compared to a correlation curve of analyte signal to analyte concentration determined from a standard sample to provide an analyte concentration.

Detection of

The various signals, such as background signals and analyte signals, may be detected by any means known in the art. The type of device may depend on the type of signal. When the signal is a luminescent signal, a photometer or a CCD camera is generally used. Other useful detectors include photographic film, X-ray film, scintillation counters, light gauges, transmittance detectors (e.g., UV/Vis and IR detectors), and the like. Most commonly, the detection is performed in a cuvette or in a multiwell plate in a luminometer or in front of a CCD camera. The use of a multi-well plate and a CCD camera may be convenient, since in this case the determination and detection of a plurality of test samples and standard samples may be performed simultaneously in different wells of the multi-well plate. Detection may be performed with any of a number of commercially available photometers, CCD cameras, and the like, or known in the art.

Use of

The assays described herein can be used in a variety of systems. ELISA systems are one use, but assays involving nucleic acid ligand binding and other non-immunogen specific binding partners may also be employed in accordance with the teachings herein and the knowledge of the skilled artisan. Examples include solution hybridization assays, southern blot assays, northern blot assays, DNA or RNA sequencing, DNA or RND fingerprinting, colony hybridization, and plaque assays, all of which are known in the art. Any analyte that can produce at least one specific binding partner can be analyzed. Examples include antigens, toxins, venoms, nucleic acids, nucleotides, polynucleotides, drugs, steroids, haptens, antibodies, peptides, peptide fragments, hormones, receptors, primers, small molecules, and the like.

List of exemplary embodiments

This list of embodiments is intended to aid in the understanding of particular aspects of the invention. And are not intended to be limiting.

1. A method of determining an analyte in a sample, the method comprising

Forming a reaction mixture, and

mixing a trigger solution with the reaction mixture; wherein

The analyte to be determined may be present in the sample,

the reaction mixture is an aqueous solution comprising

A chemiluminescent-labeled specific binding partner comprising a chemiluminescent label irreversibly bound to the first specific binding partner, the chemiluminescent-labeled specific binding partner capable of binding to the analyte to form an analyte-bound chemiluminescent-labeled specific binding complex,

an activator-labeled specific binding partner comprising an activator label irreversibly bound to a second specific binding partner, and

a selective signal inhibitor; and wherein

The reaction mixture further comprises an unbound chemiluminescent substrate, and

the trigger solution is capable of generating a background signal from the unbound chemiluminescent substrate, and

in the presence of an analyte, the trigger solution is capable of generating a detectable analyte signal that is related to the amount of the analyte in the sample.

2. The method of embodiment 1, wherein the reaction mixture further comprises an enhancer.

3. The method of embodiment 1, wherein the trigger solution further comprises an enhancer.

4. The method of any one of the preceding embodiments, wherein the selective signal inhibitor causes a ratio of signals resulting from the reaction between the chemiluminescent-labeled specific binding partner and the activator-labeled specific binding partner in the complex with the analyte to exceed a ratio of signals from the reaction between the chemiluminescent-labeled specific binding partner and activator-labeled specific binding partner when the complex with the analyte is not present.

5. The method according to any one of the preceding embodiments, wherein the selective signal inhibitor is selected from the group consisting of aromatic compounds having at least two hydroxyl groups in ortho or para orientation, aromatic compounds having at least one hydroxyl group and an amino group ortho or para to one or more of the at least one hydroxyl group, compounds having at least two hydroxyl groups substituted on an ethylenically unsaturated group and a nitrogen heterocyclic group.

6. The method according to any one of the preceding embodiments, wherein the selective signal inhibitor is selected from ascorbic acid, erythorbic acid, 6-hydroxy-2, 5,7, 8-tetramethylchroman-2-carboxylic acid, ascorbic acid 6-palmitate, 5, 6-isopropylidene-ascorbic acid, butylated hydroxytoluene, glutathione, uric acid, one or more tocopherols, and catechins.

7. The method of any one of the preceding embodiments, wherein the selective signal inhibitor is ascorbic acid.

8. The method according to any one of the preceding embodiments, wherein the specific binding partner of the chemiluminescent label comprises a chemiluminescent label linked directly or indirectly to a specific binding pair member, wherein the chemiluminescent label is selected from the group consisting of aromatic cyclic dialdehyde hydrazide, trihydroxyaromatic compound, 9, 10-dihydroacrylketene dithioacetal compound, 9, 10-dihydroacridinium ester, 9, 10-dihydroacridinium thioester, 9, 10-dihydroacridol, and compounds having the formula

Wherein

R¹Selected from alkyl, alkenyl, alkynyl, aryl and aralkyl groups of 1 to 20 carbons or independently selected from carbonyl, trialkylsilyl, SO₃-、-OSO₃Sugar radical, PO₃-、-OPO₃Halogen, hydroxy, mercapto, amino, quaternary ammonium or quaternary phosphoniumAny of the foregoing groups partially substituted with 1 to 3 groups of (a);

x is selected from C1-C8 alkyl, aryl, aralkyl, alkyl or alkylcarbonyl having 1 to 20 carbon atoms, trialkylsilyl, SO₃-, a glycosyl group and PO (OR ') (OR'), wherein R 'and R' are independently selected from the group consisting of C1-C8 alkyl, cyanoalkyl, cyanoaryl, cyanoaralkyl, trialkylsilyl, alkali metal cation, alkaline earth metal cation, ammonium cation and trialkyiA cation;

Z¹and Z²Independently selected from O and S atoms; and is

R²And R³Independently selected from H and C1-C8 alkyl.

9. The method according to any one of embodiments 1 to 7, wherein the specific binding partner of the chemiluminescent label comprises a chemiluminescent label directly or indirectly linked to the specific binding partner, wherein the chemiluminescent label has the formula

Wherein

The wavy line represents the site of attachment to the specific binding partner or to a linker linking the compound to the specific binding partner;

R¹、R²and R³Independently selected from the group consisting of substituted alkyl, unsubstituted alkyl, substituted alkenyl, unsubstituted alkenyl, substituted alkynyl, unsubstituted alkynyl, substituted aralkyl and unsubstituted aralkyl groups of 1 to 20 carbon atoms, wherein when R is¹Or R²When substituted, it is substituted with 1 to 3 substituents, each substituent independently selected from carbonyl, carboxyl, tri (C1-C8) alkylsilyl, -SO₃ ^-、-OSO₃ ^2-Sugar radical, -PO₃ ^-、-OPO₃ ^2-Halogen, hydroxy, mercapto, amino, C (O) NHNH₂Quaternary ammonium and quaternary phosphonium salts

10. The method of any one of the preceding embodiments, wherein the activator-labeled specific binding partner comprises an activator-labeling compound directly or indirectly linked to a specific binding pair member, wherein the activator-label is selected from the group consisting of a transition metal salt, a transition metal complex, and an enzyme, and wherein the activator-label has peroxidase activity.

11. The method of any one of the preceding embodiments, wherein the activator-label is a peroxidase.

12. The method of embodiment 11, wherein the activator label is horseradish peroxidase.

13. The method according to any one of the preceding embodiments, wherein at least one of the chemiluminescent-labeled specific binding partner and the activator-labeled specific binding partner comprises an auxiliary substance selected from the group consisting of soluble proteins, streptavidin, avidin, neutravidin, biotin, cationized BSA, fos, jun, soluble synthetic dendrimers, soluble synthetic polymers, polysaccharides, dextran, organic nucleotides, nucleosides, nucleic acid ligands, liposomes, and micelles.

14. The method of any one of embodiments 2 to 12, wherein the enhancer is one or more compounds that promote the catalytic conversion of an activator having peroxidase activity.

15. The method of embodiment 13, wherein the enhancer is selected from the group consisting of phenolic compounds, aromatic amines, benzolsOxazoles, hydroxybenzothiazoles, arylboronic acids, and mixtures of any of the foregoing.

16. The method of any one of the preceding embodiments, wherein the trigger solution comprises an oxidizing agent or a reducing agent.

17. The method of any one of the preceding embodiments, wherein the trigger solution comprises a peroxide.

18. The method of embodiment 17, wherein the peroxide is selected from the group consisting of alkyl peroxides (especially wherein the alkyl group is ethyl or methyl), alkyl hydroperoxides (especially wherein the alkyl group is ethyl or methyl), aromatic peroxides (especially benzyl peroxides), lipid hydroperoxides (especially hydroperoxides of eicosapentaenoic acid, docosahexaenoic acid, or linoleic acid), peroxy acids (e.g., m-chloroperoxybenzoic acid), hydrogen peroxide, urea peroxide, peroxycarbamates, and perborates.

19. The method of embodiment 18, wherein the peroxide is hydrogen peroxide.

20. The method of any one of the preceding embodiments, wherein the trigger solution comprises an enhancer selected from the group consisting of phenolic compounds, aromatic amines, benzolsOxazoles, hydroxybenzothiazoles, arylboronic acids, and mixtures of any of the foregoing.

21. The method of any one of the preceding embodiments, wherein all of the components of the trigger solution and the reaction mixture and the analyte are water soluble.

22. The method of any one of the preceding embodiments, wherein none of the trigger solution, the assay solution, or the reaction mixture comprises a material that is directly or indirectly conjugated to a solid phase species.

23. The method according to any one of the preceding embodiments, wherein the selective signaling inhibitor comprises a compound selected from the group consisting of: aromatic compounds having at least two hydroxyl moieties in the ortho or para orientation, aromatic compounds having a hydroxyl moiety and an amino moiety in the ortho or para orientation, compounds having at least two vinyl hydroxyl groups and a nitrogen heterocycle.

24. The method according to any one of the preceding embodiments, wherein the selective signal inhibitor comprises ascorbic acid, and wherein the ascorbic acid is especially L-ascorbic acid.

25. The method of any one of the preceding embodiments, wherein the selective signal inhibitor comprises 6-hydroxy-2, 5,7, 8-tetramethylchroman-2-carboxylic acid.

26. The method of any one of the preceding embodiments, wherein the selective signal inhibitor comprises a 2-aminophenol.

27. The method according to any one of the preceding embodiments, wherein the selective signaling inhibitor comprises 3-amino-tyrosine, in particular 3-amino-L-tyrosine.

28. The method of any one of the preceding embodiments, wherein the selective signal inhibitor comprises 4-chlorocatechol.

29. The method of any one of the preceding embodiments, wherein the selective signaling inhibitor comprises a thiopheneAnd (3) an oxazine.

30. The method according to any one of the preceding embodiments, wherein the selective signal inhibitor comprises 2-bromobenzo-1, 4-diol.

31. The method of any one of the preceding embodiments, wherein the selective signal inhibitor comprises 5, 6-isopropylidene ascorbic acid.

32. The method of any one of the preceding embodiments, wherein the selective signal inhibitor comprises 6-palmitate.

33. The method of any one of the preceding embodiments, wherein the chemiluminescent-labeled specific binding partner and the activator-labeled specific binding partner each bind to the analyte in the sample.

34. The method of any one of the preceding embodiments, wherein the chemiluminescent-labeled specific binding partner comprises a chemiluminescent-labeled compound linked to an analog of the analyte, and further wherein the analyte and the chemiluminescent-labeled specific binding partner compete for binding to the activator-labeled specific binding partner.

35. The method according to any one of the preceding embodiments, wherein the unbound chemiluminescent substrate is selected from the group consisting of cyclic hydrazides (e.g., luminol and isoluminol), imidazole compounds (e.g., lofenadine), acridine esters (e.g., lucigenin and 9, 10-dihydroacridine), phthalylhydrazides (e.g., 2, 3-dihydro-1, 4-diketophthalazine), fluorescein, and 1, 2-dioxetane-containing compounds (e.g., diketones, 3- (2' -spiroadamantane) -4-methoxy-4- (3 "-phosphoryloxy) phenyl-1, 2-dioxetane (AMPPD), and 3- (2' -spiroadamantane) -4-methoxy-4- (3" -beta-D ' -galactopyranosyloxy) phenyl-1, 2-dioxetane (AMPGD), disodium 3- (4-methoxyspiro {1, 2-dioxetane-3, 2'- (5' -chloro) tricyclo [3.3.1.13,7] decane } -4-yl) phenyl phosphate and 1, 2-dioxetanedione, adamantyl-1, 2-dioxetane), specifically luminol or isoluminol, and more specifically luminol.

36. The method of any one of the preceding embodiments, wherein the unbound chemiluminescent substrate comprises luminol.

37. The method according to any one of the preceding embodiments, wherein the wavelength of maximum luminescence intensity of the unbound chemiluminescent substrate is different from the wavelength of maximum luminescence intensity of the chemiluminescent label.

38. The method of any one of the preceding embodiments, wherein the method further comprises detecting an analyte signal and a background signal.

39. The method of embodiment 38, wherein detecting an analyte signal comprises detecting at least one standard analyte signal from at least one sample that is a standard sample having a known analyte concentration, and further comprising detecting an assay analyte signal from at least one test sample having an unknown analyte concentration.

40. The method according to embodiment 38 or 39, wherein detecting a background signal comprises detecting at least one standard background signal from at least one sample that is a standard sample having a known analyte concentration, and further comprising detecting a test background signal from at least one test sample having an unknown analyte concentration.

41. A kit for the determination of an analyte, said kit comprising

A chemiluminescent-labeled specific binding partner comprising a chemiluminescent label irreversibly bound to the first specific binding partner, the chemiluminescent-labeled specific binding partner capable of binding to the analyte to form an analyte-bound chemiluminescent-labeled specific binding complex,

an activator-labeled specific binding partner comprising an activator label irreversibly bound to a second specific binding partner, and

a selective signal inhibitor; and wherein

The trigger solution is capable of generating a background signal from the unbound chemiluminescent substrate,

and also comprises

Unbound chemiluminescent substrate; wherein

In the presence of an analyte, the trigger solution is capable of generating a detectable analyte signal that is correlated with the amount of the analyte in the sample.

42. The kit of any one of embodiments 41, wherein the trigger solution further comprises an enhancer.

43. The kit of any one of embodiments 41 or 42, wherein the selective signal inhibitor causes a ratio of signals resulting from the reaction between the chemiluminescent-labeled specific binding partner and the activator-labeled specific binding partner in the complex with the analyte to exceed a ratio of signals from the reaction between the chemiluminescent-labeled specific binding partner and activator-labeled specific binding partner when the complex with the analyte is not present.

44. The kit according to any one of embodiments 41 to 43, wherein the selective signal inhibitor is selected from the group consisting of an aromatic compound having at least two hydroxyl groups in ortho or para orientation, an aromatic compound having at least one hydroxyl group and an amino group in ortho or para position to one or more of the at least one hydroxyl group, a compound having at least two hydroxyl groups substituted on an ethylenically unsaturated group and a nitrogen heterocyclic group.

45. The kit according to any one of embodiments 41 to 44, wherein the selective signal inhibitor is selected from ascorbic acid, erythorbic acid, 6-hydroxy-2, 5,7, 8-tetramethylchroman-2-carboxylic acid, ascorbic acid 6-palmitate, 5, 6-isopropylidene-ascorbic acid, butylated hydroxytoluene, glutathione, uric acid, one or more tocopherols, and catechins.

46. The kit according to any one of embodiments 41 to 45, wherein the selective signal inhibitor is ascorbic acid.

47. The kit of any one of embodiments 41 to 46, wherein the chemiluminescent-labeled specific binding partner comprises a chemiluminescent label linked directly or indirectly to a specific binding pair member, wherein the chemiluminescent label is selected from the group consisting of an aromatic cyclic dialdehyde hydrazide, a trihydroxyaromatic compound, a 9, 10-dihydroacridineone dithioacetal compound, a 9, 10-dihydroacridinium ester, a 9, 10-dihydroacridinium thioester, a 9, 10-dihydroacridinenol, and a compound having the formula

Wherein

R¹Selected from alkyl, alkenyl, alkynyl, aryl and aralkyl groups of 1 to 20 carbons or independently selected from carbonyl, trialkylsilyl, SO₃-、-OSO₃Sugar radical, PO₃-、-OPO₃Halogen, hydroxy, mercapto, amino, quaternary ammonium or quaternary phosphoniumAny of the foregoing groups partially substituted with 1 to 3 groups of (a);

x is selected from C1-C8 alkyl, aryl, aralkyl, alkyl or alkylcarbonyl having 1 to 20 carbon atoms, trialkylsilyl, SO₃-, a glycosyl group and PO (OR ') (OR'), wherein R 'and R' are independently selected from the group consisting of C1-C8 alkyl, cyanoalkyl, cyanoaryl, cyanoaralkyl, trialkylsilyl, alkali metal cation, alkaline earth metal cation, ammonium cation and trialkyiA cation;

Z¹and Z²Independently selected from O and S atoms; and is

R²And R³Independently selected from H and C1-C8 alkyl.

48. The kit of any one of embodiments 41 to 46, wherein the chemiluminescent-labeled specific binding partner comprises a chemiluminescent label directly or indirectly linked to a specific binding partner, wherein the chemiluminescent label has the formula

Wherein

The wavy line represents the site of attachment to the specific binding partner or to a linker linking the compound to the specific binding partner;

R¹and R²Independently selected from the group consisting of substituted alkyl, unsubstituted alkyl, substituted alkenyl, unsubstituted alkenyl, substituted alkynyl, unsubstituted alkynyl, substituted aralkyl, and unsubstituted aralkyl, wherein when R is¹Or R²When substituted, it is substituted with 1 to 3 substituents, each substituent independently selected from carbonyl, carboxyl, trialkylsilyl, -SO₃Sugar radical, -PO₃Halogen, hydroxy, mercapto, amino, C (O) NHNH₂Quaternary ammonium and quaternary phosphonium salts

R³Selected from the group consisting of substituted alkyl, unsubstituted alkyl, substituted alkenyl, unsubstituted alkenyl, substituted alkynyl, unsubstituted alkynyl, substituted aralkyl, and unsubstituted aralkyl, wherein when R is¹Or R²When substituted, it is substituted with 1 to 3 substituents, each substituent independently selected from carbonyl, carboxyl, trialkylsilyl, -SO₃Sugar radical, -PO₃Halogen, hydroxy, mercapto, amino, C (O) NHNH₂Quaternary ammonium and quaternary phosphonium salts

49. The kit of any one of embodiments 41 to 48, wherein the activator-labeled specific binding partner comprises an activator-labeling compound linked directly or indirectly to a specific binding pair member, wherein the activator-label is selected from the group consisting of a transition metal salt, a transition metal complex, and an enzyme, and wherein the activator-label has peroxidase activity.

50. The kit of any one of embodiments 41-49, wherein the activator label is a peroxidase.

51. The kit of any one of embodiments 50, wherein the activator label is horseradish peroxidase.

52. The kit of any one of embodiments 41 to 50, wherein at least one of said chemiluminescent-labeled specific binding partner and said activator-labeled specific binding partner comprises an auxiliary substance selected from the group consisting of soluble proteins, streptavidin, avidin, neutravidin, biotin, cationized BSA, fos, jun, soluble synthetic dendrimers, soluble synthetic polymers, polysaccharides, dextrans, organic nucleotides, nucleosides, nucleic acid ligands, liposomes, and micelles.

53. The kit according to any one of embodiments 42 to 52, wherein the enhancer is one or more compounds that promote the catalytic conversion of an activator having peroxidase activity.

54. The kit according to any one of embodiments 53, wherein the enhancer is selected from the group consisting of phenolic compounds, aromatic amines, benzolsOxazoles, hydroxybenzothiazoles, arylboronic acids, and mixtures of any of the foregoing.

55. The kit according to any one of embodiments 41 to 54, wherein the trigger solution comprises an oxidizing agent or a reducing agent.

56. The kit of any one of embodiments 41-55, wherein the trigger solution comprises a peroxide.

57. The kit according to any one of embodiments 56, wherein the peroxide compound is selected from the group consisting of alkyl peroxides (especially wherein the alkyl group is ethyl or methyl), alkyl hydroperoxides (especially wherein the alkyl group is ethyl or methyl), aromatic peroxides (especially benzyl peroxides), lipid hydroperoxides (especially the hydroperoxides of eicosapentaenoic acid, docosahexaenoic acid or linoleic acid), peroxy acids (e.g. m-chloroperoxybenzoic acid), hydrogen peroxide, urea peroxide, peroxycarbamates and perborates.

58. The kit according to any one of embodiments 57, wherein the peroxide is hydrogen peroxide.

59. The kit according to any one of embodiments 41 to 58, wherein the trigger solution comprises an enhancer selected from the group consisting of phenolic compounds, aromatic amines, benzolsOxazoles, hydroxybenzothiazoles, arylboronic acids, and mixtures of any of the foregoing.

60. The kit according to any one of embodiments 41 to 59, wherein all of the components of the trigger solution and the reaction mixture and the analyte are water-soluble.

61. The kit according to any one of embodiments 41 to 60, wherein none of the trigger solution, the assay solution, or the reaction mixture comprises a material that is directly or indirectly conjugated to a solid phase substance.

62. The kit according to any one of embodiments 41 to 61, wherein the selective signaling inhibitor comprises a compound selected from the group consisting of: aromatic compounds having at least two hydroxyl moieties in the ortho or para orientation, aromatic compounds having a hydroxyl moiety and an amino moiety in the ortho or para orientation, compounds having at least two vinyl hydroxyl groups and a nitrogen heterocycle.

63. The kit according to any one of embodiments 41 to 62, wherein the selective signal inhibitor comprises ascorbic acid, and wherein the ascorbic acid is in particular L-ascorbic acid.

64. The kit according to any one of embodiments 41 to 63, wherein the selective signal inhibitor comprises 6-hydroxy-2, 5,7, 8-tetramethylchroman-2-carboxylic acid.

65. The kit according to any one of embodiments 41 to 64, wherein the selective signal inhibitor comprises 2-aminophenol.

66. The kit according to any one of embodiments 41 to 65, wherein the selective signal inhibitor comprises 3-amino-tyrosine, in particular 3-amino-L-tyrosine.

67. The kit according to any one of embodiments 41 to 66, wherein the selective signal inhibitor comprises 4-chlorocatechol.

68. The kit according to any one of embodiments 41-67, wherein the selective signal inhibitor comprises a thiopheneAnd (3) an oxazine.

69. The kit according to any one of embodiments 41 to 68, wherein the selective signaling inhibitor comprises 2-bromobenzo-1, 4-diol.

70. The kit according to any one of embodiments 41 to 69, wherein the selective signaling inhibitor comprises 5, 6-isopropylidene ascorbic acid.

71. The kit according to any one of embodiments 41 to 70, wherein the selective signal inhibitor comprises 6-palmitate.

72. The kit according to any one of embodiments 41 to 71, wherein said chemiluminescent-labeled specific binding partner and said activator-labeled specific binding partner are each adapted to bind to said analyte in said sample.

73. The kit of any one of embodiments 41 to 72, wherein the chemiluminescent-labeled specific binding partner comprises a chemiluminescent-labeled compound linked to an analog of the analyte, and further wherein the analyte and the chemiluminescent-labeled specific binding partner are adapted to compete for binding to the activator-labeled specific binding partner.

74. The kit according to any one of embodiments 41 to 73, wherein the unbound chemiluminescent substrate is selected from the group consisting of cyclic hydrazides, such as luminol and isoluminol, imidazole compounds, such as lofenadine, acridine esters, such as lucigenin, and 9, 10-dihydroacridine, phthalylhydrazides, such as 2, 3-dihydro-1, 4-diketophthalazine, fluorescein, and 1, 2-dioxetane-containing compounds, such as diketones, 3- (2' -spiroadamantane) -4-methoxy-4- (3 "-phosphoryloxy) phenyl-1, 2-dioxetane (AMPPD), and 3- (2' -spiroadamantane) -4-methoxy-4- (3" -beta-D ' -galactopyranosyloxy) phenyl-1, 2-dioxetane (AMPGD), disodium 3- (4-methoxyspiro {1, 2-dioxetane-3, 2'- (5' -chloro) tricyclo [3.3.1.13,7] decane } -4-yl) phenyl phosphate and 1, 2-dioxetanedione, adamantyl-1, 2-dioxetane), in particular luminol or isoluminol, and more in particular luminol.

75. The kit according to any one of embodiments 41 to 74, wherein the unbound chemiluminescent substrate comprises luminol.

76. The kit according to any one of embodiments 41 to 75, wherein the wavelength of maximum luminescence intensity of the unbound chemiluminescent substrate is different from the wavelength of maximum luminescence intensity of the chemiluminescent label.

77. A solution phase assay method for an analyte in a sample, the solution phase assay method comprising:

a) forming an aqueous reaction mixture comprising the following components by addition to an aqueous solution in any order or simultaneously:

the samples were taken from the test piece and,

a chemiluminescent-tagged specific binding partner comprising a chemiluminescent-tagged compound directly or indirectly linked to a first specific binding partner, wherein the chemiluminescent-tagged compound is a compound of the formula

Wherein

The wavy line represents the site of attachment to the specific binding partner or to a linker linking the compound to the specific binding partner;

R¹、R²and R³Independently selected from the group consisting of substituted alkyl, unsubstituted alkyl, substituted alkenyl, unsubstituted alkenyl, substituted alkynyl, unsubstituted alkynyl, substituted aralkyl and unsubstituted aralkyl groups of 1 to 20 carbon atoms, wherein when R is¹Or R²When substituted, it is substituted with 1 to 3 substituents, each substituent independently selected from carbonyl, carboxyl, tri (C1-C8) alkylsilyl, -SO₃ ^-、-OSO₃ ^2-Sugar radical, -PO₃ ^-、-OPO₃ ^2-Halogen, hydroxy, mercapto, amino, C (O) NHNH₂Quaternary ammonium and quaternary phosphonium salts

An activator-labeled specific binding partner comprising an activator-labeled compound having peroxidase activity directly or indirectly linked to a second specific binding partner, and

a selective signal inhibitor selected from the group consisting of L-ascorbic acid, 6-hydroxy-2, 5,7, 8-tetramethylchroman-2-carboxylic acid, 2-aminophenol, 3-amino-L-tyrosine, 4-chlorocatechol, thiophenOxazine, 2-bromobenzo-1, 4-diol, 5, 6-isopropylidene ascorbic acid and ascorbic acid 6-palmitate,

wherein all of said components are soluble in said aqueous solution and none of said components are immobilized to a solid support, and

wherein the chemiluminescent-labeled specific binding partner and the activator-labeled specific binding partner bind to the analyte present in the sample to form a bound complex in the aqueous solution; and

b) adding a trigger solution comprising a peroxide compound to the aqueous reaction mixture, wherein the trigger solution releases a detectable chemiluminescent signal in the presence of the selective signal inhibitor that correlates to the amount of the analyte-bound chemiluminescent-labeled specific binding partner and the analyte-bound activator-labeled specific binding partner in the aqueous reaction mixture, and wherein the selective signal inhibitor causes the ratio of the signals produced by the reaction between the chemiluminescent labeling compound and the activator labeling compound to exceed the ratio of the signals from the reaction between the chemiluminescent labeling compound and the activator labeling compound when not in such combination;

it is characterized in that

The reaction mixture further comprises an unbound chemiluminescent substrate that produces a detectable background signal that is not related to the concentration of the analyte in the sample.

78. A kit for detecting an analyte in a sample, the kit comprising:

a first specific binding partner for the analyte;

a chemiluminescent compound conjugated to the first specific binding partner;

a second specific binding partner for the analyte;

an activator compound conjugated to the second specific binding partner;

a selective signal inhibitor; and

the trigger solution is applied to the surface of the substrate,

wherein all of the aforementioned components of the kit are soluble in an aqueous solution,

wherein the chemiluminescent compound is a 9, 10-dihydroacrylketene dithioacetal compound having the formula

Wherein R is₁And R₂Each of which is selected from H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, aralkyl or substituted aralkyl when R is₁And R₂When substituted, it is most commonly substituted with 1 to 3 groups selected from carbonyl, carboxyl, tri (alkyl) silyl, glycosyl, -SO₃ ^-、-OSO₃ ^-、-PO₃ ^-、-OPO₃ ^-Halogen, hydroxy, mercapto, amino, quaternary ammonium and quaternary phosphonium salts

R₃Selected from H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, aralkyl or substituted aralkyl,

R₄to R₁₁Each of which is independently H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, aralkyl, substituted aralkyl, alkoxy, aryloxy, halogen, amino, substituted amine, carboxyl, alkoxycarbonyl, carboxamide, cyano, or sulfonic acid, wherein pairs of adjacent R are₄To R₁₁The moieties may be covalently bonded to form a five to seven membered carbocyclic or heterocyclic ring,

wherein the selective signal inhibitor is selected from glutathione, ascorbic acid, especially L-ascorbic acid, ascorbate, especially L-ascorbate, uric acid, L-ascorbic acid-6-palmitate, tocopherol, 5, 6-isopropylidene-L-ascorbic acid, erythorbic acid, including D-erythorbic acid, L-erythorbic acid, or both, sodium sulfite, diethylhydroxylamine, BHT, and combinations of the foregoing.

79. The kit of embodiment 78, wherein the chemiluminescent labeling compound is a compound of the formula

Wherein

The wavy line represents the site of attachment to the specific binding partner or to a linker linking the compound to the specific binding partner;

R¹、R²and R³Independently selected from the group consisting of substituted alkyl, unsubstituted alkyl, substituted alkenyl, unsubstituted alkenyl, substituted alkynyl, unsubstituted alkynyl, substituted aralkyl and unsubstituted aralkyl groups of 1 to 20 carbon atoms, wherein when R is¹Or R²When substituted, it is substituted with 1 to 3 substituents, each substituent independently selected from carbonyl, carboxyl, tri (C1-C8) alkylsilyl, -SO₃ ^-、-OSO₃ ^2-Sugar radical, -PO₃ ^-、-OPO₃ ^2-Halogen, hydroxy, mercapto, amino, C (O) NHNH₂Quaternary ammonium and quaternary phosphonium salts

Examples

Material

Dipotassium ethylenediaminetetraacetate (dipotassium EDTA), p-coumaric acid, tween 20, Tris base, Tris hydrochloride, sodium ascorbate, and lucigenin were purchased from Sigma Aldrich Corporation of st louis, missouri, usa.

Hydrogen peroxide (30%, analytical grade of beck) was purchased from VWR International, Radnor, PA, usa.

Ethanol (200 standard) was purchased from Decon Labs Incorporated, King of Prussia, PA.

PVP360[ poly (vinylpyrrolidone) with an average molecular weight of 360,000 ] and PEG-6000[ poly (ethylene glycol) with an average molecular weight of 6000 ] were purchased from Sigma Aldrich Corporation.

The bovine IgG SPARCL assay kit was purchased from Life Diagnostics, Inc., West Chester, Pa., West Diagnostics, West, Inc., of Wechester, Pa., USA. Bovine IgG standard solutions were prepared in PBS according to the solution formulations described in table 1.

A bovine IgG standard + lucigenin solution was prepared by mixing 20 parts by volume of a bovine IgG standard solution (table 1) with 1 part by volume of a saturated solution of lucigenin in deionized water.

Solutions containing (anti-bovine IgG) -HRP conjugate were prepared according to assay kit instructions.

Solutions containing 9, 10-dihydroacridine labeled anti-bovine IgG were prepared according to assay kit instructions and additionally supplemented with sodium ascorbate (200. mu.M).

Horse serum (16050-. Test samples with horse serum were prepared at dilution concentrations of 1e-1, 1e-2, 1e-3, 1e-4 and 1e-5 (vol/vol) in PBS.

TABLE 1 bovine IgG Standard solution

The trigger solution of example 1 was prepared as an aqueous solution of the components listed in table 2.

TABLE 2 composition of trigger solution

Components	Concentration in trigger solution (g/L)
		P-coumaric acid	1.31
EDTA dipotassium salt	0.40
		Tween 20	2.00
Hydrogen peroxide (30%)	11.33
		Ethanol (200 standard)	32.00
Tris base	1.33
		Tris HCl salt	2.22

Cortisol SPARCL assay kit was purchased from Life Diagnostics, Inc. (Life Diagnostics Incorporated).

A cortisol standard solution is prepared using a cortisol stock solution and a diluent provided in the assay kit.

A solution containing cortisol-HRP conjugate was prepared by mixing cortisol-HRP conjugate (781 microliters) from the kit with 249 microliters of diluent from the kit.

A solution containing the anti-cortisol 9, 10-dihydroacridine conjugate was prepared by mixing the anti-cortisol 9, 10-dihydroacridine conjugate (2.5 microliters) from the kit with 247.5 microliters of diluent from the kit.

Computing

The Scale Factor (SF) is determined by dividing the reported background signal for the standard sample by the reported background signal for the test sample.

A corrected analyte signal for the test sample is calculated by multiplying the analyte signal of the test sample by a Scaling Factor (SF).

Example 1

Test samples were prepared in a reflex-white 96-well plate by first adding a diluted sample of horse serum (20 microliters) to the wells in the plate. Each well contained a single test sample, and each test sample was tested in triplicate. For wells containing standard samples, 20 μ l of PBS solution was used (3 replicates). Next, 20 microliters of bovine IgG standard solution + lucigenin solution was added to each well and the plate was shaken at 300rpm for 30 seconds. An anti-bovine IgG-HRP conjugate solution (20 microliters) was added to each well and the plate was shaken at 300rpm for 30 seconds. A solution of 9, 10-dihydroacridine-labeled anti-bovine IgG conjugate (20. mu.l) was then added to each well and the plate was shaken at 300rpm for 30 seconds. 30 microliters of trigger solution was added to each well using a 3M MLS II injection photometer (available from 3M Corporation, Maplewood, MN, Maplewood, MN, usa). The instrument is set to read and integrate the stroboscopic luminescence signal (analyte signal of either the test sample or the standard sample) simultaneously for three seconds. After recording the stroboscopic light-emitting signal (relative light units, RLU), a second light-emitting signal (background signal of the test sample or standard sample) is recorded during a time period of 60 to 70 seconds after injection (integration time of one second). The RLU value of the second signal is reported as the average of the values recorded over a 10 second time frame. In tables 3 to 7, the background signal (RLU) of the test sample, the mean background signal (RLU) of the standard sample, the analyte signal (RLU) of the test sample, the mean analyte signal (RLU) of the standard sample, the calculated scaling factor and the corrected analyte signal (RLU) value of the test sample are reported for each of the replicate samples. The determination of whether the corrected analyte signal for each test sample is within ± 20% of the analyte signal of the corresponding standard sample is also recorded in the table.

For the three wells containing standard samples, the individual background signals were 643RLU, 748RLU, and 853 RLU. The scaling factor was calculated using the mean value of 748RLU of the background signal of the standard sample (n-3). The mean analyte signal (RLU) for the standards was calculated as 38015RLU (with three individual standard analyte signals being 35903RLU, 38075RLU, and 40067 RLU).

TABLE 3 corrected analyte Signal for test samples (IgG) with 1e-5 horse serum as added interferent Measurement of (2)

TABLE 4 corrected analyte Signal for test samples (IgG) with 1e-4 horse serum as added interferent Measurement of (2)

TABLE 5 corrected analyte Signal for test samples (IgG) with 1e-3 horse serum as added interferent Measurement of (2)

TABLE 6 corrected analyte Signal for test samples (IgG) with 1e-2 horse serum as added interferent Measurement of (2)

TABLE 7 corrected analyte Signal for test samples (IgG) with 1e-1 horse serum as added interferent Measurement of (2)

Comparative example A

The same procedure as described in example 2 was followed except that the bovine milk IgG standard solution + the lucigenin solution (20 microliters) was replaced with a bovine milk IgG standard solution (20 microliters). In tables 8 to 12, the analyte signal (RLU) of the test sample and the average analyte signal (RLU) of the standard sample are reported for each of the replicate samples. The mean analyte signal (RLU) for the standard was calculated as 37694RLU (where the three individual standard analyte signals were 36955RLU, 37062RLU, and 39066 RLU). The determination as to whether the analyte signal of each test sample is within ± 20% of the analyte signal of the corresponding standard sample is also recorded in the table.

TABLE 8 analyte signals of test samples and of standard samples with 1e-5 horse serum as added interferent Comparison of analyte signals

TABLE 9 analyte signals of test samples and of standard samples with 1e-4 horse serum as added interferent Comparison of analyte signals

TABLE 10 analyte signals of test samples and of standard samples with 1e-3 horse serum as added interferent Analyte signalComparison of

TABLE 11 analyte signals of test samples and of standard samples with 1e-2 horse serum as added interferent Comparison of analyte signals

TABLE 12 analyte signals of test samples and of standard samples with 1e-1 horse serum as added interferent Comparison of analyte signals

Comparative example B

Test samples of horse serum (dilutions 1e-1 to 1 e-5) were prepared as described above. The standard sample was PBS solution without added horse serum. The test samples and standard samples were analyzed using a bovine IgG ELISA kit (catalog No. IgG-11, available from Life Diagnostics Incorporated) according to the manufacturer's instructions. The absorbance value of each well was determined using a Bio Tek POWERWAVE 340 plate reader (Bio Tek Instruments Incorporated, Winooski, VT) from Wenksky, Vernonia, U.S.A.) (A450). The absorbance (a450) values for each replicate (test and standard) are reported in table 13 and compared to the average a450 value calculated for the standard. The average a450 value of the three standard samples was 0.551.

Table 13.

Example 2

The dry feed corn was ground using a Romer series II grinder (Romer Labs, getterdorf, Austria) and passed through a #20 sieve. Screened corn (2g) was added to 8mL of buffer from the cortisol SPARCL assay kit (available from Life Diagnostics Incorporated). The resulting slurry was mixed by repeated inversion for 3 minutes. The supernatant from the sample was filtered through a 0.45 micron syringe filter (Thermo Fischer Scientific, Waltham MA USA) by seimer femtolite technologies, Waltham USA. Five series of filtrates were prepared using the buffer from the assay kit. (2x, 4x, 6x, 8x and 10 x). Eight individual test samples were prepared for the undiluted sample and each dilution series. Each test sample was doped with different concentrations of cortisol (25nM, 12.5nM, 6.25nM, 3.13nM, 1.56nM, 0.78nM, 0.39nM or 0.20 nM). A corresponding series of standard samples were prepared from cortisol solution standards. Lucigenin (5 microliters of a 10mg/mL aqueous solution) was added to each sample (test sample and standard sample).

For competitive format assays, samples were prepared in 96-well plates provided in the kit by first adding cortisol-doped samples (50 microliters) to wells in the plate. Each well contains a single test sample or standard sample. Next, 25 microliters of cortisol-HRP conjugate solution was added to each well and the plate was shaken at 300rpm for 30 seconds. An anti-cortisol-9, 10-dihydroacridine conjugate solution (25 microliters) was then added to each well and the plate was shaken at 300rpm for 30 seconds.

30 microliters of trigger solution (from the assay kit) was added to each well of the assay plate using a Turner BioSystems TD 20/20n luminometer (Promega Corporation, Madison, WI, Madison, usa). The instrument was set to read and integrate the luminescence signal simultaneously for sixty seconds (integration time 0.1 second). The analyte signal (of the test sample or of the standard sample) is measured during the first 20 seconds and averaged. After recording the flash emission signal, a second emission signal (background signal of the test sample or the standard sample) is recorded during a period of 58 seconds and 59 seconds and averaged.

In tables 14 to 19, the background signal (RLU) of the test sample, the background signal (RLU) of the standard sample, the analyte signal (RLU) of the test sample, the analyte signal (RLU) of the standard sample, the calculated scale factor and the corrected analyte signal (RLU) value of the test sample are reported for each sample.

The determination as to whether the corrected analyte signal for each test sample is within ± 7.5% of the analyte signal of the corresponding standard sample is recorded in table 26.

TABLE 14 corrected fractions of test samples (Cortisol) with 10 Xdilution of corn extract as interferent Determination of analyte Signal (RLU)

TABLE 15 corrected analysis of test samples (Cortisol) with 8 Xdilution of corn extract as interferent Determination of substance Signal (RLU)

TABLE 16 corrected analysis of test samples (Cortisol) with 6 Xdilution of corn extract as interferent Determination of substance Signal (RLU)

TABLE 17 corrected analysis of test samples (Cortisol) with 4 Xdilution of corn extract as interferent Determination of substance Signal (RLU)

TABLE 18 corrected analysis of test samples (Cortisol) with 2 Xdilution of corn extract as interferent Determination of substance Signal (RLU)

TABLE 19 corrected analysis of test samples (Cortisol) with undiluted corn extract as interferent Determination of substance Signal (RLU)

Comparative example C

The same procedure as described in example 2 was followed except that no shine essence was added to any of the test samples. In tables 20 to 25, the analyte signal (RLU) of the test sample and the analyte signal (RLU) of the standard sample for each sample are reported. The determination as to whether the analyte signal of each test sample is within ± 7.5% of the analyte signal of the corresponding standard sample is recorded in table 27.

TABLE 20 analyte Signal of test samples (Cortisol) with 10 Xdilution of corn extract as interferent Comparison of analyte signals with a Standard sample (Cortisol)

TABLE 21 analyte signals and of test samples (Cortisol) with 8 Xdilution of corn extract as interferent Comparison of analyte signals for Standard samples (Cortisol)

TABLE 22 analyte signals and of test samples (Cortisol) with 6 Xdilution of corn extract as interferent Comparison of analyte signals for Standard samples (Cortisol)

TABLE 23 analyte signals and of test samples (Cortisol) with 4 Xdilution of corn extract as interferent Comparison of analyte signals for Standard samples (Cortisol)

TABLE 24 analyte signals and of test samples (Cortisol) with 2 Xdilution of corn extract as interferent Comparison of analyte signals for Standard samples (Cortisol)

TABLE 25 analyte signals and of test samples (Cortisol) with undiluted corn extract as interferent Comparison of analyte signals for Standard samples (Cortisol)

TABLE 26 results of example 2

TABLE 27 results of comparative example C

Example 3

The effect of lucigenin (unbound chemiluminescent substrate) on solution phase luminescence assays was evaluated. The cortisol SPARCL assay (Life Diagnostics Incorporated) was used in a competitive format. Four different samples a to D were prepared. In sample a, a cortisol standard (50 μ l) was prepared with a cortisol concentration of 25nM using the diluent from the assay kit. In sample B, a control standard (50 μ l) was prepared without cortisol (i.e. only the diluent of sample a). In sample C, 50 microliters of a cortisol standard sample was prepared as in sample A and supplemented with 5 microliters of a 10mg/mL aqueous lucigenin solution. In sample D, 50 microliters of a control standard sample was prepared as in sample B and supplemented with 5 microliters of a 10mg/mL aqueous lucigenin solution. Samples a to D (50 microliters each) were analyzed individually according to the manufacturer's instructions provided with the assay kit. A Turner BioSystems TD 20/20n luminometer (Promega Corporation, Madison, WI, Madison, usa) was used to add a trigger solution (100 μ L) to each well of the assay plate. The instrument was set to read the luminescence signal at time points of 1 second, 2 seconds, 5 seconds, 10 seconds, 15 seconds, 20 seconds, 30 seconds, 40 seconds, and 60 seconds after the addition of the trigger solution. Each reported luminescence value (RLU) is recorded as the average of the first ten 0.1 second integrations. The results are reported in table 28.

Table 28.