Methods and reagents for increasing chemiluminescence signals - Patents.com

Abstract

The present invention relates to a method for increasing the chemiluminescent signal in a chemiluminescent reaction of a chemiluminescent compound in a chemiluminescent reaction mixture, comprising oxidizing the chemiluminescent compound in the presence of an effective amount of an enhancer to obtain an increased chemiluminescent signal, and to the use of a compound selected from the group consisting of quaternary amine cationic materials of formula (I) for increasing the chemiluminescent signal in a chemiluminescent reaction of an acridinium compound, preferably an acridinium ester or an acridinium sulfonamide, preferably in a chemiluminescent immunoassay, and to chemiluminescent compositions and kits related thereto.

Description

[Technical field]

[0001] The present invention relates to a method for increasing the chemiluminescent signal in a chemiluminescent reaction of a chemiluminescent compound in a chemiluminescent reaction mixture, comprising oxidizing the chemiluminescent compound in the presence of an effective amount of an enhancer to obtain an increased chemiluminescent signal, to the use of a compound selected from the group consisting of quaternary amine cationic materials of formula (I) for increasing the chemiluminescent signal in a chemiluminescent reaction of an acridinium compound, preferably an acridinium ester or an acridinium sulfonamide, preferably in a chemiluminescent immunoassay, and to related chemiluminescent compositions and kits. [Background technology]

[0002] As a non-radioactive immunoassay technique, chemiluminescent immunoassay has developed very rapidly worldwide after enzyme immunoassay (EIA), radioimmunoassay, and fluorescent immunoassay techniques. Chemiluminescent immunoassay is a technique with high sensitivity, wide detection range, simple and fast operation, stable labeling, and low contamination, which makes it a desirable quantitative immunoassay method (Cinquanta et al., “Chemiluminescent immunoassay technology: what does it change in autoantibody detection?,” Auto Immun Highlights, 8:9, 24 June 2017).

[0003] Nowadays, chemiluminescence immunoassays are often preferred over traditional detection methods such as radioimmunoassays for the quantification of various biological substances, such as small molecules, nucleic acids, hormones, and antibodies. It is characterized by the generation of light by chemiluminescent substances upon external stimulation by chemical or enzymatic trigger substances. Well-known examples of chemiluminescent substances are luminol, 1,2-dioxetane aryl phosphates, tris-(bipyridyl)ruthenium(II) complexes or acridinium-based probes. Typically, chemiluminescence reactions can be divided into flash-type and glow-type chemiluminescence, depending on the nature of the chemiluminescent substrate, the trigger substance added, and the type of light output obtained. As described by Tannous et al. (Tannous et al., "Combined flash and glow-type chemiluminescent reactions for high-throughput genotyping of biallelic polymorphisms," Analytical Biochemistry, Volume 320, Issue 2, Pages 266-272, 15 September 2003), flash-type reactions result in bright chemiluminescent signals in the range of seconds to minutes, whereas the light output in glow-type reactions is less bright, but it can last up to hours. Depending on the method of choice, flash-type and glow-type immunoassays can be designed accordingly.

[0004] Flash-type acridinium-based probes are often used in chemiluminescence immunoassays for fast and ultrasensitive signal generation when high sample throughput is required. Well-known examples are 10-methyl-2,6-dimethyl-acridinium-NHS ester (Me-DMAE-NHS), N-sulfopropyl-dimethyl-acridinium-N-hydroxysuccinimide ester (NSP-DMAE-NHS), N-sulfopropyl-acridinium-sulfonamide-N-hydroxysuccinimide ester (NSP-SA-NHS) and 10-methyl-2,6-dimethyl-acridinium-(polyethylene oxide)4-N-hydroxysuccinimide ester (MeAE-PEO4-NHS), and these molecules and their variants are described, for example, in EP 1273917, EP 1539702, etc. All of these molecules generate light upon external stimulation with alkaline hydrogen peroxide. Typically, these reagents are added stepwise to trigger solutions A and B. Hydrogen peroxide is part of Trigger A, usually combined with an acid such as nitric acid. Sodium hydroxide is then injected into Trigger B.

[0005] For optimal signal generation, Trigger B is further said to require detergent molecules that can form micellar structures in aqueous solutions. Chang and Miller, in US Pat. No. 4,927,769, state that cationic detergents based on quaternary amines are particularly advantageous. Although nonionic or anionic detergents are also possible, it is said that the positive charge in the head group of the cationic detergent brings the mostly hydrophobic acridinium-based probe and the negatively charged hydrogen peroxide anion into spatial proximity at the micelle interface, thus promoting chemiluminescence signal output, as also described by Natrajan et al. (Natrajan et al., “Effect of surfactants on the chemiluminescence of acridinium dimethylphenyl ester labels and their conjugates,” Org. Biomol. Chem., 9, pp. 5092-5103, 13 April 2011). A well-known example of such a cationic detergent, included in US Pat. No. 4,927,769, is cetyltrimethylammonium chloride (CTAC).

[0006] However, further improvements in chemiluminescence immunoassay technology are needed to allow quantification of ultralow concentrations of analytes and to facilitate the discrimination between signal and noise in general. These improvements will likely require the enhancement of chemiluminescence by optimized enhancer substances.

[0007] The problem underlying the present invention is to provide methods and associated reagents and uses in chemiluminescence immunoassay techniques that can be used for improved quantification of analytes, particularly at ultra-low concentrations, and that can be used to make it easier to distinguish between signal and noise in general.

[0008] Another problem underlying the present invention is to provide substances capable of increasing the chemiluminescent signal of chemiluminescent compounds, preferably acridinium compounds, e.g. as acridinium esters or acridinium sulfonamides, in chemiluminescent immunoassays. Summary of the Invention

[0009] In a first aspect, the problem underlying the present invention is a method for increasing a chemiluminescent signal in a chemiluminescent reaction of a chemiluminescent compound in a chemiluminescent reaction mixture, comprising the step of oxidizing the chemiluminescent compound in the presence of an effective amount of an enhancer to obtain an increased chemiluminescent signal, wherein the chemiluminescent compound is an acridinium compound, preferably an acridinium ester or an acridinium sulfonamide, and said enhancer is a compound or a mixture of two, three or more compounds, wherein said enhancer compound or each of said enhancer compounds is a quaternary amine cationic material of formula (I): [ka] (In the formula, R 1 is C3~C 20 is alkyl or alkenyl; R 2 and R 3 are each independently a C1-C4 alkyl or alkenyl; R 4 , R 5 , R 6 , R 7 and R 8 are each independently hydrogen, alkyl, or alkenyl; X 1- is selected from the group consisting of a halide ion or a hydroxyl ion, preferably a chloride ion or a bromide ion.

[0010] An effective amount of the enhancer of the present invention is provided by one, two or more enhancer compounds, each of which is selected from the group consisting of quaternary amine cationic materials of formula (I) disclosed herein.

[0011] Each enhancer compound of the present invention has an aromatic head group. Possible anionic counterions to the cationic portion of the enhancer compounds of the present invention include, but are not limited to, chloride, bromide, and hydroxide.

[0012] In a preferred embodiment, the method of the present invention includes the step of adding an enhancer to a chemiluminescent reaction mixture that includes a chemiluminescent compound, ie, an acridinium compound, such as an acridinium ester or an acridinium sulfonamide.

[0013] In a preferred embodiment, the enhancer compound or each of the enhancer compounds according to the invention comprises R 1 But C3~C 18 Alkyl or alkenyl, preferably C5-C 18 Alkyl or alkenyl; more preferably C8-C 16 Alkyl or alkenyl; most preferably C 12 ~C 14 is alkyl or alkenyl; R 2 and R 3 are each independently methyl, ethyl, propyl or butyl, preferably methyl; R 4 , R 5 , R 6 , R 7 and R 8 are, independently of each other, hydrogen, alkyl, alkenyl, preferably hydrogen, methyl or ethyl; and / or X 1- is selected from the group consisting of the quaternary amine cationic materials of formula (I) disclosed herein which are chloride or bromide ions, preferably chloride ions.

[0014] In a preferred embodiment, one enhancer compound, at least one compound of the enhancer or each compound of the enhancer according to the invention comprises: - benzyl dimethyl isocyl ammonium chloride, - benzyl dimethyl nonadecyl ammonium chloride, - benzyl dimethyl octadecyl ammonium chloride, - benzyl dimethyl heptadecyl ammonium chloride, - benzyl dimethyl hexadecyl ammonium chloride, - benzyl dimethyl pentadecyl ammonium chloride, - benzyl dimethyl tetradecyl ammonium chloride, - benzyl dimethyl tridecyl ammonium chloride, - benzyl dimethyl dodecyl ammonium chloride, - benzyldimethylundecyl ammonium chloride, - benzyl dimethyl decyl ammonium chloride, - benzyl dimethyl nonyl ammonium chloride, - benzyldimethyloctylammonium chloride, - benzalkonium chloride, - benzyl dimethylheptyl ammonium chloride, - benzyldimethylhexyl ammonium chloride, - benzyldimethylpentyl ammonium chloride, - benzyl dimethyl butyl ammonium chloride, - benzyldimethylpropylammonium chloride, - benzyltripropylammonium chloride, - benzyldipropylethylammonium chloride, - benzyldiethylpropylammonium chloride, - benzyldipropylmethylammonium chloride, - benzylpropylethylmethylammonium chloride, - benzyltributylammonium chloride, - benzyl dibutylpropyl ammonium chloride, - benzyl dipropyl butyl ammonium chloride, - benzyl butyl propyl ethyl ammonium chloride, - benzyl butyl propyl methyl ammonium chloride, - benzyl butyl ethyl methyl ammonium chloride, - benzyl diethyl butyl ammonium chloride, - C 12~14 -Alkyldimethyl(ethylbenzyl)ammonium chloride, - dodecyl(ethylbenzyl)dimethylammonium chloride, - tetradecyldimethyl(ethylbenzyl)ammonium chloride, - Octadecyldimethyl(ar-ethylbenzyl)ammonium chloride and each of the above compounds containing bromide instead of chloride is selected from the group consisting of:

[0015] A list of exemplary enhancer compounds (cationic substances with aromatic head groups suitable as chemiluminescence enhancers) containing molecular structures according to the present invention is presented in Table 1. To clarify the identity of the listed substances despite possible ambiguities in chemical nomenclature, Chemical Abstracts Service (CAS) registration numbers and / or compound records (CIDs) are listed in Table 1 if this information can be found and is publicly available. CAS registration numbers from the Division of the American Chemical Society can be accessed at https: / / www.cas.org or https: / / commonchemistry.cas.org. CID records can be found in the National Institutes of Health's PubChem database at https: / / pubchem.ncbi.nlm.nih.gov. If a CAS and / or CID was not assigned to a compound or the information was not publicly available, the respective compound was defined as "nd."

[0016] [Table 1] JPEG2024527110000004.jpg255165JPEG2024527110000005.jpg255164JPEG2024527110000006.jpg255165 JPEG2024527110000007.jpg255164JPEG2024527110000008.jpg255165JPEG2024527110000009.jpg255165

[0017] The enhancer of the present invention may be comprised of a mixture of two, three or more of the compounds listed above in Table 1, which may also be commercially available as individual products. An example of a mixture of two, three or more of the compounds listed above in Table 1 that is commercially available as individual products is, for example, benzalkonium chloride or benzalkonium bromide. Benzalkonium chloride is a mixture of alkylbenzyldimethylammonium chlorides (ABDAC), whose alkyl moieties range from C8 to C 18 Depending on the composition of the mixture, numerous commercial products and CAS numbers can be found for benzalkonium chloride, respectively.

[0018] If the enhancer compound according to the invention contains a chiral C atom, the enhancer compound discloses and includes all isomeric forms of said compound.

[0019] In a preferred embodiment, the effective amount of said enhancer in the chemiluminescence reaction mixture is 0.001% or more by weight per volume (w / v), preferably 0.01% by w / v, more preferably 0.1% by w / v, and most preferably 0.2% by w / v, based on the total volume of the chemiluminescence reaction mixture in each case.In general, the effective amount of enhancer required to increase the chemiluminescence signal of the chemiluminescent compound in the chemiluminescence reaction mixture in the chemiluminescence reaction varies depending on the enhancer selected and can be determined empirically.As a general rule, the effective amount of enhancer in the chemiluminescence reaction mixture is more than 0.005% by weight per volume, preferably more than 0.01% by w / v, based on the total volume of the chemiluminescence reaction mixture in each case. In a preferred embodiment, the amount of enhancer that results in an increased chemiluminescence signal contained in the chemiluminescence reaction mixture is in the range of 0.005% w / v to 3% w / v, preferably in the range of 0.01% w / v to 2% w / v, also preferably in the range of 0.05% w / v to 1% w / v, more preferably in the range of 0.08% w / v to 0.8% w / v, even more preferably in the range of 0.1% w / v to 0.6% w / v, and most preferably in the range of 0.2% w / v to 0.5% w / v. In a particularly preferred embodiment, the amount of enhancer that results in an increased chemiluminescence signal contained in the chemiluminescence reaction mixture is in the range of 0.08% w / v to 0.4% w / v, in each case based on the total volume of the chemiluminescence reaction mixture.

[0020] Preferably, the chemiluminescence reaction according to the method or use of the present invention should be carried out at a temperature of 20° C. to 37° C. and at a pH in the range of 10 to 13.5. The enhancer according to the present invention is preferably diluted before use in the method of the present invention. Suitable diluents include water, an aqueous acid solution or an aqueous base solution.

[0021] In a preferred embodiment, the enhancers of the present invention are soluble in the chemiluminescent reaction mixture.

[0022] In a preferred embodiment, the method according to the invention is carried out by or is part of an immunoassay, which preferably comprises a step of detecting the presence or absence of an antigen, an antibody or an autoantibody. The immunoassay can preferably be selected from the group comprising competitive assay, capture bridge assay, immunometric assay, direct or indirect class capture assay. The principles of each of these formats are detailed in The Immunoassay Handbook, 3rd edition, edited by David Wild, Elsevier, 2005.

[0023] In a preferred embodiment, the chemiluminescent reaction of the present invention is a flash-type chemiluminescent reaction. Flash-type chemiluminescent reactions result in a bright chemiluminescent signal in the range of seconds to minutes.

[0024] In a further preferred embodiment, the method of increasing a chemiluminescent signal in a chemiluminescent reaction of a chemiluminescent compound in a chemiluminescent reaction mixture according to the present invention comprises a flash-type chemiluminescent reaction as the chemiluminescent reaction.

[0025] In a preferred embodiment, an enhancer according to the present invention is combined with a chemiluminescent compound in a chemiluminescent reaction mixture prior to, simultaneously with, or immediately after the initiation of the chemiluminescent reaction, preferably the enhancer is combined with a chemiluminescent compound in a chemiluminescent reaction mixture simultaneously with the initiation of the chemiluminescent reaction.

[0026] In a preferred embodiment, the chemiluminescent signal produced by the chemiluminescent reaction of the present invention is detected for a certain duration, preferably, the chemiluminescent reaction mixture is formed, the chemiluminescent reaction is initiated and the signal is detected simultaneously, more preferably, the duration of detection ranges from 0.01 to 360 seconds, even more preferably from 0.1 to 30 seconds, even more preferably from 0.3 to 15 seconds, and most preferably from 0.5 to 10 seconds, wherein in each case, the duration of detection begins with the initiation of the chemiluminescent reaction.

[0027] In a preferred embodiment, the method of the present invention comprises combining an enhancer of the present invention with a chemiluminescent compound in a chemiluminescent reaction mixture simultaneously with the initiation of the chemiluminescent reaction and / or detecting the chemiluminescent signal produced by the chemiluminescent reaction of the present invention for a period of time of 0.1 to 10 seconds beginning with the initiation of the chemiluminescent reaction.

[0028] According to the present invention, the chemiluminescent reaction is preferably initiated by initiating the oxidation of the chemiluminescent compound. Preferably, the initiation step of the oxidation of the chemiluminescent compound is carried out by adding an oxidizing compound, preferably hydrogen peroxide.

[0029] In a preferred embodiment, preferably according to at least one of the preceding embodiments, the chemiluminescent reaction mixture further comprises hydrogen peroxide, an acid, preferably nitric acid, and / or an alkali hydroxide, preferably sodium hydroxide.

[0030] In a preferred embodiment, preferably according to at least one of the preceding embodiments, the chemiluminescent reaction mixture comprises a trigger solution A and a trigger solution B. Preferably, trigger solution A comprises an acid, preferably nitric acid, and hydrogen peroxide, and trigger solution B comprises an alkali hydroxide, preferably sodium hydroxide, and an enhancer according to the present invention.

[0031] In a preferred embodiment, preferably according to at least one of the preceding embodiments, a chemiluminescent reaction mixture is provided by mixing a trigger solution A, preferably comprising nitric acid and hydrogen peroxide, and a trigger solution B, preferably comprising sodium hydroxide and an enhancer according to the present invention, with a chemiluminescent compound, which is an acridinium compound, preferably an acridinium ester or an acridinium sulfonamide.

[0032] The chemiluminescent compounds of the present invention are acridinium compounds, preferably acridinium esters or acridinium sulfonamides.

[0033] It is well known in the art that acridinium compounds exist in equilibrium between acridinium and pseudobase forms in the aqueous media in which virtually all immunoassays are performed. The pseudobase form of the acridinium compound cannot react with hydrogen peroxide and therefore cannot produce chemiluminescence. The equilibrium between the chemiluminescent acridinium form and the non-chemiluminescent pseudobase form is strongly influenced by the pH of the medium. An acidic pH favors the formation of the acridinium form, and a basic pH favors the formation of the pseudobase form.

[0034] In heterogeneous assay formats, chemiluminescence from acridinium compounds or biologically active molecules labeled with acridinium compounds, referred to as tracers or conjugates, is usually triggered by the sequential addition of two reagents. An initial treatment of the acridinium compound with a strong acid containing peroxide is required to convert the pseudobase form of the acridinium compound to the acridinium form. Subsequent treatment with an alkaline solution then neutralizes the acid and raises the pH of the reaction medium to allow light emission to occur by ionization of hydrogen peroxide.

[0035] Preferably, the chemiluminescent compound used in the present invention is an acridinium ester according to formula (II): [ka] (In the formula, R 1 is alkyl, alkenyl, alkynyl, aryl or aralkyl, sulfoalkyl, carboxyalkyl, and oxoalkyl; R 3 From R 15 are each independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, aryl or aralkyl, amino, amido, acyl, alkoxyl, hydroxyl, carboxyl, halogen, halide, nitro, cyano, sulfo, sulfoalkyl, sulfamoyl, carboxyalkyl, succinimidyl ester and oxoalkyl or any other leaving group; optionally, when present, X 1- is an anion); or an acridinium sulfonamide according to formula (III) [ka] (In the formula, R 1 is selected from the group consisting of alkyl, alkenyl, alkynyl, aryl or aralkyl, sulfoalkyl, carboxyalkyl, and oxoalkyl; R 2 From R 15 are each independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, aryl or aralkyl, amino, amido, acyl, alkoxyl, hydroxyl, carboxyl, halogen, halide, nitro, cyano, sulfo, sulfoalkyl, sulfamoyl, carboxyalkyl, succinimidyl ester and oxoalkyl or any other leaving group; optionally, when present, X 1- is an anion).

[0036] In a preferred embodiment, preferably according to at least one of the preceding embodiments, the chemiluminescent compound further comprises R 12 and R 14 are each independently selected from the group consisting of hydrogen, alkyl, amino, carboxyl, hydroxyl, alkoxyl, nitro, or halide; R 11 and R15 are each independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, aryl, alkoxyl, amino, amido, sulfonamido or sulfide; R 13 is the following substituent R 13 =LR 16 -R 17 (wherein L is sulfamoyl or is omitted; R 16 is omitted or is alkyl, aryl, aralkyl, z=1 to 10, preferably z=1 or 2 (amide) z or x=1 to 10, preferably x=4 (polyethylene oxide) x ;R 17 teeth, [ka] SO2Cl or any other leaving group; where R is alkyl, aryl, or aralkyl; and X is CH3SO4, OSO2F, a halide, OSO2CF3, OSO2C4F9, or [ka] ), R 12 , R 13 and R 14 The substituent positions are interchangeable in the acridinium esters according to formula (II) disclosed herein.

[0037] In a preferred embodiment, preferably according to at least one of the preceding embodiments, the chemiluminescent compound further comprises R 12 , R 13 and R 14 are each independently selected from the group consisting of hydrogen, alkyl, amino, carboxyl, hydroxyl, alkoxyl, nitro, or halide; R 11 and R 15are each independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, aryl, alkoxyl, amino, amido, sulfonamido or sulfide; R 2 is the following substituent R 2 =LR 16 -R 17 (wherein L is sulfamoyl or omitted; R 16 is omitted or is alkyl, aryl, aralkyl, z=1 to 10, preferably z=1 or 2 (amide) z or x=1 to 10, preferably x=4 (polyethylene oxide) x ;R 17 teeth [ka] Halide SO2Cl or any other leaving group; where R is alkyl, aryl, or aralkyl; and X is CH3SO4, OSO2F, a halide, OSO2CF3, OSO2C4F9, or [ka] ), R 11 From R 15 The substituent positions are interchangeable and are acridinium sulfonamides according to formula (III) disclosed herein.

[0038] Acridinium compounds useful in the present invention are further described in U.S. Patent Application No. 915,527, filed October 6, 1986, and Natrajan et al., “A comparison of chemiluminescent acridinium dimethylphenyl ester labels with different conjugation sites”, Org Biomol Chem, 13(9):2622-337, March 2015.

[0039] In a preferred embodiment, preferably according to at least one of the preceding embodiments, the chemiluminescent compound is - N-sulfopropyl-dimethylacridinium-(polyethylene oxide) where x=1-10 x -N-hydroxysuccinimide ester (NSP-DMAE-PEO x -NHS), preferably N-sulfopropyl-dimethylacridinium-(polyethylene oxide) 4-N-hydroxysuccinimide ester (NSP-DMAE-PEO4-NHS); - 2,6-(dimethyl)-3-chlorosulfonylphenyl-N-(3-sulfopropyl)-acridinium-9-carboxylate (SPAE); - 2,6-(dimethyl)-3-sulfamoylphenyl-N-(3-sulfopropyl)-acridinium-9-carboxylate-(polyethylene oxide) 4-pentafluorophenyl ester (SPAE-PEO4-PFP); - 2,6-(dimethyl)-3-chlorosulfonylphenyl-N-methyl-acridinium-9-carboxylate triflate (MeAE); - 2,6-(dimethyl)-3-sulfamoylphenyl-N-methyl-acridinium-9-carboxylate triflate-(polyethylene oxide) 4-N-hydroxysuccinimide ester (MeAE-PEO4-NHS), - 2,3,4,5,6-pentafluorophenyloxy-1-carbonyl-4,7,10,13-tetraoxapentadecyl-(15-amino)-3'-sulfonyl-2',6'-dimethylphenyl-N-(3-sulfopropyl)-acridinium-9-carboxylate, - 10-Methyl-2,6-dimethyl-acridinium-NHS ester methylsulfate (Me-DMAE-NHS) - N-sulfopropyl-dimethylacridinium-N-hydroxysuccinimide (NSP-DMAE-NHS); and - N-Sulfopropyl-acridinium-sulfonamide-N-hydroxysuccinimide (NSP-SA-NHS) is selected from the group consisting of:

[0040] A list of exemplary acridinium compounds according to formula (II) or (III) as defined herein that are suitable as chemiluminescent compounds according to the present invention is presented in Table 2. To clarify the identity of the listed substances despite possible ambiguities in chemical nomenclature, Chemical Abstracts Service (CAS) registration numbers and / or compound records (CIDs) are listed in Table 2, if this information can be found and is publicly available. CAS registration numbers from the Division of the American Chemical Society can be accessed at https: / / www.cas.org or https: / / commonchemistry.cas.org. CID records can be found in the National Institutes of Health's PubChem database at https: / / pubchem.ncbi.nlm.nih.gov. If a CAS and / or CID was not assigned to a compound or the information was not publicly available, the respective compound was defined as "nd."

[0041] [Table 2] JPEG2024527110000017.jpg198170JPEG2024527110000018.jpg255164JPEG2024527110000019.jpg194170

[0042] The acridinium compounds of the present invention can be oxidized by any oxidant that will react with the compound and cause excitation of the compound such that the compound emits light in a chemiluminescent reaction. A preferred oxidant is hydrogen peroxide in dilute alkali.

[0043] In a preferred embodiment, the chemiluminescent reaction mixture of the present invention comprises an oxidant in an amount of 0.01% to 1% by volume based on the total volume of the chemiluminescent reaction mixture, preferably in an amount of 0.05% to 0.5% by volume based on the total volume of the chemiluminescent reaction mixture, and most preferably in an amount of 0.1% to 0.3% by volume based on the total volume of the chemiluminescent reaction mixture.

[0044] The emitted light can be quantified using standard measuring devices such as the LB 960 Centro Microplate Luminometer (Berthold Technologies GmbH & Co. KG, Germany), the Model 810 Luminometer (Ciba-Corning Diagnostics Corp., Medfield, MA) or the RA Analyzer 10 (Euroimmun, a PerkinElmer subsidiary, Germany).

[0045] In a preferred embodiment, the chemiluminescent compound capable of emitting chemiluminescence emits luminescence having a wavelength of 400 nm or more, preferably from 400 nm to 690 nm, more preferably from 400 nm to 550 nm, more preferably from 405 nm to 500 nm, and most preferably from 410 to 490 nm.

[0046] In a second aspect, the problem underlying the present invention is to provide a quaternary amine cationic substance of formula (I) for increasing the chemiluminescence signal of an acridinium compound, preferably an acridinium ester or an acridinium sulfonamide, more preferably according to the present invention, in a chemiluminescence reaction, preferably in a chemiluminescence immunoassay. [ka] (In the formula, R 1 is C3~C 20 is alkyl or alkenyl; R 2 and R 3 are each independently a C1-C4 alkyl or alkenyl; R 4 , R 5 , R 6 , R 7 and R 8 are each independently hydrogen, alkyl, or alkenyl; X 1- is solved by the use of a compound selected from the group consisting of halide ions or hydroxyl ions, preferably chloride ions or bromide ions.

[0047] In a preferred embodiment, the compound is R 1 But, C3~C 18 Alkyl or alkenyl, preferably C5-C 18 Alkyl or alkenyl; more preferably C8-C 16 Alkyl or alkenyl; most preferably C 12 ~C 14 is alkyl or alkenyl; R 2 and R 3 are each independently methyl, ethyl, propyl or butyl, preferably methyl; R 4 , R 5 , R 6 , R 7 and R 8 are, independently of one another, hydrogen, alkyl, alkenyl, preferably hydrogen, methyl or ethyl; and / or X 1-is selected from the group consisting of quaternary amine cationic materials of formula (I) as defined herein, wherein is a chloride ion or a bromide ion, preferably a chloride ion.

[0048] Preferably, one, two or more compounds selected from the group consisting of quaternary amine cationic substances of formula (I) disclosed above are used to increase the chemiluminescent signal of a chemiluminescent compound, preferably an acridinium ester or an acridinium sulfonamide, preferably those according to the present invention, in a chemiluminescent reaction, preferably in a chemiluminescent immunoassay.

[0049] According to the present invention, the compound is an enhancer compound according to the present invention having an aromatic head group.

[0050] In a third aspect, the problem underlying the present invention is to provide a quaternary amine cationic material of formula (I) [ka] (In the formula, R 1 is C3~C 20 is alkyl or alkenyl; R 2 and R 3 are each independently a C1-C4 alkyl or alkenyl; R 4 , R 5 , R 6 , R 7 and R 8 are each independently hydrogen, alkyl, or alkenyl; X 1-is a halide or hydroxyl ion, preferably a chloride or bromide ion), and at least one chemiluminescent compound, which is an acridinium compound, preferably an acridinium ester or an acridinium sulfonamide, more preferably an acridinium ester or an acridinium sulfonamide of formula (II) or formula (III) as disclosed herein.

[0051] In accordance with the present invention, at least one compound in the chemiluminescent compositions of the present invention is an enhancer compound of the present invention having an aromatic head group.

[0052] In a preferred embodiment, the chemiluminescent composition of the present invention further comprises a fluorescent agent.

[0053] The chemiluminescent composition of the present invention may further include one or more fluorescent agents, such as sodium fluorescein, rhodamine B and / or rhodamine 6G. The fluorescent agent will emit a fluorescent light after receiving light energy from the chemiluminescent signal of the chemiluminescent compound. There is a possibility that the quantum yield of fluorescein can be much higher than that of the chemiluminescent compound, so the quantum yield of the chemiluminescent compound can be indirectly improved. For example, sodium fluorescein (disodium 9-(o-carboxyphenyl)-6-hydroxy-3H-xanthene-3-ketone) is a water-soluble fluorescent agent, with excitation and emission wavelengths of 494 nm and 518 nm, respectively. The quantum yield of sodium fluorescein is up to 0.97. Similarly, the rhodamine series of fluorescent agents also have very high fluorescence quantum yields. The fluorescent agent is preferably used in the chemiluminescent composition of the present invention at a concentration of about 0.1 mg / L to about 1 g / L.

[0054] In a preferred embodiment, the chemiluminescent composition of the present invention is a chemiluminescent reaction mixture according to the method of the present invention. Preferably, the chemiluminescent composition of the present invention further comprises nitric acid, hydrogen peroxide and / or sodium hydroxide.

[0055] The chemiluminescent compositions of the present invention, preferably according to the disclosed embodiments, can be included in a kit by packaging the components of the chemiluminescent composition separately in a multi-package system. In other embodiments according to the present invention, some components can be combined in one container while other components are stored in separate containers to form a multi-package system. Alternatively, the components can be mixed and then packaged as a single mixture.

[0056] In a fourth aspect, the problem underlying the present invention is solved by a kit for preferably increasing the chemiluminescence of a chemiluminescent compound, comprising at least one chemiluminescent compound which is an acridinium compound, preferably an acridinium ester or an acridinium sulfonamide, more preferably an acridinium ester or an acridinium sulfonamide of formula (II) or formula (III) disclosed above, and one, two or more compounds selected from the group consisting of a quaternary amine cationic material of formula (I) disclosed above. The problem is preferably solved by a kit for use in increasing the chemiluminescence of a chemiluminescent compound, wherein the chemiluminescent compound is an acridinium compound, preferably an acridinium ester or an acridinium sulfonamide, more preferably an acridinium ester or an acridinium sulfonamide of formula (II) or formula (III) disclosed above, and also including one, two or more compounds selected from the group consisting of quaternary amine cationic materials of formula (I) disclosed above.

[0057] Preferably, one, two or more compounds selected from the group consisting of quaternary amine cationic substances of formula (I) are enhancers according to the present invention as described above.

[0058] In a preferred embodiment, the components of the kit according to the invention are stored individually or in the form of one, two or more mixtures.

[0059] In a preferred embodiment, at least one chemiluminescent compound, preferably one, two or more compounds selected from the group consisting of a compound of formula (II) or formula (III) disclosed herein and a quaternary amine cationic material of formula (I) disclosed herein, are stored together in a mixture in the kit of the present invention, optionally further together with sodium hydroxide. Trigger solution A, preferably comprising nitric acid and hydrogen peroxide, is preferably stored separately in the kit of the present invention.

[0060] In a preferred embodiment, the kit according to the present invention, preferably according to at least one of the disclosed preferred embodiments, comprises a diagnostically useful carrier, preferably a solid carrier, comprising a means for capturing antigens, antibodies or autoantibodies in a liquid solution and a means for detecting the antigens, antibodies or autoantibodies bound to the carrier, the means for detecting the antigens, antibodies or autoantibodies bound to the carrier being labeled with at least one chemiluminescent compound, i.e. an acridinium compound, preferably an acridinium ester of formula (II) according to the present invention or an acridinium sulfonamide of formula (III). According to the present invention, it is further preferred that the diagnostically useful carrier is selected from the group comprising beads, preferably paramagnetic beads, test strips, microtiter plates, membranes, preferably from the group comprising Western blots, line blots and dot blots, lateral flow devices, glass surfaces, slides for microscopy, microarrays and biochips. In a further preferred embodiment, the diagnostically useful carrier is a line blot, a biochip or beads, most preferably beads. Preferably, the diagnostically useful carrier is provided by beads. It is further preferred according to the present invention that at least one chemiluminescent compound, which is an acridinium compound, preferably an acridinium ester or an acridinium sulfonamide, more preferably an acridinium ester or an acridinium sulfonamide of formula (II) or formula (III) according to the present invention, is bound to the beads. The binding can be by non-covalent protein-protein interactions cross-linked by protein-protein interactions or by covalent bonds.

[0061] In a preferred embodiment, the kit according to the present invention further comprises a diagnostically useful carrier as described above, a trigger solution A and a trigger solution B, wherein trigger solution A comprises nitric acid and hydrogen peroxide and trigger solution B comprises sodium hydroxide and one, two or more compounds selected from the group consisting of quaternary amine cationic substances of formula (I) as defined above, and at least trigger solution A and trigger solution B are stored separately.

[0062] In a preferred embodiment, the kit according to the invention further comprises a means for detecting the antigen, antibody or autoantibody bound to the carrier, which means for detecting the antigen, antibody or autoantibody bound to the carrier can be a secondary antibody. Preferably, the secondary antibody is an antibody that specifically binds to all antibodies of an antibody class, preferably a mammalian antibody class, more preferably a human antibody class such as IgG. A secondary antibody typically recognizes the constant domain of said class, but may also recognize other epitopes shared by antibodies of the class of interest, such as conformational epitopes across the three-dimensional structure. A wide range of them are commercially available, for example from Thermo Fisher. It can be a monoclonal or a polyclonal antibody. In a preferred embodiment, the term "recognized" as used herein means that the secondary antibody specifically binds to the antigen, antibody / antibodies or autoantibody / autoantibodies to be detected. The secondary antibody can specifically bind to all isotypes of the antibody class. For example, a secondary antibody for an IgG class antibody can bind to IgG1, IgG2, IgG3 and IgG4 isotypes. This can be achieved by using as a secondary antibody against that class, preferably against IgG class antibodies, a mixture containing antibodies that specifically bind to each IgG isotype or a single antibody that reacts with all isotypes of interest. The use of secondary antibodies is explained in Kruger, NJ, Detection of Polypeptides on Blots Using Secondary Antibodies, in The Protein Protocols Handbook (ed. JM Alker), page 967, volume 1996, Springer. Briefly, such secondary antibodies can be produced by immunizing laboratory animals with the antibody to be recognized or with a mixture of antibodies to be recognized.

[0063] In a preferred embodiment, the term "specifically binds" as used herein preferably refers to a binding reaction that specifically binds to a 1×10 antibody or antibody fragment as measured by surface plasmon resonance in PBS buffer at pH 7 at 25° C. using a Biacore instrument. -5M, more preferably 1×10 -7 M, more preferably 1×10 -8 M, more preferably 1×10 -9 M, more preferably 1×10 -10 M, more preferably 1×10 -11 M, more preferably 1×10 -12 This means that the binding reaction is stronger than the one characterized by the dissociation constant M.

[0064] In a preferred embodiment, the chemiluminescent composition, chemiluminescent reaction mixture and / or kit according to the present invention further comprises one or more, preferably all, reagents from the group comprising stabilizers, preferably a set of stabilizers, washing buffers, anti-degradation agents.

[0065] The chemiluminescent compositions, chemiluminescent reaction mixtures and / or kits of the present invention may further comprise a buffer to maintain the pH of the reaction system. Suitable buffers include carbonate buffers, diethanolamine buffers, 2-amino-2-methyl-1-propanol, etc. The buffers may be used at a concentration of about 10 to about 500 mM.

[0066] The chemiluminescent composition, chemiluminescent reaction mixture and / or kit of the present invention may further include a preservative to facilitate the preservation and long-term storage of the reagent. There is no limitation on the type of preservative, and commercially available preservatives such as Proclin 300, sodium azide, Kathon, and gentamicin may be used. The preservative may be used at any concentration that does not affect the chemiluminescent reaction.

[0067] In a preferred embodiment, the kit according to the invention further comprises one or more, preferably all, reagents from the group comprising a calibrator, preferably a set of calibrators, a positive control and a negative control.

[0068] According to further different detection applications, the kit according to the present invention may further comprise a corresponding enzyme reagent, a labeled enzyme reagent, a solid-phase antibody and / or a manual to instruct the operator.

[0069] In a fifth aspect, the problem underlying the present invention is solved by the use of a chemiluminescent composition or a chemiluminescent reaction mixture according to the present invention for the manufacture of a kit, a medical device, preferably a diagnostic device for the diagnosis of a disease. According to the present invention, the medical device is preferably selected from the group comprising glass slides, preferably for microscopy, biochips, microtiter plates, lateral flow devices, test strips, membranes, preferably line blots, chromatography columns and beads, preferably magnetic or fluorescent beads.

[0070] In another aspect, the present invention provides an immunoassay and / or method for detecting an analyte of interest in a test sample, comprising: (a) contacting a test sample suspected of containing an analyte of interest with a first antibody that binds to at least one epitope on the analyte of interest to form a first antibody-analyte complex, wherein the first antibody is immobilized on a solid phase, and further wherein at least one autoantibody in the test sample binds to at least one epitope on the analyte of interest to form an autoantibody-analyte complex, said autoantibody being bound to the solid phase; (b) contacting said mixture comprising the first antibody-analyte complex and the autoantibody-analyte complex with a second antibody conjugated to a detectable label to form a first antibody-analyte-second antibody complex and an autoantibody-analyte-second antibody complex, wherein the second antibody binds to at least one epitope on the analyte of interest and further wherein the detectable label is at least one acridinium compound, more preferably an acridinium ester according to formula (II) or an acridinium sulfonamide of formula (III) according to the present invention as described above; (c) generating or providing a source of hydrogen peroxide and an enhancer according to formula (I) of the present invention to the mixture of step (b); (d) adding a basic solution to the mixture of step (c) to produce a light signal; and (e) measuring the optical signal generated or emitted in step (d) to detect the analyte of interest in the test sample. The present invention relates to immunoassays and / or methods comprising the steps of:

[0071] In the above immunoassays or methods, the test sample can be whole blood, serum, or plasma.

[0072] In a further aspect the problem underlying the present invention is solved by a chemiluminescent composition according to the invention or a kit according to the invention for use in the diagnosis of a disease.

[0073] The present invention is based on the inventors' surprising discovery that an enhancer (compound) selected from the group consisting of quaternary amine cationic materials of formula (I) disclosed herein can be used to provide an increased chemiluminescent signal of a chemiluminescent compound that is an acridinium compound, preferably an acridinium ester or an acridinium sulfonamide, in a chemiluminescent reaction mixture in a chemiluminescent reaction.

[0074] The inventors have surprisingly found that the addition of an aromatic moiety in the positively charged head group of a quaternary amine cationic material further increases the chemiluminescence signal output compared to conventional materials such as cetyltrimethylammonium chloride (CTAC). Furthermore, the inventors have surprisingly found that a length of at least 3 carbon atoms in the hydrophobic tail of such quaternary amine cationic materials is additionally important for the increase in chemiluminescence signal output compared to conventional materials such as cetyltrimethylammonium chloride (CTAC) and compared to quaternary amine cationic materials having an aromatic moiety in the positively charged head group but lacking a length of at least 3 carbon atoms in the hydrophobic tail.

[0075] Thus, the new methods described according to the invention (and related uses, compositions, mixtures and / or substances) allow for even more sensitive quantification of ultra-low concentrations of analytes, but are also advantageous for signal discrimination in chemiluminescence immunoassays in general.

[0076] The chemiluminescence enhancer compounds of the present invention comprise simple components that have desirable enhancing effects for the chemiluminescence of chemiluminescent compounds, particularly the chemiluminescent compounds of formula (II) and / or formula (III) of the present invention. The chemiluminescent compositions of the present invention provide stable, long-lasting and highly enhanced chemiluminescence signals, and can be used in chemiluminescence immunoassays, DNA probe detection, and chemiluminescence analysis of biological membrane protein blotting. The chemiluminescent compositions of the present invention can be widely used in the fields of clinical diagnosis, scientific research, environmental and sanitary detection, and forensic identification. In general, the chemiluminescence enhancer compounds of the present invention are commercially available and inexpensive. Moreover, they are suitable for the manufacture of chemiluminescent compositions on a large scale. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0077] Unless otherwise indicated, terms used herein have the following meanings:

[0078] As used herein, the term "enhancing" means that the total luminescence of a chemiluminescent reaction and / or the signal to background noise ratio of a chemiluminescent reaction is higher when an enhancer according to the present invention is used than in the absence of an enhancer according to the present invention and / or compared to the so-called enhancers that have been used up to the present invention (i.e., hexadecyltrimethylammonium chloride; CTAC).

[0079] The term "alkyl," as used herein, individually or in combination with other groups, refers to a straight or branched chain alkyl group containing from 1 to 20 carbon atoms, such as from 1 to 12, 1 to 8, and 1 to 6 carbon atoms. Reference to a single straight chain alkyl, such as "n-propyl," specifically refers to a straight chain alkyl group, and reference to a single branched chain alkyl, such as "isopropyl," specifically refers to a branched chain alkyl group. For example, "C 1~6 Alkyl" is C 1~4 Alkyl, C 1~3 Alkyl includes methyl, ethyl, n-propyl, isopropyl and tert-butyl. The same rule applies to other groups used throughout this specification. Representative examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, secbutyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl and n-decyl.

[0080] The term "alkoxy" as used herein means an alkyl group, as defined herein, appended to the parent molecular moiety through an oxygen atom. Representative examples of alkoxy include, but are not limited to, methoxy, ethoxy, propoxy, 2-propoxy, butoxy, tert-butoxy, pentyloxy, and hexyloxy.

[0081] The term "aryl" as used herein refers to a phenyl group or a bicyclic or tricyclic fused ring system, where one or more of the fused rings are phenyl groups. A bicyclic fused ring system is exemplified by a phenyl group fused to a cycloalkenyl group, a cycloalkyl group, or another phenyl group. A tricyclic fused ring system is exemplified by a bicyclic fused ring system fused to a cycloalkenyl group, a cycloalkyl group, or another phenyl group, as defined herein. Representative examples of aryl include, but are not limited to, anthracenyl, azulenyl, fluorenyl, indanyl, indenyl, naphthyl, phenyl, and tetrahydronaphthyl. The aryl group of the present disclosure can be optionally substituted with 1, 2, 3, 4, or 5 substituents independently selected from the group consisting of alkoxy, alkyl, carboxyl, halo, and hydroxyl.

[0082] As used herein, the term "acyl" refers to a a is hydrogen, alkyl, cycloalkyl, cycloalkylalkyl, phenyl or phenylalkyl; a Representative examples of acyl include, but are not limited to, formyl, acetyl, cyclohexylcarbonyl, cyclohexylmethylcarbonyl, benzoyl, benzylcarbonyl, and the like.

[0083] The term "alkenyl" as used herein refers to a straight or branched chain hydrocarbon containing 2 to 20 carbons and containing at least one carbon-carbon double bond formed by the removal of two hydrogens. Representative examples of alkenyl include, but are not limited to, ethenyl, 2-propenyl, 2-methyl-2-propenyl, 3-butenyl, 4-pentenyl, 5-hexenyl, 2-heptenyl, 2-methyl-1-heptenyl, and 3-decenyl.

[0084] The term "alkynyl" as used herein refers to a straight or branched chain hydrocarbon group containing from 2 to 20 carbon atoms and containing at least one carbon-carbon triple bond. Representative examples of alkynyl include, but are not limited to, acetylenyl, 1-propynyl, 2-propynyl, 3-butynyl, 2-pentynyl, and 1-butynyl.

[0085] The term "amide," as used herein, refers to an amino group attached to the parent molecular moiety through a carbonyl group, where the term "carbonyl group" refers to a -C(O)- group.

[0086] As used herein, the term "amino" refers to R b and R c is independently selected from the group consisting of hydrogen, alkyl, and alkylcarbonyl; b R c means...

[0087] The term "aralkyl" as used herein means an aryl group appended to the parent molecular moiety by an alkyl group, as defined herein. Representative examples of arylalkyl include, but are not limited to, benzyl, 2-phenylethyl, 3-phenylpropyl, and 2-naphth-2-ylethyl.

[0088] As used herein, the term "carboxy" or "carboxyl" refers to -CO2H or -CO2.

[0089] As used herein, the term "carboxyalkyl" refers to -(CH), where n is 1 to 20. n CO2H or -(CH2) n Refers to the CO2- group.

[0090] As used herein, the term "cyano" refers to a -CN group.

[0091] As used herein, the term "cycloalkenyl" refers to a non-aromatic cyclic or bicyclic ring system having 3 to 10 carbon atoms and 1 to 3 rings, where each 5-membered ring has one double bond, each 6-membered ring has one or two double bonds, each 7- and 8-membered ring has one to three double bonds, and each 9- to 10-membered ring has one to four double bonds. Representative examples of cycloalkenyl groups include cyclohexenyl, octahydronaphthalenyl, norbornylenyl, and the like. Cycloalkenyl groups can be optionally substituted with 1, 2, 3, 4, or 5 substituents independently selected from the group consisting of alkoxy, alkyl, carboxyl, halo, and hydroxyl.

[0092] As used herein, the term "cycloalkyl" refers to a saturated monocyclic, bicyclic, or tricyclic hydrocarbon ring system having from 3 to 12 carbon atoms. Representative examples of cycloalkyl groups include cyclopropyl, cyclopentyl, bicyclo[3.1.1]heptyl, adamantyl, and the like. The cycloalkyl groups of the present disclosure can be optionally substituted with 1, 2, 3, 4, or 5 substituents independently selected from the group consisting of alkoxy, alkyl, carboxyl, halo, and hydroxyl.

[0093] As used herein, the term “cycloalkylalkyl” refers to a cycloalkyl group represented by R d is an alkylene group, and R e -R is a cycloalkyl group d R e Representative examples of cycloalkylalkyl groups include cyclohexylmethyl and the like.

[0094] As used herein, the term "halide" includes fluoride, chloride, bromide, and iodide.

[0095] As used herein, the term "hydroxyl" means an --OH group.

[0096] As used herein, the term "nitro" means a --NO.sub.2 group.

[0097] As used herein, the term "oxoalkyl" refers to an alkyl group represented by R a is hydrogen, alkyl, cycloalkyl, cycloalkylalkyl, phenyl or phenylalkyl, and n is 1 to 20; n C(O)R a Refers to...

[0098] As used herein, the term "phenylalkyl" refers to an alkyl group substituted with a phenyl group.

[0099] As used herein, the term "sulfo" refers to -SO3H or -SO3 - means a group.

[0100] As used herein, the term "sulfoalkyl" refers to -(CH), where n is 1 to 20. n SO3H or -(CH2) n SO3 - Refers to the base.

[0101] As used herein, the term "soluble" refers to the absence of precipitate.

[0102] As used herein, the term "sulfamoyl" refers to -SO2-NR1-, where R1 is selected from the group consisting of hydrogen, alkyl, aryl, and alkylcarbonyl.

[0103] As used herein, the term "sulfonamide" refers to any amide of a sulfonic acid.

[0104] The term "chemiluminescence" as used herein refers to the light that occurs in certain reactions of chemical substances. When high-energy intermediates decompose in a chemical reaction, singlet molecules are excited to form, and then the excited singlet molecules return to the ground state, and part of the energy is released in the form of luminescence. Thus, a chemiluminescence reaction includes two processes, an excitation process and a luminescence process. Some molecular energy will also be dissipated in the excited state due to intersystem and intrasystem crossing.

[0105] In a preferred embodiment, the term "diagnosis" as used herein shall be used in its broadest possible sense and may refer to any kind of procedure that aims to obtain information that will help evaluate whether a patient, known or an anonymous subject from a cohort, has suffered or may suffer from a particular disease or disorder in the past, at the time of diagnosis or in the future, or is more likely to suffer from a particular disease or disorder than comparable subjects with the average or preferably similar symptoms, in order to find out how a disease is progressing or may progress in the future, or to evaluate the responsiveness of the patient or patients in general to a particular treatment, such as the administration of an immunosuppressant drug, or to find out whether a sample is from such a patient. Such information may be used in clinical diagnosis, but may also be obtained by experimental and / or research laboratories for general research purposes, such as to determine the proportion of subjects suffering from a disease in a patient cohort or population. In other words, the term "diagnosis" includes not only diagnosing, but also predicting and / or monitoring the course of a disease or disorder, including monitoring the response of one or more patients to the administration of a drug or candidate drug to determine, for example, the efficacy of the drug. For clinical diagnostic applications the results may be assigned to a particular patient and communicated to the physician or institution treating said patient, however this is not necessarily the case for other applications, e.g. diagnostics for research purposes, in which case it may be sufficient to assign the results to anonymized patient-derived samples.

[0106] In a preferred embodiment, the term "diagnosis" may also refer to the method or agent used to select the most promising treatment regimen for a patient.In other words, the method or agent may be related to selecting a treatment regimen for a subject.For example, the detection of autoantibodies may indicate that an immunosuppressive therapy should be selected, which may include administering one or more immunosuppressive drugs to the patient.

[0107] The method according to the invention is preferably an in vitro method.

[0108] According to the present invention, the kit may include instructions on how to practice the methods of the present invention.

[0109] In a preferred embodiment, any method or use according to the invention may be for a non-diagnostic use, e.g. determining the presence of autoantibodies for a use other than diagnosing a patient. For example, the method or use may be for testing in vitro the efficiency of a medical device designed to remove autoantibodies from the blood of a patient, where the test is performed on a fluid other than the patient's blood.

[0110] In another preferred embodiment, the method may be for confirming the reliability of a diagnostic assay and may comprise detecting antibodies against the polypeptide.

[0111] In a preferred embodiment, any method or use according to the invention may be for identifying subjects suffering from or at risk of developing a disease and / or tumor. [Brief description of the drawings]

[0112] [Figure 1]Chemiluminescence kinetics of 1 nM NSP-DMAE-NHS in the presence of 0.5% (w / v) of three different cationic compounds, each with an aromatic head group (compound 1: benzyldimethylhexadecylammonium chloride; compound 2: benzalkonium chloride; compound 3: alkyl(C12-C14)dimethylethylbenzyl-ammonium chloride), or in the presence of 0.5% (w / v) of the cationic compound CTAC, which lacks an aromatic head group. [Diagram 2] Chemiluminescence kinetics of 1 nM NSP-SA-NHS in the presence of 0.5% (w / v) of three different cationic compounds, each with an aromatic head group (compound 1: benzyldimethylhexadecylammonium chloride; compound 2: benzalkonium chloride; compound 3: alkyl(C12-C14)dimethylethylbenzyl-ammonium chloride), or in the presence of 0.5% (w / v) of the cationic detergent CTAC, which lacks an aromatic head group. [Diagram 3] Chemiluminescence kinetics of 0.1 μg / mL anti-human IgG-Fcγ-NSP-DMAE in the presence of 0.5% (w / v) of three different cationic compounds, each with an aromatic head group (compound 1: benzyldimethylhexadecylammonium chloride; compound 2: benzalkonium chloride; compound 3: alkyl(C12-C14)dimethylethylbenzyl-ammonium chloride), or in the presence of 0.5% (w / v) of the cationic detergent CTAC, which lacks an aromatic head group. [Figure 4] Chemiluminescence kinetics of 0.1 μg / mL anti-human IgG-Fcγ-NSP-SA in the presence of 0.5% (w / v) of three different cationic compounds, each with an aromatic head group (compound 1: benzyldimethylhexadecylammonium chloride; compound 2: benzalkonium chloride; compound 3: alkyl(C12-C14)dimethylethylbenzyl-ammonium chloride), or in the presence of 0.5% (w / v) of the cationic detergent CTAC, which lacks an aromatic head group. [Diagram 5]Chemiluminescence kinetics of 1 nM NSP-DMAE-NHS in the presence of 0.5% (w / v) of four different cationic compounds, each with an aromatic head group and a long hydrophobic tail with at least 3 carbon atoms (compound 1: benzyl dimethyl hexadecyl ammonium chloride; compound 2: benzalkonium chloride; compound 3: alkyl (C12-C14) dimethyl ethyl benzyl-ammonium chloride; compound 4: benzyl dimethyl dodecyl ammonium chloride), or in the presence of 0.5% (w / v) benzyl triethyl ammonium chloride (BTEAC), which contains an aromatic head group but lacks a long hydrophobic tail, or in the presence of 0.5% (w / v) CTAC, which lacks an aromatic head group but contains a long hydrophobic tail. [Figure 6] Chemiluminescence kinetics of 1 nM NSP-SA-NHS in the presence of 0.5% (w / v) of four different cationic compounds, each with an aromatic head group and a long hydrophobic tail with at least 3 carbon atoms (compound 1: benzyldimethylhexadecylammonium chloride; compound 2: benzalkonium chloride; compound 3: alkyl(C12-C14)dimethylethylbenzyl-ammonium chloride; compound 4: benzyldimethyldodecylammonium chloride), or in the presence of 0.5% (w / v) of BTEAC, which contains an aromatic head group but lacks a long hydrophobic tail, or in the presence of 0.5% (w / v) of CTAC, which lacks an aromatic head group but contains a long hydrophobic tail. [Figure 7] Shown are the chemiluminescence kinetics of 0.1 µg / mL anti-human IgG-Fcγ-NSP-DMAE in the presence of 0.5% (w / v) of four different cationic compounds, each with an aromatic head group and a long hydrophobic tail with at least 3 carbon atoms (compound 1: benzyl dimethyl hexadecyl ammonium chloride; compound 2: benzalkonium chloride; compound 3: alkyl (C12-C14) dimethyl ethyl benzyl-ammonium chloride; compound 4: benzyl dimethyl dodecyl ammonium chloride), or in the presence of 0.5% (w / v) BTEAC, which contains an aromatic head group but lacks a long hydrophobic tail, or in the presence of 0.5% (w / v) CTAC, which lacks an aromatic head group but contains a long hydrophobic tail. [Figure 8] Chemiluminescence kinetics of 0.1 µg / mL anti-human IgG-Fcγ-NSP-SA in the presence of 0.5% (w / v) of four different cationic compounds, each with an aromatic head group and a long hydrophobic tail with at least 3 carbon atoms (compound 1: benzyl dimethyl hexadecyl ammonium chloride; compound 2: benzalkonium chloride; compound 3: alkyl (C12-C14) dimethyl ethyl benzyl-ammonium chloride; compound 4: benzyl dimethyl dodecyl ammonium chloride) or in the presence of 0.5% (w / v) BTEAC, which contains an aromatic head group but lacks a long hydrophobic tail, or in the presence of 0.5% (w / v) CTAC, which lacks an aromatic head group but contains a long hydrophobic tail. EXAMPLES

[0113] The present invention is further illustrated by the following non-limiting examples from which further features, embodiments, aspects and advantages of the present invention may be understood.

[0114] Preparation of acridinium label and conjugate solutions: Labeling stock solutions of 1 mg / mL of N-sulfopropyl-dimethyl-acridinium-N-hydroxysuccinimide (NSP-DMAE-NHS; PerkinElmer Inc., Finland) and N-sulfopropyl-acridinium-sulfonamide-N-hydroxysuccinimide (NSP-SA-NHS; BOC Sciences, USA) were each prepared in 100% (v / v) N,N-dimethylformamide (Th. Geyer Hamburg GmbH & Co. Kg, Germany). AffiniPure goat anti-human IgG, Fcγ fragment specific (anti-human IgG-Fcγ; Jackson ImmunoResearch Europe Ltd., UK) was diluted to a final concentration of 0.33 mg / mL in phosphate-buffered saline, pH 7.6, and then incubated with a 10-fold molar excess of NSP-DMAE-NHS or with a 2.5-fold molar excess of NSP-SA-NHS for 2 h at room temperature in an Intelli-Mixer, type RM-2M (Hassa GmbH, Germany) at 10 rpm. The labeling reaction was stopped by the addition of 17.4 mM L-lysine (VWR International GmbH, Germany) for 30 min at room temperature during rotation at 10 rpm. Excess free label was removed from the antibody conjugate during ultrafiltration at 3,800 g in a Heraeus Megafuge 40R centrifuge (Fisher Scientific GmbH, Germany) using Vivaspin 20, molecular weight cut-off 10 kDa concentrators (VWR International GmbH, Germany) at 4° C., and the buffer was changed to phosphate-buffered saline, pH 7.4, containing 0.045% (w / v) sodium azide (Merck KgaA, Germany).The purified antibody conjugate was stored in phosphate buffered saline containing 0.045% (w / v) sodium azide, 1% (w / v) bovine serum albumin (Th. Geyer Hamburg GmbH & Co. Kg, Germany), 0.3% (w / v) Kolliphor® P 188 (Th. Geyer Hamburg GmbH & Co. Kg, Germany) and 0.5 mM ethylenediaminetetraacetic acid disodium salt (Gerbu Biotechnik GmbH, Germany).

[0115] Preparation of chemiluminescence trigger solution: Chemiluminescence trigger solution A containing 100 mM nitric acid (VWR International GmbH, Germany) and 0.2% (v / v) hydrogen peroxide (Roth GmbH & Co. KG, Germany) and trigger solution B containing 0.5 M sodium hydroxide (Gerbu Biotechnik GmbH, Germany) were prepared. Trigger solution B was used without detergent or supplemented with 0.05, 0.1, 0.25 or 0.5% (w / v) of hexadecyltrimethylammonium chloride (Merck KgaA, Gemany), hexadecyltrimethylammonium bromide (Merck KgaA, Gemany) or tetradecyltrimethylammonium chloride (Merck KgaA, Gemany), all cationic compounds lacking aromatic head groups. Alternatively, trigger solution B was supplemented with 0.05, 0.1, 0.25 or 0.5% (w / v) of the cationic compounds hexadecylbenzyldimethylammonium chloride (Merck KgaA, Gemany; CAS 122-18-9), benzalkonium chloride (Merck KgaA, Gemany; CAS 63449-41-2), alkyl (C12-C14) dimethylethylbenzyl-ammonium chloride (Alfa Chemistry, USA; CAS 85409-23-0) or benzyldimethyldodecylammonium chloride (VWR International GmbH, Germany; CAS 139-07-1), all of which contain aromatic head groups, or benzyltriethylammonium chloride (Merck KgaA, Gemany), which contains an aromatic head group but lacks a long hydrophobic tail of at least 3 carbon atoms.

[0116] Measurement of chemiluminescence signal intensity: NSP-DMAE-NHS and NSP-SA-NHS labeling stock solutions were diluted to a final concentration of 1 nM, and antibody conjugates were diluted to a final concentration of 0.1 μg / mL in phosphate-buffered saline, pH 7.4. 10 μL of each of these dilutions were transferred to wells of a 96-well LumiNunc™ plate (VWR International GmbH, Germany), and chemiluminescence signal intensity was measured in a LB 960 Centro Microplate Luminometer (Berthold Technologies GmbH & Co.KG, Germany) in the presence of 100 μL each of chemiluminescence trigger A solution and different trigger B solutions with different cationic compound identities and concentrations. After injection of trigger A at medium speed, the mixture was incubated for 1 second during mixing at normal speed in double orbit mode. Chemiluminescence intensity in relative luminescence units (RLU) was recorded every 50 ms for a total time of 1.5 seconds simultaneously with the addition of different trigger B solutions with low injection speed. All samples were measured in four technical replicates. In a control experiment, all the different trigger B solutions were injected in combination with trigger A into empty wells of a 96-well plate. The latter experiment reflects the signal background caused by the different trigger solutions.

[0117] Data Analysis: For graphical representation of the observed chemiluminescence kinetics, a representative data set was selected from four technical replicates and the signal intensity in RLU was plotted against time. Furthermore, the total signal intensity was calculated by addition of all RLU values ​​observed during a 1.5 second time window. The background signal intensities of the different Trigger B solutions were subtracted from the total signal intensity and all background-corrected total signal intensities were compared.

[0118] [Example 1] Three representative enhancer compounds according to the present invention (compounds 1-3) were selected for analysis. These molecules are all based on cationic quaternary ammonium compounds containing an aromatic moiety adjacent to the quaternary ammonium. The enhancer compounds according to the present invention were tested against the conventional enhancer substance CTAC (e.g., as proposed in U.S. Pat. No. 4,927,769). In contrast, CTAC does not contain an aromatic moiety adjacent to the quaternary ammonium.

[0119] Four representative analytes were selected to investigate the effect of the enhancer compounds of the present invention on the chemiluminescence signals generated by these analytes in comparison with conventional enhancer substances already known in the prior art (Figures 1-4). The selected analytes include the acridinium compounds NSP-DMAE-NHS and NSP-SA-NHS in free form (Figures 1-2) and in antibody-bound form (Figures 3-4). A goat-derived polyclonal anti-human IgG-Fcγ antibody was selected for conjugation with both types of acridinium labels.

[0120] The acridinium label, conjugate solution and chemiluminescence trigger solution were obtained by the methods disclosed above. Measurements of chemiluminescence signal intensity were performed and the data were analyzed as described above.

[0121] result: A 43% increase in signal output using benzyldimethylhexadecylammonium chloride (compound 1) with the analyte NSP-DMAE-NHS (FIG. 1, Table 3) was observed compared to the enhancer substance CTAC for the same analyte.

[0122] Benzalkonium chloride (compound 2) and alkyl (C 12 ~C 14 ) Dimethylethylbenzylammonium chloride (compound 3) also provided a 19% and 15% signal increase, respectively, compared to CTAC with NSP-DMAE-NHS as the analyte.

[0123] Similar results were obtained when the analyte was changed to NSP-SA-NHS (Figure 2, Table 3).

[0124] Compound 1 (benzyldimethylhexadecylammonium chloride) resulted in a 67% increased signal compared to the substance CTAC. Furthermore, a 58% and 40% signal increase with compounds 2 and 3, respectively, was observed for this analyte compared to CTAC.

[0125] Furthermore, it was found that the increased signal output observed in both cases with free acridinium-based probes could also be observed when the probes were covalently bound to antibodies, as is typically required for immunoassays.

[0126] In this scenario, compound 1 (benzyldimethylhexadecylammonium chloride) resulted in a 45% and 66% signal increase for the analytes anti-human IgG-Fcγ-NSP-DMAE (FIG. 3, Table 3) and anti-human IgG-Fcγ-NSP-SA (FIG. 4, Table 3), respectively, when compared to the signal generation of those analytes in the presence of CTAC. Compound 2 (benzalkonium chloride) also increased the signal of these two antibody conjugates by 40% and 52%, respectively. Furthermore, compound 3 (alkyl(C 12 ~C 14 A 25% and 35% signal increase in the presence of dimethylethylbenzylammonium chloride) was observed compared to signal generation in the presence of CTAC.

[0127] [Table 3]

[0128] [Example 2] explanation: To test the effect of chain length in the hydrophobic tail of the material on chemiluminescence enhancement, further experiments were carried out with compounds 1-3 (as described in Example 1), with the regular enhancer CTAC, with an additional cationic enhancer according to the invention (compound 4; benzyldimethyldodecylammonium chloride), and with an additional material BTEAC (benzyltriethylammonium chloride) containing a chain length of less than 3 carbon atoms in the hydrophobic tail (Figures 5-8). These further experiments were carried out as described above and in Example 1, respectively.

[0129] result: Similar to compounds 1-3, compound 4 enhanced the chemiluminescence of 1 nM NSP-DMAE-NHS (Figure 5), 1 nM NSP-SA-NHS (Figure 6), 0.1 μg / mL anti-human IgG-Fcγ-NSP-DMAE (Figure 7), and 0.1 μg / mL anti-human IgG-Fcγ-NSP-SA (Figure 8) compared to the conventional enhancer CTAC. In this experiment, compounds 1–3 increased the chemiluminescence of 1 nM NSP-DMAE-NHS by 45–79%, 1 nM NSP-SA-NHS by 62–102%, 0.1 μg / mL anti-human IgG-Fcγ-NSP-DMAE by 46–63%, and 0.1 μg / mL anti-human IgG-Fcγ-NSP-SA by 47–50% compared to the conventional enhancer CTAC (Table 4). Similarly, compound 4 increased the chemiluminescence of 1 nM NSP-DMAE-NHS by 50%, 1 nM NSP-SA-NHS by 47%, 0.1 μg / mL anti-human IgG-Fcγ-NSP-DMAE by 50%, and 0.1 μg / mL anti-human IgG-Fcγ-NSP-SA by 40% compared to the regular enhancer CTAC (Table 4).

[0130] For BTEAC, a decrease in chemiluminescence was observed with all four analytes compared to the conventional enhancer CTAC (Figures 5-8 and Table 4). Although this material contains an aromatic head group, it does not function as an equivalent chemiluminescence enhancer, presumably, and without being bound by any theory, because the chain length of the hydrophobic tail is too short to form micellar structures.

[0131] [Table 4]

[0132] Overall, the data obtained in the described examples show that all four tested compounds, which represent examples of enhancers presented by the present invention, were able to increase the signal generated by all tested acridinium-based analytes compared to the signal generated by the same acridinium-based analytes in the presence of a conventional enhancer as CTAC.

[0133] Moreover, these new chemiluminescence enhancers all share a molecular structure that includes a cationic quaternary ammonium compound linked to an aromatic moiety, i.e., an aromatic head group, and a minimum chain length of at least 3 carbon atoms in the hydrophobic tail of the enhancer. It is possible that all substances that include these molecular moieties, i.e., a minimum chain length of at least 3 carbon atoms in the aromatic head group and the hydrophobic tail, will similarly increase the chemiluminescence generated by the acridinium-based analyte, compared to enhancer substances that lack these molecular moieties, i.e., a minimum chain length of at least 3 carbon atoms in the aromatic head group and the hydrophobic tail. Substances with shorter chain lengths in the hydrophobic tail do not similarly increase the chemiluminescence, presumably because they do not form micellar structures that are believed to favor chemiluminescence enhancement. Without being bound by theory, it is further believed that the newly proposed cationic chemiluminescence enhancers according to the present invention favor the chemiluminescence reaction process by direct interaction with the acridinium ring, forming so-called hydrophobic π-π stacking interactions. In this way, the acridinium label can be efficiently mobilized and positioned for reaction with hydrogen peroxide anions attracted by the positive charge of the adjacent cationic ammonium ion.

[0134] The data, figures, devices, reagents, and steps herein should be understood to be illustrative, not limiting. Although the present disclosure has been described with reference to the above specific embodiments, many modifications and variations will be apparent to those skilled in the art. All modifications and variations are within the spirit and scope of the present disclosure.

Claims

1. A method for increasing a chemiluminescence signal in a chemiluminescence reaction of a chemiluminescent compound in a chemiluminescent reaction mixture, comprising: oxidizing the chemiluminescent compound in the presence of an effective amount of an enhancer to obtain an increased chemiluminescence signal, wherein the chemiluminescent compound is an acridinium compound, preferably an acridinium ester or an acridinium sulfonamide, the enhancer is one compound or a mixture of two, three or more compounds, and each of the one enhancer compound or the plurality of enhancer compounds is a quaternary amine cationic substance of formula (I) 【Chemical 1】 (wherein, R １ is C ３ - C ２０ alkyl or alkenyl; R ２ and R ３ are, independently of each other, C １ - C ４ alkyl or alkenyl; R ４ , R ５ , R ６ , R ７ and R ８ are, independently of each other, hydrogen, alkyl, alkenyl; X １－ is a halide ion or a hydroxyl ion, preferably a chloride ion or a bromide ion) selected from the group consisting of. A method.

2. -R １ is C ５ -C １８ alkyl or alkenyl; -R ２ and R ３ are, independently of one another, methyl, ethyl, propyl or butyl, preferably methyl; -R ４ 、R ５ 、R ６ 、R ７ and R ８ are, independently of one another, hydrogen, alkyl, alkenyl, preferably hydrogen, methyl or ethyl; and / or -X １－ The method according to claim 1, wherein X is a chloride ion or a bromide ion, preferably a chloride ion.

3. The enhancer, at least one compound of the enhancer or each compound of the enhancer is - Benzyldimethylicosylammonium chloride, - Benzyldimethylnonadecylammonium chloride, - Benzyldimethyloctadecylammonium chloride, - Benzyldimethylheptadecylammonium chloride, - Benzyldimethylhexadecylammonium chloride, - Benzyldimethylpentadecylammonium chloride, - Benzyldimethyltetradecylammonium chloride, - Benzyldimethyltridecylammonium chloride, - Benzyldimethyldodecylammonium chloride, - Benzyldimethylundecylammonium chloride, - Benzyldimethyldecylammonium chloride, - Benzyldimethylnonylammonium chloride, - Benzyldimethyloctylammonium chloride, - Benzalkonium chloride, - Benzyldimethylheptylammonium chloride, - Benzyldimethylhexylammonium chloride, - Benzyldimethylpentylammonium chloride, - Benzyldimethylbutylammonium chloride, - Benzyldimethylpropylammonium chloride, - Benzyltripropylammonium chloride, - Benzyldipropylethylammonium chloride, - Benzyldiethylpropylammonium chloride, - Benzyldipropylmethylammonium chloride, - Benzylpropylethylmethylammonium chloride, - Benzyltributylammonium chloride, - Benzyl dibutyl propyl ammonium chloride, - Benzyl dipropyl butyl ammonium chloride, - Benzyl butyl propyl ethyl ammonium chloride, - Benzyl butyl propyl methyl ammonium chloride, - Benzyl butyl ethyl methyl ammonium chloride, - Benzyl diethyl butyl ammonium chloride, -C １２～１４ -Alkyldimethyl(ethylbenzyl)ammonium chloride, - Dodecyl (ethylbenzyl) dimethyl ammonium chloride, - Tetradecyl dimethyl (ethylbenzyl) ammonium chloride, - Octadecyl dimethyl (ar-ethylbenzyl) ammonium chloride, and the method according to claim 1, selected from the group consisting of each of the above-mentioned compounds containing bromide instead of chloride.

4. The effective amount of the enhancer in the chemiluminescence reaction mixture is 0.001% or more by weight per volume (w / v), preferably 0.01% w / v, more preferably 0.1% or more w / v, most preferably 0.2% or more w / v, based on the total volume of the chemiluminescence reaction mixture in each case. The method according to claim 1.

5. The enhancer is combined with the chemiluminescent compound in the chemiluminescence reaction mixture before, simultaneously, or immediately after the start of the chemiluminescence reaction. Preferably, the enhancer is combined with the chemiluminescent compound in the chemiluminescence reaction mixture simultaneously with the start of the chemiluminescence reaction. The method according to claim 1.

6. The chemiluminescence signal generated by the chemiluminescence reaction is detected for a certain duration. Preferably, the chemiluminescence reaction mixture is formed, the chemiluminescence reaction is started, and the signal is detected simultaneously. More preferably, the duration of the detection is in the range of 0.01 to 360 seconds, even more preferably 0.1 to 30 seconds, even more preferably 0.3 to 15 seconds, and most preferably 0.5 to 10 seconds. In each case, the duration of the detection starts with the start of the chemiluminescence reaction. The method according to claim 1.

7. The chemiluminescence reaction mixture further contains hydrogen peroxide, nitric acid, and / or sodium hydroxide. The method according to claim 1.

8. The chemiluminescent compound is an acridinium ester according to formula (II) 【Chemical 2】 (In the formula, R １ is alkyl, alkenyl, alkynyl, aryl or aralkyl, sulfoalkyl, carboxyalkyl and oxoalkyl; R ３ from R １５ are each independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, aryl or aralkyl, amino, amide, acyl, alkoxyl, hydroxyl, carboxyl, halogen, halide, nitro, cyano, sulfo, sulfoalkyl, sulfamoyl, carboxyalkyl, succinimidyl ester and oxoalkyl or any other leaving group; Optionally, if present, X １－ is an anion); or an acridinium sulfonamide according to formula (III) [Chemical 3] (wherein, R １ is selected from the group consisting of alkyl, alkenyl, alkynyl, aryl or aralkyl, sulfoalkyl, carboxyalkyl and oxoalkyl, and R ２ from R １５ is independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, aryl or aralkyl, amino, amide, acyl, alkoxyl, hydroxyl, carboxyl, halogen, halide, nitro, cyano, sulfo, sulfoalkyl, sulfamoyl, carboxyalkyl, succinimidyl and oxoalkyl or any other leaving group; Optionally, if present, X １－ is an anion) The method according to claim 1, which is such.

9. A quaternary amine cationic substance of formula (I) for increasing the chemiluminescence signal in the chemiluminescence reaction of an acridinium compound, preferably an acridinium ester or an acridinium sulfonamide, preferably in a chemiluminescence immunoassay [Chemical Formula 4] (wherein R １ is C ３ - C ２０ alkyl or alkenyl; R ２ and R ３ are, independently of each other, C １ - C ４ alkyl or alkenyl; R ４ , R ５ , R ６ , R ７ and R ８ are, independently of each other, hydrogen, alkyl, alkenyl; X １－ is a halide ion or a hydroxyl ion, preferably a chloride ion or a bromide ion) for use of a compound selected from the group consisting of.

10. -R １ is C ５ ~C １８ is alkyl or alkenyl; -R ２ and R ３ are, independently of one another, methyl, ethyl, propyl or butyl, preferably methyl, -R ４ , R ５ , R ６ , R ７ and R ８ are, independently of one another, hydrogen, alkyl, alkenyl, preferably hydrogen, methyl or ethyl; and / or - X １－ The use according to claim 9, wherein X is a chloride ion or a bromide ion, preferably a chloride ion.

11. The quaternary amine cationic substance of formula (I) ​ (wherein R １ is C ３ - C ２０ alkyl or alkenyl; R ２ and R ３ are each independently C １ - C ４ alkyl or alkenyl; R ４ , R ５ , R ６ , R ７ and R ８ are each independently hydrogen, alkyl, alkenyl; X １－ is a halide ion or a hydroxyl ion, preferably a chloride ion or a bromide ion) and at least one compound selected from the group consisting of At least one chemiluminescent compound, which is an acridinium compound, preferably an acridinium ester or an acridinium sulfonamide, more preferably an acridinium ester or an acridinium sulfonamide according to formula (II) or formula (III) as described in any one of claims 1 to 10, and A chemiluminescent composition comprising the same.

12. At least one chemiluminescent compound, which is an acridinium compound, preferably an acridinium ester or an acridinium sulfonamide, more preferably an acridinium ester or an acridinium sulfonamide according to formula (II) or formula (III) as described in any one of claims 1 to 11, and The quaternary amine cationic substance of formula (I) [Chemical Formula 6] (wherein, R １ is C ３ - C ２０ alkyl or alkenyl; R ２ and R ３ are, independently of each other, C １ - C ４ alkyl or alkenyl; R ４ , R ５ , R ６ , R ７ and R ８ are, independently of each other, hydrogen, alkyl, alkenyl; X １－ is a halide ion or a hydroxyl ion, preferably a chloride ion or a bromide ion) and one, two or more compounds selected from the group consisting of A kit, preferably for increasing the chemiluminescence of the chemiluminescent compound, comprising the same.

13. A diagnostically useful carrier, preferably a solid carrier, comprising means for capturing an antigen, an antibody or an autoantibody in a liquid solution and means for detecting the antigen, the antibody or the autoantibody bound to the carrier, The kit according to claim 12, wherein the means for detecting the antigen, antibody or autoantibody bound to the carrier is labeled with the at least one chemiluminescent compound.

14. The chemiluminescent composition according to claim 11 or the kit according to claim 12 or 13 for use in the diagnosis of a disease.

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