Peroxidase chemiluminescent substrate with extended shelf life

Abstract

A kit for performing an analysis for determining an analyte in a sample, the kit having an extended shelf life, wherein the kit comprises a chemiluminescent cyclic dihydrazide, an enhancer, a co-enhancer and a peroxide oxidant. The kit can be used in blot analysis and immunoassay for detecting protein and nucleic acid molecules.

Description

Technical Field

[0001] The present invention relates to a chemiluminescent substrate for use in assaying peroxidase in an assay for assaying an analyte in a sample, wherein the chemiluminescent substrate has an extended shelf life. Background Art

[0002] The chemiluminescent oxidation of luminol catalyzed by horseradish peroxidase (HRP) is widely used in analytical tests for antigens, antibodies and nucleic acids, especially blot tests such as dot blots and western blots (proteins), Southern blots and Northern blots (nucleic acids) and enzyme-linked immunosorbent assays (EIA, for proteins or nucleic acids).

[0003] It is known that the HRP-catalyzed chemiluminescent oxidation of luminol can be made faster and more efficient by adding electron mediators or enhancers, as described, for example, in Kricka LJ (1991), Clinical Chemistry, 37: 1472-1481 or Kricka LJ, Voyta JC and Bronstein I in "Chemiluminescent Methods for Detecting and Quantitating Enzyme Activity" (2000), Methods Enzymol; 305: 370-390.

[0004] The mechanism of the enhanced chemiluminescence reaction (ECL) in which luminol and an enhancer are oxidized simultaneously has been described as follows [Lind J, Merenyi G and Eriksen TE (1983), J Am Chem Soc, 105:7655-7661]. In the first step of ECL, the enhancer (E), which is a substrate for HRP with a higher activity than luminol, is oxidized by hydrogen peroxide in the presence of HRP according to a "ping-pong" mechanism:

[0005] HRP+H2O2→HRP-I (1)

[0006] HRP-I+E→HRP-II+E ● (2)

[0007] HRP-II+E→HRP+E ● (3)

[0008] Where E is the enhancer, E ● is the free radical product of the one-electron oxidation of the enhancer. HRP is horseradish peroxidase in the Fe(III) resting state. HRP-I and HRP-II are the oxidative intermediates of peroxidase, which are two and one oxidation equivalents higher than the resting state, respectively. Then, the free radical product of the enhancer (E● ) and the luminol molecule (LH - ) reacts reversibly [Easton PM, Simmonds AC, Rakishev A, Egorov AM and Candeias LP (1996), J Am Chem Soc; 118: 6619-6624]:

[0009]

[0010] Thermodynamically, the position of the redox equilibrium (4) is determined by the difference between the reduction potentials of the enhancer and the luminol radical under the experimental conditions. Once formed, the two luminol radicals (L ●- ) is disproportionated to luminol anion (LH - ) and diazaquinone intermediate (L):

[0011] 2L ●- →LH - +L(5)

[0012] The diazaquinone intermediate (L) reacts with hydrogen peroxide to generate luminol peroxide (LO2 2- ), which collapses into an excited state of 3-aminophthalic acid ([AP 2- ]*); [AP 2- ]*Then return to the ground state (AP 2- ) and emits 425nm photons (hν):

[0013] L+H2O2→LO2 2- →[AP 2- ]*+N2→AP 2- +hν (6)

[0014] The intensity of the emitted light is related to the luminol free radical (L ●- ) is proportional to the square of the rate of generation. This quadratic relationship is the result of the excited species generation mechanism, which involves the disproportionation of two luminol radicals (reaction 5). In turn, the rate of generation of luminol radicals is given by the enzyme conversion rate, which after redox equilibrium (4) gives luminol radicals (L ●- ). The enzyme conversion rate is controlled by the rate-limiting step (reduction of HRP-II to orthoferroenzyme, reaction (3)). In summary, the enhancement of chemiluminescence can be approximately described by considering (a) the acceleration of enzyme conversion by the reaction of enhancer with HRP-II and (b) the reversible electron transfer reaction between enhancer radicals and luminol.

[0015] Many compounds have been successfully used to enhance HRP-induced chemiluminescence, including fluorescein, 6-hydroxybenzotriazoles, p-iodophenol, p-coumaric acid and other phenolic enhancers (Thorpe GHG and Kricka LJ (1986) Methods Enzymol 133:331); aromatic amines (U.S. Pat. No. 4,279,950); acetanilides (European Patent Application No. 603953); N-substituted phenothiazines (U.S. Pat. No. 5,171,688 and U.S. Pat. No. 6,432,662); and boric acids (U.S. Pat. No. 5,629,168).

[0016] In addition, as described in U.S. Pat. No. 7,803,573, further greater enhancement of chemiluminescent light emission is obtained with certain 4-dialkylaminopyridines, such as 4-morpholino-pyridine (MORP), 4-dimethylaminopyridine (DMAP) and 4-pyrrolidinylpyridine (PPY). These compounds belonging to the 4-aminopyridine class provide further enhancement of light output only when used in combination with primary electron transfer enhancers. Therefore, they can be described as secondary enhancers or co-enhancers, as described in Vdovenko MM, Della Ciana L and Sakharov IY (2009) Anal Biochem 392:54. Addition of 4-dialkylaminopyridines to a substrate solution containing an SPTZ enhancer significantly increases the rate of generation of SPTZ cation radicals, reactions (2) and (3), thereby increasing the HRP conversion rate, as described in Sakharov IY and Vdovenko MM (2013) Anal Biochem 434:12. Another group of compounds useful as co-enhancers is described in US 9,040, 252. These co-enhancers belong to the class of N-azoles, the most useful being imidazole, 1-methyl-imidazole, 1,2,3-triazole and 1,2,4-triazole.

[0017] In practice, the user mixes two separate, previously prepared buffer solutions, one containing luminol and an enhancer, and the other containing a peroxide oxidant. While the two separate solutions can be stored for long periods of time, the final "working solution" obtained by mixing the two partial solutions has limited stability, ranging from a few hours to a few days at most.

[0018] It would therefore be desirable to eliminate the need to mix the individual components when conducting a testing procedure without degrading or compromising the performance of the substrate.

[0019] Previous attempts to provide intact peroxidase substrates with extended storage stability have had very limited success. For example, Giri disclosed a formulation with extended stability in U.S. Patent No. 6,602,679, which includes an enhancer and a peroxide stabilizer. However, no stability data was reported. Similarly, Woerner described the use of stannous halides as stabilizers for hydrogen peroxide under alkaline conditions for chemiluminescent, fluorescent and colorimetric detection of peroxidase-based assays in U.S. Patent Application 2007 / 0264647. Although it is claimed that "by stabilizing and / or enhancing hydrogen peroxide in a buffer system, the composition used for such analysis can have a higher shelf life", no experimental evidence is provided.

[0020] Therefore, despite those previously reported, there remains a need for improved substrate formulations for enhanced chemiluminescent detection of peroxidases, in which all components are mixed in a buffered solution and have an extended shelf life. Summary of the invention

[0021] An object of the present invention is to provide a novel chemiluminescent substrate for quantitative determination of peroxidase in an assay for detecting an analyte in a sample, wherein the chemiluminescent substrate has an extended shelf life.

[0022] According to the invention, the above objects are achieved by the kit specified in the subsequent claims, which are to be understood as forming an integral part of the present description.

[0023] According to one embodiment, the present disclosure relates to a kit for performing an assay for determining an analyte in a sample, wherein the kit comprises a chemiluminescent cyclic diacylhydrazide, a peroxide oxidant, an enhancer, and a co-enhancer, wherein the enhancer is an anionic N-alkylphenoxazine, and the co-enhancer is selected from 4-dialkylaminopyridine and N-azole.

[0024] According to another embodiment, the present disclosure relates to a method for performing a chemiluminescent assay for detecting an analyte in a sample, wherein the method employs a kit disclosed herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The present invention will now be described, by way of example only, with reference to the accompanying drawings, in which:

[0026] - Figure 1 The structure of the cyclic dihydrazide of the present invention is shown.

[0027] - Figure 2 Shown are the structures of anionic N-alkylphenoxazine enhancers of the present invention.

[0028] - Figure 3A graph showing the oxidation kinetics of SPTZ.

[0029] - Figure 4 A diagram showing the oxidation kinetics of SPOX.

[0030] - Figure 5 The correlation of the chemiluminescent signal with the SPOX concentration in the luminol / perborate / SPOX substrate is shown.

[0031] - Figure 6 The pH dependence of the chemiluminescent signal in the luminol / perborate / SPOX substrate is shown.

[0032] - Figure 7 The correlation of the chemiluminescent signal with the MORP concentration in the luminol / perborate / SPOX / MORP substrate is shown.

[0033] - Figure 8 The correlation of the chemiluminescent signal with the imidazole concentration in the luminol / perborate / SPOX / imidazole substrate is shown.

[0034] - Fig. 9 The pH dependence of the chemiluminescent signal in the luminol / perborate / SPOX / MORP and luminol / perborate / SPOX / imidazole substrates is shown.

[0035] - Fig.10 The correlation of the chemiluminescent signal to the SPOX concentration in the L-012 / perborate / SPOX substrate is shown.

[0036] - Fig.11 The pH dependence of the chemiluminescent signal in the L-012 / perborate / SPOX substrate is shown.

[0037] - Fig.12 The correlation of the chemiluminescent signal with the MORP concentration in the L-012 / perborate / SPOX / MORP substrate is shown.

[0038] - Fig.13 Shown are Western blots of human IKBα obtained with luminol / SPOX and L-012 / SPOX substrates vs. luminol / SPTZ substrate.

[0039] - Fig.14 Shown are Western blots of human IKBα obtained with luminol / SPOX and L-012 / SPOX substrates vs. luminol / SPTZ substrate.

[0040] - Fig.15 Shown is a graph of the chemiluminescent signal of the substrate L / SPOX6 prepared as described in Example 11 versus storage time at 4°C.

[0041] - Fig.16 Shown is a graph of the chemiluminescent signal of the substrate L012 / SPOX prepared as described in Example 11 versus storage time at 4°C. DETAILED DESCRIPTION

[0042] In the following description, numerous specific details are given to provide a thorough understanding of the embodiments. The embodiments may be implemented without one or more of the specific details, or may be implemented using other methods, components, materials, etc. In other cases, well-known structures, materials, or operations are not shown or described in detail to avoid obscuring various aspects of the embodiments.

[0043] Reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the phrases "in one embodiment" or "in an embodiment" appearing in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0044] The headings provided herein are for convenience only and do not interpret the scope or meaning of the embodiments.

[0045] The present invention relates to a kit for performing an analysis for determining an analyte in a sample, wherein the kit comprises a chemiluminescent dihydrazide, a peroxide oxidant, an enhancer and a co-enhancer, wherein the enhancer is an anionic N-alkylphenoxazine, and the co-enhancer is selected from 4-dialkylaminopyridine and N-azole.

[0046] In one embodiment, the chemiluminescent dihydrazide is selected from luminol, isoluminol, 5-amino-6,8-dimethyl-2,3-dihydrophthalazine-1,4-dione (DML), 5-amino-6,8-diethyl-2,3-dihydrophthalazine-1,4-dione (DEL), and 8-amino-5-chloro-7-phenylpyrido[3,4-d]pyridazine-1,4(2H,3H)-dione (L-012). The chemical structures of these chemiluminescent dihydrazides are shown in Figure 1 Preferably, the chemiluminescent dihydrazide is selected from luminol and 8-amino-5-chloro-7-phenylpyrido[3,4-d]pyridazine-1,4(2H,3H)-dione (L-012).

[0047] In one embodiment, the anionic N-alkylphenoxazines are selected from 3-(10H-phenoxazin-10-yl)propane-1-sulfonic acid (SPOX), 4-(10H-phenoxazin-10-yl)butane-1-sulfonic acid (SBOX), 3-(10H-phenoxazin-10-yl)propionic acid (CPOX) and 4-(10H-phenoxazin-10-yl)butanoic acid (CBOX) and salts thereof. The chemical structures of these N-alkylphenoxazines are shown in Figure 2 Preferably, the anionic N-alkylphenoxazine is selected from 3-(10H-phenoxazine-10-yl)propane-1-sulfonic acid (SPOX) and 3-(10H-phenoxazine-10-yl)propionic acid (CPOX).

[0048] The co-enhancers of the present invention belong to two different classes of compounds, 4-dialkylaminopyridines (as described in US7,803,573) and N-azoles (as described in US9,040,252). Among the 4-dialkylaminopyridines, preferred compounds are 4-morpholino-pyridine (MORP), 4-dimethylaminopyridine (DMAP) and 4-pyrrolidinylpyridine (PPY). Among the N-azoles, preferred compounds are imidazole and 1-methyl-imidazole.

[0049] The peroxide oxidant according to the present invention can be hydrogen peroxide or any complex of hydrogen peroxide (which releases hydrogen peroxide when dissolved in an aqueous solution), such as perborate, percarbonate or urea / hydrogen peroxide complex in a molar ratio of 1: 1. In a preferred embodiment, the peroxide oxidant is selected from hydrogen peroxide, urea / hydrogen peroxide complex, perborate, percarbonate. Preferably, perborate and percarbonate are alkali metal or alkaline earth metal salts.

[0050] In a further embodiment, the kit further comprises a peroxidase.

[0051] The peroxidase is any peroxidase suitable for chemiluminescent analysis. According to one embodiment, the peroxidase is selected from horseradish peroxidase (eg Sigma type VIA or IX) or anionic peroxidases such as soybean peroxidase and sweet potato peroxidase.

[0052] The peroxidase may or may not be conjugated to a detection reagent for the analyte to be detected.The peroxidase (or its conjugate) may be added by the end user.

[0053] The limited storage stability of the peroxidase enhanced chemiluminescent substrate can be explained by two main reasons: (1) oxidation of the enhancer; (2) decomposition of hydrogen peroxide. Of these two reasons, the first, oxidation of the enhancer, is the most important. The other components of the substrate, such as the cyclic dihydrazide, the co-enhancer and the buffer are also generally stable to oxidation by molecular oxygen or peroxide, even in alkaline solutions.

[0054] Oxidation of the enhancer can occur by reaction with molecular oxygen or peroxides. These oxidation reactions generally accelerate with increasing pH and are catalyzed by trace metal ions, especially iron and copper.

[0055] Certain types of enhancers, such as phenolic enhancers, are even rapidly oxidized by molecular oxygen. In fact, solutions containing luminol and phenolic enhancers must be stored at 4°C, since they lose most of their activity if stored at room temperature for even one day.

[0056] N-alkylphenothiazines, such as 3-(10′-phenothiazinyl)propane-1-sulfonic acid (SPTZ), are stable to oxidation by molecular oxygen for a long time even in alkaline solutions maintained at room temperature. However, they decompose slowly in the presence of peroxides ( Figure 3 ). A simultaneous decrease in enhancer and peroxide concentrations resulted in a decrease in performance.

[0057] In contrast, the present inventors have found that anionic N-alkylphenoxazine enhancers are extremely stable to oxidation by peroxides even in alkaline solutions ( Figure 4 ).

[0058] This finding strongly suggests that the main factor affecting the storage stability of enhanced chemiluminescent substrates is the stability to oxidation of the enhancer itself. In contrast, Giri in U.S. Patent No. 6,602,679 and Woerner in U.S. Patent Application 2007 / 0264647 only rely on the addition of peroxide stabilizers to improve the storage stability of enhanced chemiluminescent substrates, while ignoring the role of enhancers.

[0059] On this basis, the present inventors developed chemiluminescent substrates in which anionic N-alkyl phenothiazines as enhancers were replaced by anionic N-alkyl phenoxazine, Examples 2-10. Example 11 discloses an optimized formulation of chemiluminescent substrates based on luminol or L-012, in which 3-(10′-phenoxazinyl)propane-1-sulfonic acid (SPOX) was used as an enhancer, and MORP or imidazole was used as a co-enhancer. Fig.13 ) and Western blot immunoassay ( Fig.14 ), the performance of these substrates was similar to or better than the reference substrates with SPTZ as enhancer, L / SPTZ1 and L / SPTZ2 (Example 2).

[0060] The stability study of the substrate of Example 11 was carried out at 4°C and 23°C. The initial chemiluminescent signal was recorded immediately after mixing all components. The results are shown in Tables I and Fig.13(4°C) and Table II (23°C. In both cases, the substrate containing SPOX as enhancer was much more stable than the substrate containing SPTZ.

[0061] Table I

[0062]

[0063] Table II

[0064]

[0065] The most promising substrates were monitored for longer periods of time. Fig.15 The results for L / SPXO6 are summarized. After storage for 211 days (7 months) at 4°C, the signal for this substrate decreased by about 15%. Even better storage stability was found for the L012 / SPOX substrate, see Fig.16 In this case, after 585 days (nearly 20 months) of storage at 4°C, a signal drop of only 8% was observed.

[0066] In one embodiment, the kit comprises a chemiluminescent cyclic dihydrazide, a peroxide oxidant, an enhancer and a co-enhancer, wherein the chemiluminescent cyclic dihydrazide is present in a first vial, and the peroxide oxidant is present in a second vial, and wherein the enhancer and co-enhancer are present in the first vial or the second vial or both. The second vial containing the peroxide oxidant does not contain any stabilizer for the peroxide oxidant. The kit reagents are present in the vials in a liquid or solid state. If the kit reagents are solid, they can be present in the form of powders or tablets and must be reconstituted in a liquid phase by adding water or a buffer solution when used. Enhancers and co-enhancers, as well as other additives such as chelating agents and stabilizers, can be added to the chemiluminescent cyclic dihydrazide or peroxide oxidant vials or both, provided that the stabilizer is not a stabilizer for the peroxide oxidant. The two vials may also contain a buffer substance, which generates a chemiluminescent substrate or "working solution" having a pH value suitable for performing the analysis when mixed. The working solutions obtained by mixing the above listed compounds are stable for a long time (typically 6 months or more) and do not contain any stabilizers for peroxide oxidizing agents.

[0067] In one embodiment, the chemiluminescent cyclic dihydrazide, the peroxide oxidant, the enhancer, the co-enhancer are formulated in a single vial, and any stabilizer for the peroxide oxidant is absent.

[0068] The peroxidase or its conjugate (if present in the kit) is not stored together with its substrate component (ie, the peroxide oxidizing agent), but rather in a separate vial.

[0069] According to further embodiments, the present disclosure relates to a method for performing a chemiluminescent assay for detecting an analyte in a sample.

[0070] The term "analysis" refers to the detection, semi-quantification and quantification of an analyte. Typically, the implementation of the analysis requires correlating the light output with the amount of peroxidase used. Thus, light emission is detected or measured such that the presence and / or amount of the analyte is correlated with the generation of light.

[0071] According to the present disclosure, the implementation of the analysis requires that the light output generated by the chemiluminescent reaction of the chemiluminescent cyclic dihydrazide, enhancer, co-enhancer and peroxide oxidant reacted with the peroxidase is correlated with the amount of any reaction partner (i.e., any one of the chemiluminescent cyclic dihydrazide, peroxidase, peroxide oxidant, enhancer and co-enhancer). According to a preferred embodiment, the light output is correlated with the amount of peroxidase used.

[0072] The method for performing a chemiluminescent assay for detecting an analyte in a sample comprises the following steps:

[0073] (i) obtaining a chemiluminescent substrate by mixing together a chemiluminescent cyclic dihydrazide, an enhancer, a co-enhancer and a peroxide oxidant, wherein the enhancer is an anionic N-alkylphenoxazine, and the co-enhancer is selected from 4-dialkylaminopyridine and N-oxazole, and wherein the chemiluminescent substrate does not contain any stabilizer for the peroxide oxidant,

[0074] (ii) reacting a solid support on which the target analyte has been immobilized with a chemiluminescent substrate,

[0075] (iii) adding a detection reagent for the target analyte to the solid support, wherein the detection reagent is conjugated to a peroxidase, and

[0076] (iv) recording the output light signal generated by the chemiluminescent reaction of the peroxidase with the chemiluminescent substrate, which signal is correlated with the presence / amount of the target analyte.

[0077] In one embodiment, the chemiluminescent dihydrazide is selected from luminol, isoluminol, 5-amino-6,8-dimethyl-2,3-dihydrophthalazine-1,4-dione (DML), 5-amino-6,8-diethyl-2,3-dihydrophthalazine-1,4-dione (DEL) and 8-amino-5-chloro-7-phenylpyrido [3,4-d] pyridazine-1,4 (2H, 3H) -dione (L-012). Preferably, the chemiluminescent dihydrazide is selected from luminol and 8-amino-5-chloro-7-phenylpyrido [3,4-d] pyridazine-1,4 (2H, 3H) -dione (L-012).

[0078] In one embodiment, the anionic N-alkylphenoxazines are selected from 3-(10H-phenoxazin-10-yl)propane-1-sulfonic acid (SPOX), 4-(10H-phenoxazin-10-yl)butane-1-sulfonic acid (SBOX), 3-(10H-phenoxazin-10-yl)propionic acid (CPOX) and 4-(10H-phenoxazin-10-yl)butanoic acid (CBOX) and salts thereof. Preferably, the anionic N-alkylphenoxazines are selected from 3-(10H-phenoxazin-10-yl)propane-1-sulfonic acid (SPOX) and 3-(10H-phenoxazin-10-yl)propionic acid (CPOX).

[0079] Among the 4-dialkylaminopyridines, preferred co-enhancers are 4-morpholino-pyridine (MORP), 4-dimethylaminopyridine (DMAP) and 4-pyrrolidinylpyridine (PPY). Among the N-azoles, preferred co-enhancers are imidazole and 1-methyl-imidazole.

[0080] The peroxide oxidant according to the present invention can be hydrogen peroxide or any complex of hydrogen peroxide (which releases hydrogen peroxide when dissolved in an aqueous solution), such as perborate, percarbonate or urea / hydrogen peroxide complex in a molar ratio of 1: 1. In a preferred embodiment, the peroxide oxidant is selected from hydrogen peroxide, urea / hydrogen peroxide complex, perborate, percarbonate. Preferably, perborate and percarbonate are alkali metal or alkaline earth metal salts.

[0081] The peroxidase is any peroxidase suitable for use in chemiluminescent assays. According to one embodiment, the peroxidase is selected from horseradish peroxidase (eg Sigma VIA or IX type) or anionic peroxidases such as soybean peroxidase and sweet potato peroxidase.

[0082] According to one embodiment, the concentration of the chemiluminescent cyclic dihydrazide in the chemiluminescent substrate is between 0.01 mM and 10 mM, preferably between 0.05 mM and 5.0 mM.

[0083] According to one embodiment, the concentration of the anionic N-alkylphenoxazine enhancer in the chemiluminescent substrate is between 0.01-3 mM, preferably between 0.03-1.0 mM.

[0084] According to one embodiment, the concentration of the co-enhancer in the chemiluminescent substrate is between 0.05 and 25 mM, preferably between 0.1 and 12 mM.

[0085] According to one embodiment, the concentration of peroxide in the chemiluminescent substrate is between 0.1 and 8 mM, preferably between 0.25 and 4 mM.

[0086] According to one embodiment, the pH of the chemiluminescent substrate is between 6.0 and 9.5, preferably between 6.5 and 9.0.

[0087] According to a preferred embodiment, the chemiluminescent substrate (without any stabilizer for the peroxide oxidant), having a pH value between 6.0 and 9.5, preferably between 6.5 and 9.0, comprises:

[0088] i) a chemiluminescent cyclic dihydrazide at a concentration between 0.01 mM and 10 mM, preferably between 0.05 mM and 5 mM;

[0089] ii) an anionic N-alkylphenoxazine enhancer at a concentration of 0.01-3 mM, preferably 0.03-1 mM;

[0090] iii) a co-enhancer at a concentration of 0.05-25 mM, preferably 0.1-12 mM; and

[0091] iv) a peroxide oxidizing agent at a concentration between 0.1 and 8 mM, preferably between 0.25 and 4.0 mM.

[0092] When the analysis (whether qualitative or quantitative) of the target analyte is detected, the peroxidase is in the form of a conjugate with a detection reagent for the analyte to be detected. Depending on the type of analyte to be detected, the detection reagent can be selected from proteins, antibodies, nucleotides, oligonucleotides or nucleic acid molecules. The detection reagent conjugated with the peroxidase can be (a) a detection reagent capable of specific binding to the analyte, or (b) a secondary detection reagent capable of being combined with a detection reagent for the specific binding to the analyte. In the latter case, the detection reagent for the specific binding to the analyte is added to a solid support to which a chemiluminescent substrate or a target analyte has been fixed, and then a secondary detection reagent capable of combining the detection reagent is added to the solid support.

[0093] The chemiluminescent reaction of the present invention can be applied to the detection and quantification of analytes, for example, by utilizing the formation of a bond between an analyte (e.g., a protein or nucleic acid molecule) and a solid support (e.g., a membrane or microplate), and using peroxidase as a tracer. The luminescent reaction is initiated by adding a chemiluminescent substrate disclosed herein to a solid support (previously reacted with a sample containing the analyte), and then adding a solution containing peroxidase to the solid support. The emission of light continues and can be measured by film, a camera, or other instruments.

[0094] Chemiluminescent assays based on chemiluminescent substrates according to the present disclosure include dot blot and western blot assays for proteins and southern and northern blot assays for nucleic acids.

[0095] Blot analysis based on chemiluminescent substrates according to the present disclosure uses gel electrophoresis to separate the target analyte (protein or nucleic acid molecule) from other components (other proteins or other nucleic acid molecules) present in the sample to be tested. The analyte and other components are then transferred to a membrane where they are probed (detected) using a chemiluminescent substrate disclosed herein and a detection reagent that is capable of specifically binding to the target analyte, wherein the detection reagent is conjugated to a peroxidase.

[0096] Chemiluminescent assays based on the chemiluminescent substrates of the present invention also include enzyme immunoassays (EIA), such as ELISA assays. Enzyme immunoassays are particularly suitable for detecting analytes present in very small amounts in samples, such as tumor markers, thyroid hormones, viral proteins (e.g., HIV, HCV, HPV proteins) or steroid hormones (e.g., estradiol, aldosterone). Performing an ELISA assay for detecting an analyte involves at least one detection reagent that is specific for the target analyte. The analyte in the sample is fixed to a solid support (usually a polystyrene microplate) nonspecifically (by adsorption to the solid support surface) or specifically (by another detection reagent specific for the same analyte captured on the solid support surface, in a "sandwich ELISA"). After the analyte is fixed to the solid support surface, a detection reagent is added to form a complex with the analyte. The detection reagent can be covalently linked to the peroxidase as a tracer, or it can itself be detected by a secondary detection reagent that is linked to the peroxidase by (bio) conjugation (e.g., by biotin or streptavidin). Between each step, the plate is typically washed with a mild detergent solution to remove any non-specifically bound proteins or antibodies. After the final wash step, the plate is developed by adding a chemiluminescent substrate as described herein and (in the case where the detection reagent is not directly conjugated to the peroxidase) a solution containing the peroxidase conjugated to a secondary detection reagent to produce a light signal that indicates the amount of analyte in the sample.

[0097] The following examples are used to illustrate specific aspects of the present invention. However, they are not intended to limit the present invention.

[0098] Example

[0099] All reagents used in this application were purchased from Sigma-Aldrich, TCI Europe and Panreac.

[0100] 5-Amino-6,8-dimethyl-2,3-dihydrophthalazine-1,4-dione (DML) and 5-amino-6,8-diethyl-2,3-dihydrophthalazine-1,4-dione (DEL) were synthesized according to Neumann H, Klaus S, Klawonn M, Struebing D, Huebner S, Goerdes D, von Wangeliin AJ, Lalk M and Beller M (2004) Zeitschrift für Naturforschung B, 59:431-438.

[0101] SPOX and CPOX enhancers were synthesized according to Kulys J, Vidziunaite R, Janciene R and Palaima A (2006) Electroanalysis 18: 1771-1777.

[0102] Spectrophotometric measurements were performed on a UV-VIS Varian-Cary 100BIO. Chemiluminescence measurements were performed in luminescence mode (without emission filter) using a microplate, multilabel spectrometer, Victor3 (Perkin-Elmer). Black 96-well microplates (Optiplate-96F) were used.

[0103] Prepare HRP test solution by diluting 15 μL of horseradish peroxidase (HRP) stock solution (20 mg / L) to 50 mL (concentration: 6 ng / mL) with buffer. Add 28 μL of HRP to each well (228 μL final well volume). Therefore, the final amount of HRP in each well is 168 pg.

[0104] Example 1 - Kinetics of SPTZ and SPOX Oxidation by Sodium Perborate

[0105] The following solutions were prepared:

[0106] SPTZ Solution

[0107] [SPTZ]=0.05mM

[0108] [Sodium perborate] = 4 mM

[0109] In 300 mM Tris buffer, pH 8.73

[0110] SPOX Solution

[0111] [SPOX]=0.05mM

[0112] [Sodium perborate] = 4 mM

[0113] In 300 mM Tris buffer, pH 8.73

[0114] A 2 mL aliquot of either solution was added to a 1 cm quartz cuvette. The cuvette was inserted into a thermostatic cuvette holder maintained at 35°C. The oxidation kinetics were monitored spectrophotometrically for 64 hours at 254 nm (SPTZ) and 312 nm (SPOX). The results are shown in Figure 3 (SPTZ solution) and Figure 4 (SPOX solution).

[0115] Example 2 - Reference chemiluminescent substrate

[0116] Two chemiluminescent reference substrates were prepared as follows:

[0117] L / SPTZ1 substrates

[0118] [Luminol] = 5.0 mM

[0119] [Sodium perborate] = 4 mM

[0120] [SPTZ]=1.5mM

[0121] In 150 mM Tris buffer, pH 9.0.

[0122] The initial signal is set to 100 RLU (Relative Light Unit)

[0123] L / SPTZ2 substrate

[0124] [Luminol] = 5.0 mM

[0125] [Sodium perborate] = 4.0 mM

[0126] [SPTZ]=3.0mM

[0127] [MORP]=3.0mM

[0128] In 125 mM Tris buffer, pH 9.0.

[0129] The initial signal of L / SPTZ2 was 10-fold higher than that of L / SPTZ1.

[0130] Example 3 - Dependence of the chemiluminescent signal on the SPOX concentration in the luminol / perborate / SPOX substrate

[0131] Prepare a series of chemiluminescent substrates with the following compositions:

[0132] [Luminol] = 5.0 mM

[0133] [Sodium perborate] = 4 mM

[0134] [SPOX] = 0-3.0 mM [enhancer]

[0135] In 125 mM Tris buffer, pH 9.0

[0136] 200 μl of these substrates were placed in a black 96-well microplate. 30 μl of HRP solution (1 / 3700 ​​of a 20 mg / L solution) was added to each well and the plate was placed in a Wallac Victor-3 plate reader.

[0137] The initial chemiluminescent signal normalized with the reference substrate L / SPTZ1 (Example 2) set at 100 RLU was plotted against the SPOX concentration, as shown in Figure 5 shown.

[0138] Example 4 - pH Dependence of Chemiluminescent Signal in Luminol / Perborate / SPOX Substrate

[0139] Prepare a series of chemiluminescent substrates with the following compositions:

[0140] [Luminol] = 5.0 mM

[0141] [Sodium perborate] = 4.0 mM

[0142] [SPOX] = 0.3 mM (enhancer)

[0143] pH 7.80-9.30 125mM Tris buffer

[0144] 200 μl of these substrates were placed in a black 96-well microplate. 30 μl of HRP solution (1 / 3700 ​​of a 20 mg / L solution) was added to each well and the plate was placed in a Wallac Victor-3 plate reader.

[0145] The initial chemiluminescent signal normalized with the reference substrate L / SPTZ1 (Example 2) set at 100 RLU was plotted against pH, as shown in Figure 6 shown.

[0146] Example 5 - Correlation of chemiluminescent signal with MORP concentration in luminol / perborate / SPOX / MORP substrate

[0147] Prepare a series of chemiluminescent substrates with the following compositions:

[0148] [Luminol] = 5.0 mM

[0149] [Sodium perborate] = 4.0 mM

[0150] [SPOX] = 3.0 mM (enhancer)

[0151] [MORP] = 0-10 mM (co-enhancer)

[0152] In pH 9.0, 125 mM Tris buffer

[0153] 200 μl of these substrates were placed in a black 96-well microplate. 30 μl of HRP solution (1 / 3700 ​​of a 20 mg / L solution) was added to each well and the plate was placed in a Wallac Victor-3 plate reader.

[0154] The initial chemiluminescent signal normalized with the reference substrate L / SPTZ1 (Example 2) set at 100 RLU was plotted against the MORP concentration, as shown in Figure 7 shown.

[0155] Example 6 - Correlation of chemiluminescent signal with imidazole concentration in luminol / perborate / SPOX / imidazole substrate

[0156] Prepare a series of chemiluminescent substrates with the following compositions:

[0157] [Luminol] = 0.15 mM

[0158] [Sodium perborate] = 4.0 mM

[0159] [SPOX] = 0.3 mM (enhancer)

[0160] [Imidazole] = 0-20 mM (co-enhancer)

[0161] In pH 9.0, 125 mM Tris buffer

[0162] 200 μl of these substrates were placed in a black 96-well microplate. 30 μl of HRP solution (1 / 3700 ​​of a 20 mg / L solution) was added to each well and the plate was placed in a Wallac Victor-3 plate reader.

[0163] The initial chemiluminescent signal normalized with the reference substrate L / SPTZ1 (Example 2) set at 100 RLU was plotted against the imidazole concentration, as shown in Figure 8 shown.

[0164] Example 7 - pH Dependence of Chemiluminescent Signals in Luminol / Perborate / SPOX / MORP and Luminol / Perborate / SPOX / Imidazole Substrates

[0165] Two series of chemiluminescent substrates were prepared with the following compositions:

[0166] Co-enhancer = MORP

[0167] [Luminol] = 5.0 mM

[0168] [Sodium perborate] = 4.0 mM

[0169] [SPOX] = 0.3 mM (enhancer)

[0170] [MORP] = 3.0 mM (co-enhancer)

[0171] pH 7.80-9.40 125mM Tris buffer

[0172] Co-enhancer = Imidazole

[0173] [Luminol] = 5.0 mM

[0174] [Sodium perborate] = 4.0 mM

[0175] [SPOX] = 0.3 mM (enhancer)

[0176] [Imidazole] = 8.0 mM (co-enhancer)

[0177] pH 7.80-9.40 125mM Tris buffer

[0178] 200 μL of these substrates were placed in a black 96-well microplate. 30 μL of HRP solution (1 / 3700 ​​of a 20 mg / L solution) was added to each well and the plate was placed in a Wallac Victor-3 plate reader.

[0179] The initial chemiluminescent signal normalized with the reference substrate L / SPTZ1 (Example 2) set at 100 RLU was plotted against pH, as shown in Fig. 9 shown.

[0180] Example 8 - Correlation of chemiluminescent signal with SPOX concentration in L-012 / perborate / SPOX substrate

[0181] A series of chemiluminescent substrates with the following compositions were prepared:

[0182] [L-012] = 0.15 mM

[0183] [Sodium perborate] = 4.0 mM

[0184] [SPOX] = 0-0.4 mM (enhancer)

[0185] In pH 6.9, 125 mM Tris buffer

[0186] 200 μL of these substrates were placed in a black 96-well microplate. 30 μL of HRP solution (1 / 3700 ​​of a 20 mg / L solution) was added to each well and the plate was placed in a Wallac Victor-3 plate reader.

[0187] The initial chemiluminescent signal normalized with the reference substrate L / SPTZ1 (Example 2) set at 100 RLU was plotted against the imidazole concentration, as shown in Fig.10 shown.

[0188] Example 9 - pH Dependence of Chemiluminescent Signal in L-012 / Perborate / SPOX Substrate

[0189] Prepare a series of chemiluminescent substrates with the following compositions:

[0190] [L-012] = 0.15 mM

[0191] [Sodium perborate] = 4.0 mM

[0192] [SPOX] = 0.3 mM (enhancer)

[0193] pH 6.30-7.60 125mM Tris buffer

[0194] 200 μL of these substrates were placed in a black 96-well microplate. 30 μL of HRP solution (1 / 3700 ​​of a 20 mg / L solution) was added to each well and the plate was placed in a Wallac Victor-3 plate reader.

[0195] The initial chemiluminescent signal normalized with the reference substrate L / SPTZ1 (Example 2) set at 100 RLU was plotted against pH, as shown in Fig.11 shown.

[0196] Example 10 - Correlation between chemiluminescent signal and MORP concentration in L-012 / perborate / SPOX / MORP substrate

[0197] Prepare a series of chemiluminescent substrates with the following compositions:

[0198] [L-012] = 0.15 mM

[0199] [Sodium perborate] = 4.0 mM

[0200] [SPOX] = 0.15 mM (enhancer)

[0201] [MORP] = 0-3.0 mM

[0202] pH 6.9, 125 mM Tris buffer

[0203] 200 μL of these substrates were placed in a black 96-well microplate. 30 μL of HRP solution (1 / 3700 ​​of the 20 mg / L solution) was added to each well and the plate was placed in a Wallac Victor-3 plate reader. The initial chemiluminescent signal normalized with the reference substrate L / SPTZ1 (Example 2) set to 100 RLU was plotted against the imidazole concentration, as shown in Figure 2. Fig.12 shown.

[0204] Example 11 - Luminol / SPOX and L-012 / SPOX vs. Luminol / SPTZ Substrate

[0205] Three chemiluminescent substrates based on luminol with SPOX as enhancer were prepared as follows:

[0206] 1. L / SPOX2

[0207] [Luminol] = 4.0 mM

[0208] [Sodium perborate] = 4.0 mM

[0209] [SPOX] = 1.0 mM (enhancer)

[0210] [MORP] = 5.0 mM (co-enhancer)

[0211] In pH 9.00, 150 mM Tris buffer

[0212] Co-enhancer = Imidazole

[0213] 2. L / SPOX6

[0214] [Luminol] = 3.8 mM

[0215] [Sodium perborate] = 1.6 mM

[0216] [SPOX] = 0.34 mM (enhancer)

[0217] [Imidazole] = 12.0 mM (co-enhancer)

[0218] In pH 9.00, 150 mM Tris buffer

[0219] 3. L / SPOX10

[0220] [Luminol] = 3.8 mM

[0221] [Sodium perborate] = 1.6 mM

[0222] [SPOX] = 0.34 mM (enhancer)

[0223] [Imidazole] = 12.0 mM (co-enhancer)

[0224] In pH 8.20, 150 mM Tris buffer

[0225] 4. L / SPOX16

[0226] [Luminol] = 1.6 mM

[0227] [Sodium perborate] = 1.6 mM

[0228] [SPOX] = 0.34 mM (enhancer)

[0229] [Imidazole] = 12.0 mM (co-enhancer)

[0230] In pH 8.85, 1 M phosphate / hydrogen phosphate buffer

[0231] 5. L012 / SPOX

[0232] [L-012] = 0.6 mM

[0233] [Sodium perborate] = 1.75 mM

[0234] [SPOX] = 0.74 mM (enhancer)

[0235] [MORP] = 1.75 mM (co-enhancer)

[0236] In pH 7.6 50mM / 30mM Tris / hydrogen phosphate buffer

[0237] The initial chemiluminescent signal level was recorded for each of these substrates as well as the luminol / SPTZ reference substrates (L / SPTZ1 and L / SPTZ2) of Example 2. The results are plotted in a bar graph as Fig.13 shown.

[0238] Example 12 - Comparison of the performance of luminol / SPOX, L-012 / SPOX and luminol / SPTZ chemiluminescent substrates in Western blot analysis

[0239] A series of luminol / SPOX chemiluminescent substrates were compared to luminol / SPTZ substrates. The substrates were tested in a western blot analysis to detect HDAC-1 in HeLa cell lysates. A series of 2-fold dilutions of cell lysates (5 to 0.078 μg) / (2.5 to 0.039 μg) were run on a 4-20% mini-PROTEAN TGX precast gel and stained using Turbo TMTransfer system and blot it on nitrocellulose membrane. Use 2% ECL TM The membrane was blocked with blocking reagent (GE Healthcare), probed with anti-HDAC-1 rabbit polyclonal antibody (Cat No. ab19845, Abcam), and incubated with secondary goat anti-rabbit HRP-conjugated antibody (Cat No. ab6721, Abcam). The chemiluminescent substrate was incubated with the blot at room temperature for 1 min 30 s and then imaged with ImageQuant TM LAS 4000 (GE Healthcare) imaging. Images were acquired at 120 and 180 seconds. Results are shown in Fig.14 .

[0240] Of course, while the principle of the invention remains the same, the details of construction and embodiments may vary widely with respect to what has been described and illustrated by way of example, without departing from the scope of the present invention.

Claims

1. A kit for performing an assay for determining an analyte in a sample, wherein the kit comprises a chemiluminescent cyclic dihydrazide, a peroxide oxidant, an enhancer and a co-enhancer, wherein the enhancer is 3-(10H-phenoxazin-10-yl)propane-1-sulfonic acid, and the co-enhancer is selected from imidazole and 4-morpholino-pyridine.

2. The kit according to claim 1, wherein the kit further comprises peroxidase or a conjugate thereof.

3. The kit according to claim 1 or 2, wherein the chemiluminescent cyclic dihydrazide is selected from luminol, isoluminol, 5-amino-6,8-dimethyl-2,3-dihydrophthalazine-1,4-dione, 5-amino-6,8-diethyl-2,3-dihydrophthalazine-1,4-dione and 8-amino-5-chloro-7-phenylpyrido[3,4-d]pyridazine-1,4(2H,3H)-dione.

4. The kit according to claim 2, wherein the peroxidase is selected from the group consisting of horseradish peroxidase, soybean peroxidase and sweet potato peroxidase.

5. The kit according to claim 1 or 2, wherein the peroxide oxidizing agent is selected from hydrogen peroxide, urea / hydrogen peroxide complex, perborate, percarbonate.

6. The kit of claim 1 or 2, wherein the chemiluminescent cyclic dihydrazide is present in a first vial, and the peroxide oxidant is present in a second vial, and wherein the enhancer and the co-enhancer are present in the first vial or the second vial or both.

7. The kit of claim 1 or 2, wherein the chemiluminescent cyclic dihydrazide, the peroxide oxidant, the enhancer, and the co-enhancer are all present in a single vial.

8. A method for performing a chemiluminescent analysis for detecting an analyte in a sample, wherein the method comprises the following steps: (i) obtaining a chemiluminescent substrate by mixing together a chemiluminescent cyclic dihydrazide, an enhancer, a co-enhancer and a peroxide oxidant, wherein the enhancer is 3-(10H-phenoxazin-10-yl)propane-1-sulfonic acid, and the co-enhancer is selected from imidazole and 4-morpholino-pyridine, and wherein the chemiluminescent substrate does not contain any stabilizer for the peroxide oxidant, (ii) reacting the solid support on which the target analyte has been immobilized with the chemiluminescent substrate, (iii) adding a detection reagent for the target analyte to the solid support, wherein the detection reagent is directly or indirectly conjugated to a peroxidase, and (iv) recording an output light signal generated by the chemiluminescent reaction of the peroxidase with the chemiluminescent substrate, wherein the output light signal is correlated with the presence / amount of the target analyte.

9. The method according to claim 8, wherein the concentration of the chemiluminescent cyclic dihydrazide in the chemiluminescent substrate is between 0.01 mM and 10 mM.

10. The method according to claim 8 or claim 9, wherein the concentration of the enhancer in the chemiluminescent substrate is between 0.01 and 3 mM.

11. The method according to claim 8 or claim 9, wherein the concentration of the co-enhancer in the chemiluminescent substrate is between 0.05 and 25 mM.

12. The method of claim 11, wherein the concentration of the co-enhancer in the chemiluminescent substrate is between 0.1 and 12 mM.

13. The method of claim 8 or claim 9, wherein the concentration of the peroxide in the chemiluminescent substrate is between 0.1 and 8 mM.

14. The method of claim 8 or claim 9, wherein the pH value of the chemiluminescent substrate is between 6.0 and 9.

5.

15. The method of claim 14, wherein the pH value of the chemiluminescent substrate is between 6.5 and 9.

0.

16. The method of claim 8 or claim 9, wherein the chemiluminescent substrate at a pH between 6.0 and 9.5 comprises: i) said chemiluminescent cyclic dihydrazide at a concentration between 0.01 mM and 10 mM; ii) said enhancer at a concentration between 0.01 and 3 mM; iii) said co-enhancer at a concentration between 0.05 and 25 mM; and iv) said peroxide oxidizing agent at a concentration between 0.1 and 8 mM.

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