Methods and reagents for enhancing chemiluminescent signals

Abstract

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

Description

[0001] The present invention relates to a method for enhancing the chemiluminescent signal of a chemiluminescent compound in a chemiluminescent reaction mixture in a chemiluminescent reaction, the method comprising oxidizing the chemiluminescent compound in the presence of an effective amount of an enhancer to obtain an enhanced chemiluminescent signal, using a compound selected from the group consisting of quaternary ammonium cationic substances of formula (I) to enhance the chemiluminescent signal of an acridinium compound, preferably an acridinium ester or an acridinium sulfonamide, in a chemiluminescent reaction, preferably in a chemiluminescent immunoassay, as well as related chemiluminescent compositions and related kits.

[0002] As a non-radioactive immunoassay technology, chemiluminescent immunoassay has developed rapidly around the world following enzyme immunoassay (EIA), radioimmunoassay and fluorescent immunoassay technology. Chemiluminescent immunoassay is a technology with high sensitivity, wide detection range, simple and rapid operation, stable labeling and low pollution, making it an ideal quantitative immunoassay method (Cinquanta et al., "Chemiluminescent immunoassay technology: what does it change in autoantibody detection?", Auto Immun Highlights, 8:9, June 24, 2017).

[0003] Nowadays, chemiluminescent immunoassays are often preferred over conventional detection methods such as radioimmunoassays for quantifying various biological substances such as small molecules, nucleic acids, hormones, antibodies, etc. It is characterized in that the chemiluminescent substrate produces light due to external stimulation of a chemical or enzymatic triggering substance. Prominent examples of chemiluminescent substrates are luminol, 1,2-dioxetane aryl phosphates, tris-(bipyridyl)ruthenium(II) complexes or acridinium-based probes. In general, chemiluminescent reactions can be divided into flash-type and glow-type chemiluminescence, depending on the nature of the chemiluminescent substrate, the applied triggering substance and the final type of light output. As described by Tannouset et al. (Tannouset et al., "Combined flash and glow-type chemiluminescent reactions for high-throughput genotyping of biallelicpolymorphisms," Analytical Biochemistry, Vol. 320, No. 2, pp. 266-272, Sep. 15, 2003), flash-type reactions produce bright chemiluminescent signals in the range of seconds to minutes, while the light output in glow-type reactions is not as bright, but it can last for hours. Depending on the method selected, flash-type and glow-type immunoassays can be designed accordingly.

[0004] If high throughput of samples is required, flash probes based on acridinium salts are often used in chemiluminescent immunoassays for rapid and ultrasensitive signal generation. Prominent 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), which molecules and variants thereof are described, for example, in EP1273917 B1, EP 1539702 B1 and other literature. All of these molecules generate light under external stimulation with alkaline hydrogen peroxide. Typically, the latter reagents are added stepwise to trigger solutions A and B. Hydrogen peroxide is usually part of a combination of trigger A with an acid such as nitric acid. Thereafter, sodium hydroxide is injected into trigger B.

[0005] In order to produce an optimal signal, trigger B is additionally believed to require a detergent molecule that may form a micellar structure in aqueous solution. Chang and Miller describe in US 4,927,769 that cationic detergents based on quaternary amines are particularly advantageous. Although nonionic or anionic detergents are also possible, the positive charge in the head group of the cationic detergent is believed to bring the mostly hydrophobic acridinium-based probe and the negatively charged hydroperoxide anion into the spatial vicinity of the micelle interface, thereby promoting the chemiluminescent signal output, as also described in Natrajanet et al. (Natrajan et al., "Effect of surfactants on the chemiluminescence of acridiniumdimethylphenylester labels and their conjugates," Org. Biomol. Chem., 9, pp. 5092-5103, April 13, 2011). A prominent example of such a cationic detergent included in US 4,927,769 is hexadecyltrimethylammonium chloride (CTAC).

[0006] Nevertheless, further improvements are needed in chemiluminescent immunoassay technology to allow quantification of ultra-low concentrations of analytes and generally facilitate signal-to-noise discrimination. These improvements will most likely require enhancement of chemiluminescence by optimized enhancer substances.

[0007] The problem underlying the present invention is to provide methods and related reagents for chemiluminescent immunoassay techniques which can be used for improved quantification of analytes, in particular analytes having ultra-low concentrations, and which can generally be used to better facilitate signal-to-noise discrimination.

[0008] Another problem underlying the present invention is to provide substances which are able to enhance the chemiluminescent signal of chemiluminescent compounds, preferably acridinium compounds, such as acridinium esters or acridinium sulfonamides, in chemiluminescent immunoassays.

[0009] In a first aspect, the problem underlying the present invention is solved by a method for enhancing the chemiluminescent signal of a chemiluminescent compound in a chemiluminescent reaction mixture in a chemiluminescent reaction, said method comprising oxidizing the chemiluminescent compound in the presence of an effective amount of an enhancer to obtain an enhanced chemiluminescent signal, wherein the chemiluminescent compound is an acridinium compound, preferably an acridinium ester or an acridinium sulfonamide, wherein said enhancer is one compound or a mixture of two, three or more compounds, and wherein said enhancer compound or each of these enhancer compounds is selected from the group consisting of quaternary ammonium cationic species of formula (I):

[0010]

[0011] Where R 1 C3-C 20 alkyl or alkenyl; and R 2 and R 3 are independently C1-C4 alkyl or alkenyl; and R 4 , R 5 , R 6 , R 7 and R 8 are independently hydrogen, alkyl, alkenyl; and X 1- is a halide or hydroxide ion, preferably chloride or bromide.

[0012] An effective amount of enhancer according to the present invention is provided by one, two or more enhancer compounds, each of which is selected from the group consisting of quaternary ammonium cationic species of formula (I) disclosed herein.

[0013] Each enhancer compound according to the present invention has an aromatic head group.Possible anionic counterions for the cationic portion of the enhancer compounds according to the present invention include, but are not limited to, chloride, bromide and hydroxide.

[0014] In a preferred embodiment, the method according to the present invention comprises the step of adding an enhancer to the chemiluminescent reaction mixture containing the chemiluminescent compound (ie an acridinium compound, such as an acridinium ester or an acridinium sulfonamide).

[0015] In a preferred embodiment, the or each enhancer compound according to the present invention is selected from the group consisting of quaternary ammonium cationic species of formula (I) disclosed herein, wherein R 1 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 Alkyl or alkenyl; R 2 and R 3 are independently methyl, ethyl, propyl or butyl, preferably methyl; R 4 , R 5 , R 6 , R 7 and R 8 are independently hydrogen, alkyl, alkenyl, preferably hydrogen, methyl or ethyl; and / or X 1- It is a chloride or a bromide, preferably a chloride.

[0016] In a preferred embodiment, the enhancer compound, at least one compound of the enhancer or each compound of the enhancer according to the invention is selected from the group consisting of:

[0017] -Benzyldimethyleicosylammonium chloride,

[0018] -Benzyldimethylnonadecylammonium chloride,

[0019] -Benzyldimethyloctadecylammonium chloride,

[0020] -Benzyldimethylheptadecylammonium chloride,

[0021] -Benzyldimethylhexadecylammonium chloride,

[0022] -Benzyldimethylpentadecylammonium chloride,

[0023] -Benzyldimethyltetradecylammonium chloride,

[0024] -Benzyldimethyltridecylammonium chloride,

[0025] -Benzyldimethyldodecylammonium chloride,

[0026] -Benzyldimethylundecylammonium chloride,

[0027] -Benzyldimethyldecylammonium chloride,

[0028] -Benzyldimethylnonylammonium chloride,

[0029] -Benzyl dimethyl octyl ammonium chloride,

[0030] -Benzalkonium chloride,

[0031] -Benzyl dimethyl heptyl ammonium chloride,

[0032] -Benzyl dimethyl hexyl ammonium chloride,

[0033] -Benzyl dimethyl amyl ammonium chloride,

[0034] -Benzyl dimethyl butyl ammonium chloride,

[0035] -Benzyl dimethyl propyl ammonium chloride,

[0036] -Benzyltripropylammonium chloride,

[0037] -Benzyl dipropyl ethyl ammonium chloride,

[0038] -Benzyl diethyl propyl ammonium chloride,

[0039] -Benzyl dipropyl methyl ammonium chloride,

[0040] -Benzylpropylethylmethylammonium chloride,

[0041] -Benzyltributylammonium chloride,

[0042] -Benzyl dibutyl propyl ammonium chloride,

[0043] -Benzyl dipropyl butyl ammonium chloride,

[0044] -Benzylbutylpropylethylammonium chloride,

[0045] -Benzylbutylpropylmethylammonium chloride,

[0046] -Benzylbutylethylmethylammonium chloride,

[0047] -Benzyl diethyl butyl ammonium chloride,

[0048] -C 12-14 -Alkyl dimethyl (ethyl benzyl) ammonium chloride,

[0049] -Dodecyl(ethylbenzyl)dimethylammonium chloride,

[0050] -Tetradecyl dimethyl (ethylbenzyl) ammonium chloride,

[0051] -octadecyldimethyl(ar-ethylbenzyl)ammonium chloride and the corresponding above compounds containing bromide instead of chloride.

[0052] Table 1 shows a list of exemplary enhancer compounds (cationic substances with aromatic head groups suitable as chemiluminescence enhancers) including molecular structures according to the present invention. In order to clarify the identity of the listed substances, although there may be ambiguity in the chemical nomenclature, if the Chemical Abstracts Service (CAS) registration number and / or compound record (CID) can be found and is publicly available, the information is listed in Table 1. Chemical Abstracts Service registration numbers according to the classification of the American Chemical Society can be obtained through https: / / www.cas.org or https: / / commonchemistry.cas.org. CID records can be found in the PubChem database of the National Institutes of Health through https: / / pubchem.ncbi.nlm.nih.gov. If a CAS and / or CID is not assigned to a compound, or there is no publicly available information, the corresponding compound is defined as "nd".

[0053]

[0054]

[0055]

[0056]

[0057]

[0058]

[0059] The enhancer according to the present invention may be composed of a mixture of two, three or more of the above compounds in Table 1, and the mixture may also be commercially available as a separate product. An example of a mixture of two, three or more of the above compounds in Table 1 (which may be commercially available as a separate product) is, for example, benzalkonium chloride or benzalkonium bromide. Benzalkonium chloride is a mixture of alkylbenzyldimethylammonium chloride (ABDAC), the alkyl portion of which ranges from C8 to C 188 Chain composition. Depending on the composition of the mixture, benzalkonium chloride can be found in a variety of commercial preparations and CAS numbers.

[0060] If the enhancer compound according to the present invention comprises a chiral C atom, all isomeric forms of the enhancer compound are disclosed and included.

[0061] In a preferred embodiment, the effective amount of the enhancer in the chemiluminescent reaction mixture is 0.001% weight / volume (w / v) or more, preferably 0.01% w / v, more preferably 0.1% w / v or more, and most preferably 0.2% w / v or more, in each case based on the total volume of the chemiluminescent reaction mixture. In general, in a chemiluminescent reaction, the effective amount of enhancer required to enhance the chemiluminescent signal of the chemiluminescent compound in the chemiluminescent reaction mixture will vary depending on the enhancer selected and can be determined empirically. In general, the effective amount of enhancer in the chemiluminescent reaction mixture is greater than 0.005% weight / volume, preferably greater than 0.01% w / v, in each case based on the total volume of the chemiluminescent reaction mixture. In a preferred embodiment, the amount of the enhancer for achieving an enhanced chemiluminescent signal contained in the chemiluminescent 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, most preferably in the range of 0.2% w / v to 0.5% w / v, in each case based on the total volume of the chemiluminescent reaction mixture. In a particularly preferred embodiment, the amount of the enhancer for achieving an enhanced chemiluminescent signal contained in the chemiluminescent 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 chemiluminescent reaction mixture.

[0062] Preferably, the chemiluminescent 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 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, acidic aqueous solutions or alkaline aqueous solutions.

[0063] In a preferred embodiment, the enhancer according to the invention is soluble in the chemiluminescent reaction mixture.

[0064] In a preferred embodiment, the method according to the invention is performed by or is part of an immunoassay, wherein the immunoassay preferably includes a step of detecting the presence or absence of an antigen, an antibody or an autoantibody. The immunoassay may preferably be selected from the group comprising a competitive assay, a capture bridge assay, an immunometric assay, a direct or indirect class capture assay. The principles of each of these formats are described in detail in The Immunoassay Handbook, 3rd edition, edited by David Wild, Elsevier, 2005.

[0065] In a preferred embodiment, the chemiluminescent reaction according to the present invention is a flash-type chemiluminescent reaction. A flash-type chemiluminescent reaction produces a bright chemiluminescent signal in the range of seconds to minutes.

[0066] In a further preferred embodiment, the method according to the present invention for enhancing the chemiluminescent signal of a chemiluminescent compound in a chemiluminescent reaction mixture in a chemiluminescent reaction comprises a flash chemiluminescent reaction as the chemiluminescent reaction.

[0067] In a preferred embodiment, the enhancer according to the present invention is combined with the chemiluminescent compound in the chemiluminescent reaction mixture before, simultaneously with, or shortly after the start of the chemiluminescent reaction. Preferably, the enhancer is combined with the chemiluminescent compound in the chemiluminescent reaction mixture simultaneously with the start of the chemiluminescent reaction.

[0068] In a preferred embodiment, the chemiluminescent signal generated by the chemiluminescent reaction according to the present invention is detected for a fixed duration, preferably, a chemiluminescent reaction mixture is formed, the chemiluminescent reaction is started and the signal is detected simultaneously, more preferably, the duration of detection is 0.01 to 360 seconds, even more preferably 0.1 to 30 seconds, even more preferably 0.3 to 15 seconds, most preferably 0.5 to 10 seconds, wherein in each case the duration of detection starts at the start of the chemiluminescent reaction.

[0069] In a preferred embodiment, the method according to the present invention comprises the following steps: combining an enhancer according to the present invention with a chemiluminescent compound in a chemiluminescent reaction mixture at the same time as the start of the chemiluminescent reaction, and / or detecting the chemiluminescent signal generated by the chemiluminescent reaction according to the present invention for a fixed duration of 0.1 to 10 seconds starting from the start of the chemiluminescent reaction.

[0070] According to the present invention, the chemiluminescent reaction is preferably started by initiating oxidation of the chemiluminescent compound. Preferably, the initiation step of oxidizing the chemiluminescent compound is performed by adding an oxidizing compound, preferably hydrogen peroxide.

[0071] 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 alkaline hydroxide (preferably sodium hydroxide).

[0072] 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, the trigger solution A comprises an acid (preferably nitric acid) and hydrogen peroxide, and the trigger solution B comprises an alkaline hydroxide (preferably sodium hydrogen hydroxide) and an enhancer according to the present invention.

[0073] In a preferred embodiment, preferably according to at least one of the preceding embodiments, the 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 invention) with a chemiluminescent compound, which is an acridinium compound, preferably an acridinium ester or an acridinium sulfonamide.

[0074] The chemiluminescent compound according to the present invention is an acridinium compound, preferably an acridinium ester or an acridinium sulfonamide.

[0075] It is well known in the art that acridinium compounds exist in an equilibrium between an acridinium form and a pseudobase form in aqueous media, where virtually all immunoassays are performed. The pseudobase form of an 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 affected by the pH of the medium. Acidic pH promotes the formation of the acridinium form, and alkaline pH promotes the formation of the pseudobase form.

[0076] In a heterogeneous assay format, chemiluminescence from an acridinium compound or acridinium compound-labeled bioactive molecule (called a tracer or conjugate) is typically triggered by the sequential addition of two reagents. An initial treatment of the acridinium compound with a strong acid containing a peroxide is required to convert the pseudobase form of the acridinium compound into the acridinium form. This is followed by treatment with an alkaline solution to neutralize the acid and raise the pH of the reaction medium, ionizing the hydrogen peroxide and causing light emission to occur.

[0077] Preferably, the chemiluminescent compound used in the present invention is an acridinium ester according to formula (II):

[0078]

[0079] Where R 1 is alkyl, alkenyl, alkynyl, aryl or aralkyl, sulfoalkyl, carboxyalkyl and oxoalkyl; and wherein R 3 To R 15 each independently of the other is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, aryl or aralkyl, amino, amido, acyl, alkoxy, hydroxy, carboxyl, halogen, halide, nitro, cyano, sulfo, sulfoalkyl, sulfamoyl, carboxyalkyl, succinimidyl ester and oxoalkyl or any other leaving group; and optionally, if present, X 1- It is an anion;

[0080] or an acridinium sulfonamide according to formula (III)

[0081]

[0082] Where R 1 is selected from the group consisting of an alkyl group, an alkenyl group, an alkynyl group, an aryl or aralkyl group, a sulfoalkyl group, a carboxyalkyl group, and an oxoalkyl group, and wherein R 2 To R 15 each independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, aryl or aralkyl, amino, amido, acyl, alkoxy, hydroxy, carboxyl, halogen, halide, nitro, cyano, sulfo, sulfoalkyl, sulfamoyl, carboxyalkyl, succinimidyl ester and oxoalkyl or any other leaving group; and optionally, if present, X 1- It is an anion.

[0083] In a preferred embodiment, preferably according to at least one of the preceding embodiments, the chemiluminescent compound is an acridinium ester according to formula (II) as disclosed herein, wherein in addition R 12 and R 14 are each independently selected from the group consisting of hydrogen, alkyl, amino, carboxyl, hydroxyl, alkoxy, nitro or halide; R 11 and R 15 are each independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, aryl, alkoxy, amino, amido, sulfonamide or sulfide; and R 13 Represents the following substituents

[0084] R 13 =LR 16 -R 17

[0085] Where L is sulfamoyl chloride or is omitted, R 16 Omitted or alkyl, aryl, aralkyl, (acylamino) z , wherein z=1-10, preferably z=1 or 2, or (polyethylene oxide) x , wherein x=1-10, preferably x=4; and R 17 Selected from the group consisting of:

[0086]

[0087]

[0088] or any other leaving group,

[0089] Wherein R is alkyl, aryl or aralkyl; X is CH3SO4, OSO2F, halide, OSO2CF3, OSO2C4F9, or

[0090]

[0091] And where R 12 , R 13 and R 14 Substituent positions are interchangeable.

[0092] In a preferred embodiment, preferably according to at least one of the preceding embodiments, the chemiluminescent compound is an acridinium sulfonamide according to formula (III) as disclosed herein, wherein in addition R 12 , R 13 and R 14 are each independently selected from the group consisting of hydrogen, alkyl, amino, carboxyl, hydroxyl, alkoxy, nitro or halide; R 11 and R 15 are each independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, aryl, alkoxy, amino, amido, sulfonamide or sulfide; and R 2 Represents the following substituents

[0093] R 2 =LR 16 -R 17

[0094] Where L is sulfamoyl or is omitted, R 16 Omitted or alkyl, aryl, aralkyl, (acylamino) z , wherein z=1-10, preferably z=1 or 2, or (polyethylene oxide) x , wherein x=1-10, preferably x=4; and R 17 Selected from the group consisting of:

[0095]

[0096] or any other leaving group,

[0097] Wherein R is alkyl, aryl or aralkyl; X is CH3SO4, OSO2F, halide OSO2CF3, OSO2C4F9 or

[0098]

[0099] And where R 11 To R 15 Substituent positions are interchangeable.

[0100] Acridinium compounds suitable for use in the present invention are further described in U.S. Patent Application Serial No. 915,527 filed on October 6, 1986 and in Natrajanet et al., “A comparison of chemiluminescent acridiniumdimethylphenyl ester labels with different conjugation sites”, Org Biomol Chem, 13(9):2622-337, March 2015.

[0101] In a preferred embodiment, preferably according to at least one of the preceding embodiments, the chemiluminescent compound is selected from the group consisting of:

[0102] -N-sulfopropyl-dimethylacridinium-(polyethylene oxide) x -N-hydroxysuccinimide ester (NSP-DMAE-PEO x -NHS), wherein x=1-10, preferably N-sulfopropyl-dimethylacridinium-(polyethylene oxide) 4-N-hydroxysuccinimide ester (NSP-DMAE-PEO4-NHS);

[0103] -2,6-(Dimethyl)-3-chlorosulfonylphenyl-N-(3-sulfopropyl)-acridinium-9-carboxylate (SPAE);

[0104] -2,6-(dimethyl)-3-sulfamoylphenyl-N-(3-sulfopropyl)-acridinium-9-carboxylic acid-(polyethylene oxide) 4-pentafluorophenyl ester (SPAE-PEO4-PFP);

[0105] -2,6-(Dimethyl)-3-chlorosulfonylphenyl-N-methyl-acridinium-9-carboxylic acid trifluoromethanesulfonate (MeAE);

[0106] -2,6-(dimethyl)-3-sulfamoylphenyl-N-methyl-acridinium-9-carboxylic acid trifluoromethanesulfonate-(polyethylene oxide) 4-N-hydroxysuccinimide ester (MeAE-PEO4-NHS),

[0107] -2,3,4,5,6-pentafluorophenyloxy-1-carbonyl-4,7,10,13-tetraoxapentade-cyclo-(15-amino)-3'-sulfonyl-2',6'-dimethylphenyl-N-(3-sulfopropyl)-acridinium-9-carboxylate,

[0108] -10-Methyl-2,6-dimethyl-acridinium-NHS ester methylsulfate (Me-DMAE-NHS)

[0109] -N-sulfopropyl-dimethylacridinium-N-hydroxysuccinimide (NSP-DMAE-NHS); and

[0110] -N-sulfopropyl-acridinium-sulfonamide-N-hydroxysuccinimide (NSP-SA-NHS).

[0111] A list of exemplary acridinium compounds according to formula (II) or (III) as defined herein, suitable as chemiluminescent compounds according to the present invention, is shown in Table 2. To clarify the identity of the listed substances, despite possible ambiguity in the chemical nomenclature, if the Chemical Abstracts Service (CAS) registration number and / or compound record (CID) can be found and is publicly available, this information is listed in Table 2. Chemical Abstracts Service registration numbers according to the classification of the American Chemical Society are available through https: / / www.cas.org or https: / / commonchemistry.cas.org. CID records can be found in the PubChem database of the National Institutes of Health through https: / / pubchem.ncbi.nlm.nih.gov. If a compound is not assigned a CAS and / or CID, or there is no publicly available information, the corresponding compound is defined as "nd".

[0112] Table 2: Exemplary list of acridinium compounds of formula (II) or (III) according to the present invention.

[0113]

[0114]

[0115]

[0116]

[0117]

[0118] The acridinium compound according to the present invention may be oxidized by any oxidizing agent which reacts with the compound to cause excitation of the compound so that the compound emits light in a chemiluminescent reaction. Preferably the oxidizing agent is hydrogen peroxide in a dilute base.

[0119] In a preferred embodiment, the chemiluminescent reaction mixture according to the present invention contains an oxidant in an amount of 0.01 volume % to 1 volume % based on the total volume of the chemiluminescent reaction mixture, preferably 0.05 volume % to 0.5 volume % based on the total volume of the chemiluminescent reaction mixture, and most preferably 0.1 volume % to 0.3 volume % based on the total volume of the chemiluminescent reaction mixture.

[0120] The emitted light can be quantified using standard measuring devices such as the LB 960 Centro Microplate Photometer (Berthold Technologies GmbH & Co. KG, Germany), the Model 810 Photometer (Ciba-Corning Diagnostics Corp., Medfield, MA), or the RA Analyzer 10 (Euroimmun, a PerkinElmer company, Germany).

[0121] In a preferred embodiment, the chemiluminescent compound capable of chemiluminescent emission emits light at a wavelength of 400 nm or longer, preferably 400 nm to 690 nm, more preferably 400 nm to 550 nm, more preferably 405 nm to 500 nm, and most preferably 410 nm to 490 nm.

[0122] In a second aspect, the problem underlying the present invention is solved by using a compound selected from the group consisting of quaternary ammonium cationic substances of formula (I)

[0123]

[0124] Where R 1 C3-C 20 alkyl or alkenyl; and R 2 and R 3 are independently C1-C4 alkyl or alkenyl; and R 4 , R 5 , R 6 , R 7 and R 8 are independently hydrogen, alkyl, alkenyl; and X 1- is a halide or hydroxyl ion, preferably chloride or bromide, for enhancing the chemiluminescent signal of an acridinium compound, preferably an acridinium ester or an acridinium sulfonamide, more preferably an acridinium ester or an acridinium sulfonamide according to the present invention, in a chemiluminescent reaction, preferably in a chemiluminescent immunoassay.

[0125] In a preferred embodiment, the compound is selected from the group consisting of quaternary ammonium cationic species of formula (I) as defined herein, wherein R 1 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 Alkyl or alkenyl; R 2 and R 3 are independently methyl, ethyl, propyl or butyl, preferably methyl, R 4 , R5 , R 6 , R 7 and R 8 are independently hydrogen, alkyl, alkenyl, preferably hydrogen, methyl or ethyl; and / or X 1- It is a chloride or a bromide, preferably a chloride.

[0126] Preferably, one, two or more compounds selected from the group consisting of quaternary ammonium cationic substances of formula (I) as described above are used to enhance the chemiluminescent signal of the chemiluminescent compound, preferably an acridinium ester or an acridinium sulfonamide, preferably according to the present invention in a chemiluminescent reaction, preferably in a chemiluminescent immunoassay.

[0127] According to the invention, the compound is an enhancer compound according to the invention having an aromatic head group.

[0128] In a third aspect, the problem underlying the present invention is solved by a chemiluminescent composition comprising at least one compound selected from the group consisting of quaternary ammonium cationic substances of formula (I)

[0129]

[0130] Where R 1 C3-C 20 alkyl or alkenyl; and R 2 and R 3 are independently C1-C4 alkyl or alkenyl; and R 4 , R 5 , R 6 , R 7 and R 8 are independently hydrogen, alkyl, alkenyl; and X 1- is a halide or hydroxyl ion, preferably a chloride or bromide, and at least one 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 (III) disclosed herein.

[0131] According to the invention, at least one compound in the chemiluminescent composition according to the invention is an enhancer compound according to the invention having an aromatic head group.

[0132] In a preferred embodiment, the chemiluminescent composition according to the present invention further comprises a fluorescent agent.

[0133] The chemiluminescent composition according to the present invention may also include one or more fluorescent agents, such as sodium fluorescein, rhodamine B and / or rhodamine 6G. One or more fluorescent agents will emit a fluorescence after receiving the light energy of the chemiluminescent signal from the chemiluminescent compound. It is possible that the quantum yield of fluorescein can be much greater than the quantum yield of the chemiluminescent compound, so the quantum yield of the chemiluminescent compound can be indirectly improved. For example, sodium fluorescein (9-(o-carboxylphenyl)-6-hydroxy-3H-xanthene-3-one disodium) is a water-soluble fluorescent agent and has excitation and emission wavelengths of 494nm and 518nm, respectively. The quantum yield of sodium fluorescein is as high as 0.97. Similarly, the rhodamine series fluorescent agents also have very high fluorescence quantum yields. In the chemiluminescent composition according to the present invention, the fluorescent agent is preferably used in a concentration of about 0.1mg / L to about 1g / L.

[0134] In a preferred embodiment, the chemiluminescent composition according to the invention is a chemiluminescent reaction mixture according to the method of the invention. Preferably, the chemiluminescent composition according to the invention further comprises nitric acid, hydrogen peroxide and / or sodium hydroxide.

[0135] The chemiluminescent composition of the present invention, preferably according to the disclosed embodiments, can be included in a kit by packaging the components of the chemiluminescent composition individually into a multi-packaging 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-packaging system. Alternatively, the components can be mixed and then packaged into a single mixture.

[0136] In a fourth aspect, the problem underlying the present invention is solved by a kit, preferably for enhancing the chemiluminescence of a chemiluminescent compound, comprising at least one 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 (III) as disclosed above, and one, two or more compounds selected from the group consisting of quaternary ammonium cationic substances of formula (I) as disclosed above. The problem is preferably solved by a kit for enhancing 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 (III) as disclosed above, and one, two or more compounds selected from the group consisting of quaternary ammonium cationic substances of formula (I) as disclosed.

[0137] Preferably, one, two or more compounds selected from the group consisting of quaternary ammonium cationic substances of formula (I) are enhancers according to the present invention as described above.

[0138] 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.

[0139] In a preferred embodiment, at least one chemiluminescent compound (preferably a compound of formula (II) or formula (III) disclosed herein) and one, two or more compounds selected from the group consisting of quaternary ammonium cationic substances of formula (I) disclosed herein are stored together (optionally also with sodium hydroxide) in a mixture in a kit according to the present invention. Preferably, trigger solution A (preferably comprising nitric acid and hydrogen peroxide) is stored alone in a kit according to the present invention.

[0140] In a preferred embodiment, according to the present invention, preferably a kit according to at least one of the disclosed preferred embodiments comprises a diagnostically useful carrier, preferably a solid carrier, comprising means for capturing antigens, antibodies or autoantibodies in liquid solution and means for detecting antigens, antibodies or autoantibodies bound to the carrier, wherein the means for detecting antigens, antibodies or autoantibodies bound to the carrier are labeled with at least one chemiluminescent compound, i.e. an acridinium compound, preferably an acridinium ester of formula (II) or an acridinium sulfonamide of formula (III) according to the present invention. According to the present invention, it is further preferred that the diagnostically useful carrier is selected from the group consisting of beads, preferably paramagnetic beads, test strips, microtiter plates, membranes, preferably from the group comprising protein blots, line blots and dot blots, lateral flow devices, glass surfaces, microscope slides, microarrays and biochips. In a further preferred embodiment, the diagnostically useful carrier is a line blot, a biochip or a bead, most preferably a bead. Preferably, the diagnostically useful carrier is provided by a bead. According to the present invention, it is further preferred that at least one chemiluminescent compound is bound to the beads, 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 (III) according to the present invention. The binding may be via non-covalent protein-protein interactions, via bridging of protein-protein interactions, or via covalent binding.

[0141] In a preferred embodiment, the kit according to the present invention further comprises a diagnostically useful carrier, a trigger solution A and a trigger solution B as described above, wherein the trigger solution A comprises nitric acid and hydrogen peroxide, and the trigger solution B comprises sodium hydroxide and one, two or more compounds selected from the group consisting of quaternary ammonium cationic substances of formula (I) as defined above, and wherein at least the trigger solution A and the trigger solution B are stored separately.

[0142] In a preferred embodiment, the kit according to the present invention also includes a tool for detecting antigens, antibodies or autoantibodies combined with the carrier, wherein the tool for detecting antigens, antibodies or autoantibodies combined with the carrier can be a second antibody. Preferably, the second antibody is an antibody that specifically binds to all antibodies from an antibody class, preferably a mammalian antibody class, more preferably a human antibody class such as IgG. The second antibody usually recognizes the constant region of the class, but can also recognize other epitopes shared by the antibodies of the target class, such as conformational epitopes across 3D structures. A large part of them is commercially available, such as from Thermo Fisher. It can be a monoclonal or polyclonal antibody. In a preferred embodiment, as used herein, the term "recognized" means that the second antibody specifically binds to one or more antigens, antibodies / multiple antibodies or autoantibodies / multiple autoantibodies to be detected. The second antibody can specifically bind to all isotypes from the antibody class. For example, the second antibody of the IgG class antibody can bind to IgG1, IgG2, IgG3 and IgG4 isotypes. This can be achieved by using a mixture of antibodies that specifically bind to each IgG isotype or a single antibody that reacts with all target isotypes as a secondary antibody for the class antibody, preferably for the IgG class antibody. The use of secondary antibodies is explained in The Protein Protocols Handbook (editor JM Walker), page 967, volume 1996, Kruger, NJ, Detection of Polypeptides on Blots Using Secondary Antibodies in Springer. In short, such secondary antibodies can be produced by immunizing experimental animals with the antibody to be identified or a mixture of antibodies to be identified.

[0143] In preferred embodiments, as used herein, the term "specific binding" preferably means a binding reaction that is stronger than a specific binding reaction characterized by a dissociation constant of 1 x 10 -5 M, more preferably 1x 10 -7 M, more preferably 1x 10 -8 M, more preferably 1x 10 -9 M, more preferably 1x 10 -10 M, more preferably 1x 10 -11 M, more preferably 1x 10 -12 Binding reaction of M (as determined by surface plasmon resonance using a Biacore instrument at 25°C in PBS buffer, pH 7).

[0144] 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 group of stabilizers), washing buffers, anti-degradants.

[0145] The chemiluminescent composition, chemiluminescent reaction mixture and / or kit according to the present invention may further include a buffer for maintaining the pH of the reaction system. Suitable buffers include carbonate buffers, diethanolamine buffers, 2-amino-2-methyl-1-propanol, etc. The buffer may be used at a concentration of about 10 to about 500 mM.

[0146] The chemiluminescent composition, chemiluminescent reaction mixture and / or kit according to the present invention may also include a preservative, which is conducive to the preservation and long-term storage of the reagent. There is no restriction 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 in any concentration that does not affect the chemiluminescent reaction.

[0147] 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.

[0148] According to other different detection purposes, the kit according to the present invention may also include corresponding enzyme reagents, labeled enzyme reagents, solid-phase antibodies and / or manuals to guide operators.

[0149] In a fifth aspect, the problem underlying the present invention is solved by using the chemiluminescent composition or chemiluminescent reaction mixture according to the present invention to manufacture a kit for disease diagnosis, a medical device (preferably a diagnostic device). According to the present invention, the medical device is preferably selected from the group consisting of a slide (preferably for a microscope), a biochip, a microtiter plate, a lateral flow device, a test strip, a membrane (preferably a line blot), a chromatographic column and beads (preferably magnetic or fluorescent beads).

[0150] In another aspect, the present invention relates to an immunoassay and / or method for detecting a target analyte in a test sample, the immunoassay and / or method comprising the following steps:

[0151] (a) contacting a test sample suspected of containing a target analyte with a first antibody that binds to at least one epitope on the target analyte 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 target analyte to form an autoantibody-analyte complex, wherein the autoantibody is bound to the solid phase;

[0152] (b) contacting the 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 target analyte, 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 according to formula (III) as described above according to the present invention;

[0153] (c) producing a hydrogen peroxide source and an enhancer according to formula (I) as described above according to the present invention or providing said hydrogen peroxide source and enhancer to the mixture of step (b);

[0154] (d) adding an alkaline solution to the mixture of step (c) to generate a light signal; and

[0155] (e) measuring the optical signal generated by step (d) or emitted in step (d), and detecting the target analyte in the test sample.

[0156] In the above immunoassays or methods, the test sample may be whole blood, serum or plasma.

[0157] In a further aspect, the problem underlying the present invention is solved by a chemiluminescent composition of the present invention or a kit of the present invention for use in disease diagnosis.

[0158] The present invention is based on the surprising discovery of the inventors that an enhanced chemiluminescent signal of a chemiluminescent compound, preferably an acridinium compound, preferably an acridinium ester or an acridinium sulfonamide, can be provided in a chemiluminescent reaction mixture in a chemiluminescent reaction by using an enhancer (compound) selected from the group consisting of quaternary ammonium cationic substances of formula (I) disclosed herein.

[0159] The inventors surprisingly found that the addition of an aromatic moiety to the positively charged head group of a quaternary ammonium cationic substance further enhanced the chemiluminescent signal output compared to conventional substances such as hexadecyltrimethylammonium chloride (CTAC). In addition, the inventors surprisingly found that the length of at least three carbon atoms in the hydrophobic tail of such a quaternary ammonium cationic substance is of additional importance for the enhancement of the chemiluminescent signal output compared to conventional substances such as hexadecyltrimethylammonium chloride (CTAC), and compared to quaternary ammonium cationic substances having an aromatic moiety in the positively charged head group but losing a length of at least three carbon atoms in the hydrophobic tail.

[0160] Thus, the novel method (and related uses, compositions, mixtures and / or substances) according to the invention allows an even more sensitive quantification of analytes having ultra-low concentrations and generally facilitates signal differentiation in chemiluminescent immunoassays.

[0161] The chemiluminescent enhancer compounds according to the present invention include simple components that have the desired enhancing effect on the chemiluminescence of chemiluminescent compounds, in particular the chemiluminescent compounds of formula (II) and / or formula (III) according to the present invention. The chemiluminescent compositions of the present invention provide stable, durable and greatly enhanced chemiluminescent signals and can be used for chemiluminescent immunoassays, DNA probe detection and chemiluminescent analysis of biofilm protein blots. The chemiluminescent compositions of the present invention can be widely used in clinical diagnosis, scientific research, environmental and health testing, and forensic identification. In general, the chemiluminescent enhancer compounds according to the present invention are commercially available and inexpensive. In addition, they are suitable for large-scale production of chemiluminescent compositions.

[0162] Unless otherwise specified, the terms used herein have the following meanings:

[0163] The term "enhancement" as used herein means that when an enhancer according to the present invention is used, the total light emission of the chemiluminescent reaction and / or the ratio of the signal to background noise of the chemiluminescent reaction is greater than in the absence of the enhancer according to the present invention and / or is greater than compared to the so-called enhancer (i.e., hexadecyltrimethylammonium chloride; CTAC) that has been used prior to the present invention.

[0164] The term "alkyl" as used herein alone or in combination with other groups refers to a straight or branched chain alkyl group containing 1-20 carbon atoms, such as 1-12, 1-8, and 1-6 carbon atoms. Reference to a single straight chain alkyl group such as "n-propyl" specifically refers to a straight chain alkyl group, while reference to a single branched chain alkyl group such as "isopropyl" specifically refers to a branched chain alkyl group. For example, "C 1-6 "Alkyl" includes C 1-4 Alkyl, C 1-3 The term "alkyl" refers to a group consisting of a methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, 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.

[0165] The term "alkoxy" as used herein means an alkyl group, as defined herein, attached 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, pentoxy and hexoxy.

[0166] As used herein, the term "aryl" refers to phenyl, or a bicyclic or tricyclic fused ring system, wherein one or more of the fused rings is a phenyl. An example of a bicyclic fused ring system is a phenyl fused to a cycloalkenyl, a cycloalkyl, or another phenyl. An example of a tricyclic fused ring system is a bicyclic fused ring system fused to a cycloalkenyl, a cycloalkyl, or another phenyl as defined herein. Representative examples of aryl include, but are not limited to, anthracenyl, azulenyl, fluorenyl, indanyl, indenyl, naphthyl, phenyl, and tetrahydronaphthyl. The aryl groups of the present disclosure may be optionally substituted with one, two, three, four, or five substituents independently selected from the group consisting of alkoxy, alkyl, carboxyl, halogen, and hydroxy.

[0167] As used herein, the term "acyl" refers to -C(O)R a Group, where R a is hydrogen, alkyl, cycloalkyl, cycloalkylalkyl, phenyl or phenylalkyl. Representative examples of acyl include, but are not limited to, formyl, acetyl, cyclohexylcarbonyl, cyclohexylmethylcarbonyl, benzoyl, benzylcarbonyl, and the like.

[0168] The term "alkenyl" as used herein refers to a straight or branched hydrocarbon containing 2-20 carbons and containing at least one carbon-carbon double bond formed by removing two hydrogens. Representative examples of alkenyl include, but are not limited to, vinyl, 2-propenyl, 2-methyl-2-propenyl, 3-butenyl, 4-pentenyl, 5-hexenyl, 2-heptenyl, 2-methyl-1-heptenyl, and 3-decenyl.

[0169] The term "alkynyl" as used herein means a straight or branched hydrocarbon group containing 2 to 20 carbon atoms and containing at least one carbon-carbon triple bond. Representative examples of alkynyl include, but are not limited to, ethynyl, 1-propynyl, 2-propynyl, 3-butynyl, 2-pentynyl and 1-butynyl.

[0170] The term "amido," as used herein, refers to an amino group attached to the parent molecular moiety through a carbonyl group (wherein the term "carbonyl" refers to a -C(O)- group).

[0171] The term "amino" as used herein refers to -NR b R c , where R b and R c are independently selected from the group consisting of hydrogen, alkyl and alkylcarbonyl.

[0172] The term "aralkyl" as used herein refers to an aryl group attached to the parent molecular moiety through 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.

[0173] As used herein, the term "carboxy" or "carboxyl" refers to -CO2H or -CO2.

[0174] As used herein, the term "carboxyalkyl" refers to -(CH2) n CO2H or-(CH2) n A CO2- group, wherein n is 1 to 20.

[0175] As used herein, the term "cyano" refers to a -CN group.

[0176] 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, wherein each five-membered ring has one double bond, each six-membered ring has one or two double bonds, each seven-membered and eight-membered ring has 1 to 3 double bonds, and each nine-membered to ten-membered ring has 1 to 4 double bonds. Representative examples of cycloalkenyl include cyclohexenyl, octahydronaphthyl, norbornenyl, etc. The cycloalkenyl group may be optionally substituted with 1, 2, 3, 4 or 5 substituents independently selected from the group consisting of alkoxy, alkyl, carboxyl, halogen and hydroxy.

[0177] As used herein, the term "cycloalkyl" refers to a saturated monocyclic, bicyclic or tricyclic hydrocarbon ring system having 3 to 12 carbon atoms. Representative examples of cycloalkyl include cyclopropyl, cyclopentyl, bicyclo[3.1.1]heptyl, adamantyl, etc. The cycloalkyl of the present disclosure may be optionally substituted with one, two, three, four or five substituents independently selected from the group consisting of alkoxy, alkyl, carboxyl, halogen and hydroxyl.

[0178] As used herein, the term "cycloalkylalkyl" refers to -R d R e Group, where R d is an alkylene group, R e is a cycloalkyl group. Representative examples of cycloalkylalkyl groups are cyclohexylmethyl and the like.

[0179] As used herein, the term "halide" includes fluoride, chloride, bromide and iodide.

[0180] As used herein, the term "hydroxy" refers to an -OH group.

[0181] As used herein, the term "nitro" refers to a -NO2 group.

[0182] As used herein, the term "oxoalkyl" refers to -(CH2) n C(O)R a , where R a is hydrogen, alkyl, cycloalkyl, cycloalkylalkyl, phenyl or phenylalkyl, and wherein n is 1 to 20.

[0183] As used herein, the term "phenylalkyl" refers to an alkyl group substituted with a phenyl group.

[0184] As used herein, the term "sulfo" refers to -SO3H or -SO3 - Group.

[0185] As used herein, the term "sulfoalkyl" refers to -(CH2) n SO3H or -(CH2) n SO3 - A group wherein n is 1 to 20.

[0186] As used herein, the term "soluble" means free of precipitation.

[0187] As used herein, the term "sulfamoyl" refers to -SO2-NR1-, wherein R1 is selected from the group consisting of hydrogen, alkyl, aryl and alkylcarbonyl.

[0188] As used herein, the term "sulfonamide" refers to any amide of a sulfonic acid.

[0189] The term "chemiluminescence" as used herein is the light produced in a specific reaction of a chemical substance. Singlet molecules are excited and form high-energy intermediates that decompose in a chemical reaction, and then the excited singlet molecules return to the ground state, and part of the energy is emitted in the form of luminescence. Therefore, the chemiluminescent reaction includes two processes: the excitation process and the luminescence process. Some of the molecular energy will also be dissipated in the excited state due to inter-system and intra-system crossing.

[0190] In preferred embodiments, the term "diagnosis" as used herein is used in its broadest possible sense and may refer to any type of procedure, the purpose of which is to obtain information that helps to assess whether a patient (known or anonymous subject from a cohort) has or may have or is more likely to have a disease or condition than an average subject or comparable subject (the latter preferably has similar symptoms) in the past, at the time of diagnosis, or in the future, to find out how the disease progresses or may progress in the future, or to evaluate the overall responsiveness of one or more patients to a treatment (e.g., administration of an immunosuppressive drug), or to find out whether a sample is from such a patient. This information can be used for clinical diagnosis, but it can also be obtained by an experimental and / or research laboratory for general research purposes (e.g., determining the proportion of subjects with the disease in a patient cohort or population). In other words, the term "diagnosis" includes not only diagnosis, but also the process of predicting and / or monitoring a disease or condition, including monitoring the response of one or more patients to the administration of a drug or candidate drug, such as to determine its efficacy. While results can be assigned to a specific patient for clinical diagnostic applications and transmitted to the physician or institution treating the patient, this is not necessarily the case for other applications, such as diagnostics for research purposes, where it may be sufficient to assign results to samples from anonymous patients.

[0191] In preferred embodiments, the term "diagnosis" may also refer to a method or agent for selecting the most promising treatment regimen for a patient. In other words, the method or agent may involve selecting a treatment regimen for a subject. For example, the detection of autoantibodies may indicate that immunosuppressive therapy is to be selected, which may include administering one or more immunosuppressive drugs to the patient.

[0192] The method according to the invention is preferably an in vitro method.

[0193] According to the invention, the kit may comprise instructions how to carry out the method according to the invention.

[0194] In a preferred embodiment, any method or use according to the invention can be used for non-diagnostic purposes, such as determining the presence of autoantibodies for purposes other than diagnosing a patient. For example, the method or use can be used to test in vitro the efficacy of a medical device designed to remove autoantibodies from a patient's blood, wherein the test is performed on a fluid other than the patient's blood.

[0195] In another preferred embodiment, the method can be used to confirm the authenticity of a diagnostic assay and may include detecting antibodies against the polypeptide.

[0196] In a preferred embodiment, any method or use according to the invention can be used to identify a subject at risk of having or developing a disease and / or a tumor. BRIEF DESCRIPTION OF THE DRAWINGS

[0197] Figure 1 Three different cationic compounds (each with an aromatic head group) at 0.5% (w / v) are shown (Compound 1: benzyldimethylhexadecylammonium chloride; Compound 2: benzalkonium chloride; Compound 3: alkyl (C 12 -C 14 Figure 5. Chemiluminescence kinetics of 1 nM NSP-DMAE-NHS in the presence of (40% by weight) dimethylethylbenzyl ammonium chloride (DEMAC) or in the presence of 0.5% (w / v) of CTAC, a cationic compound lacking an aromatic head group.

[0198] Figure 2 Three different cationic compounds (each with an aromatic head group) at 0.5% (w / v) are shown (Compound 1: benzyldimethylhexadecylammonium chloride; Compound 2: benzalkonium chloride; Compound 3: alkyl (C 12 -C 14 Chemiluminescence kinetics of 1 nM NSP-SA-NHS in the presence of (40% by weight) dimethylethylbenzyl ammonium chloride) or in the presence of 0.5% (w / v) of CTAC, a cationic detergent lacking an aromatic head group.

[0199] Figure 3 Three different cationic compounds (each with an aromatic head group) at 0.5% (w / v) are shown (Compound 1: benzyldimethylhexadecylammonium chloride; Compound 2: benzalkonium chloride; Compound 3: alkyl (C 12 -C 14 Chemiluminescent kinetics of 0.1 μg / mL anti-human IgG-Fcγ-NSP-DMAE in the presence of (40% by weight) dimethylethylbenzyl ammonium chloride) or in the presence of 0.5% (w / v) of CTAC, a cationic detergent lacking an aromatic head group.

[0200] Figure 4 Three different cationic compounds (each with an aromatic head group) at 0.5% (w / v) are shown (Compound 1: benzyldimethylhexadecylammonium chloride; Compound 2: benzalkonium chloride; Compound 3: alkyl (C 12 -C 14 Chemiluminescent kinetics of 0.1 μg / mL anti-human IgG-Fcγ-NSP-SA in the presence of (40% by weight) dimethylethylbenzyl ammonium chloride) or in the presence of 0.5% (w / v) of CTAC, a cationic detergent lacking an aromatic head group.

[0201] Figure 5Four different cationic compounds (each having an aromatic head group and an extended hydrophobic tail with at least three carbon atoms) at 0.5% (w / v) are shown (Compound 1: benzyldimethylhexadecylammonium chloride; Compound 2: benzalkonium chloride; Compound 3: alkyl (C 12 -C 14 ) dimethylethylbenzyl ammonium chloride; Compound 4: benzyldimethyldodecyl ammonium chloride) or in the presence of 0.5% (w / v) benzyltriethylammonium chloride (BTEAC) containing an aromatic head group but lacking an extended hydrophobic tail or in the presence of 0.5% (w / v) CTAC lacking an aromatic head group but containing an extended hydrophobic tail.

[0202] Figure 6 Four different cationic compounds (each having an aromatic head group and an extended hydrophobic tail with at least three carbon atoms) at 0.5% (w / v) are shown (Compound 1: benzyldimethylhexadecylammonium chloride; Compound 2: benzalkonium chloride; Compound 3: alkyl (C 12 -C 14 ) dimethylethylbenzyl ammonium chloride; Compound 4: benzyldimethyldodecyl ammonium chloride) or in the presence of 0.5% (w / v) BTEAC containing an aromatic head group but lacking an extended hydrophobic tail or in the presence of 0.5% (w / v) CTAC lacking an aromatic head group but containing an extended hydrophobic tail.

[0203] Figure 7 Four different cationic compounds (each having an aromatic head group and an extended hydrophobic tail with at least three carbon atoms) at 0.5% (w / v) are shown (Compound 1: benzyldimethylhexadecylammonium chloride; Compound 2: benzalkonium chloride; Compound 3: alkyl (C 12 -C 14 ) dimethylethylbenzyl ammonium chloride; Compound 4: benzyldimethyldodecyl ammonium chloride) or in the presence of 0.5% (w / v) BTEAC containing an aromatic head group but lacking an extended hydrophobic tail or in the presence of 0.5% (w / v) CTAC lacking an aromatic head group but containing an extended hydrophobic tail. Chemiluminescent kinetics of 0.1 μg / mL anti-human IgG-Fcγ-NSP-DMAE.

[0204] Figure 8Four different cationic compounds (each having an aromatic head group and an extended hydrophobic tail with at least three carbon atoms) (Compound 1: benzyldimethylhexadecylammonium chloride; Compound 2: benzalkonium chloride; Compound 3: alkyl (C 12 -C 14 ) dimethylethylbenzyl ammonium chloride; Compound 4: benzyldimethyldodecyl ammonium chloride) or in the presence of 0.5% (w / v) BTEAC containing an aromatic head group but lacking an extended hydrophobic tail or in the presence of 0.5% (w / v) CTAC lacking an aromatic head group but containing an extended hydrophobic tail. Chemiluminescent kinetics of 0.1 μg / mL anti-human IgG-Fcγ-NSP-SA.

[0205] The invention is further illustrated by the following non-limiting examples, from which further features, embodiments, aspects and advantages of the invention can be derived. Example

[0206] Acridinium labeling and conjugate solution preparation:

[0207] 1 mg / mL labeling stocks 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 (anti-human IgG-Fcγ; Jackson ImmunoResearch Europe Ltd., UK) specific for the Fcγ fragment was diluted to a final concentration of 0.33 mg / mL in phosphate buffered saline at pH 7.6 and then incubated at 10 rpm in an RM-2M Intelli-mixer (Hassa GmbH, Germany) with a 10-fold molar excess of NSP-DMAE-NHS or a 2.5-fold molar excess of NSP-SA-NHS for 2 hours at room temperature. The labeling reaction was stopped by adding 17.4 mM L-lysine (VWR International GmbH, Germany) at room temperature for 30 minutes 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 a Vivaspin 20, 10 kDa molecular weight cutoff concentrator (VWR International GmbH, Germany) at 4°C and the buffer was exchanged into phosphate buffered saline, pH 7.4, containing 0.045% (w / v) sodium azide (Merck KgaA, Germany). The purified antibody conjugate was stored in a 1% (w / v) bovine serum albumin (Th. Geyer Hamburg GmbH & Co. Kg, Germany), 0.3% (w / v) P 188 (Th. Geyer Hamburg GmbH & Co. Kg, Germany) and 0.5 mM ethylenediaminetetraacetic acid disodium salt (Gerbu Biotechnik GmbH, Germany) in phosphate buffered saline.

[0208] Preparation of chemiluminescent trigger solution:

[0209] A chemiluminescent 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 a trigger solution B containing 0.5 M sodium hydroxide (Gerbu Biotechnik GmbH, Germany) were prepared. Trigger solution B lacking any detergent or supplemented with 0.05, 0.1, 0.25 or 0.5% (w / v) of the cationic compounds hexadecyltrimethylammonium chloride (Merck KgaA, Germany), hexadecyltrimethylammonium bromide (Merck KgaA, Germany) or tetradecyltrimethylammonium chloride (Merck KgaA, Germany), all of which have no aromatic head group, was used. 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 an aromatic head group), or benzyltriethylammonium chloride (Merck KgaA, Germany) containing an aromatic head group but lacking an extended hydrophobic tail of at least three carbon atoms.

[0210] Measurement of chemiluminescent signal intensity:

[0211] Dilute NSP-DMAE-NHS and NSP-SA-NHS labeling stocks to a final concentration of 1 nM, and dilute the antibody conjugate to a final concentration of 0.1 μg / mL in phosphate buffered saline, pH 7.4. Transfer 10 μL of each of these dilutions to a 96-well LumiNunc TMThe chemiluminescent signal intensity was measured on a LB 960 Centro microplate luminometer (Berthold Technologies GmbH & Co. KG, Germany) in the wells of a plate (VWR International GmbH, Germany) in the presence of 100 μL of each chemiluminescent trigger A and different trigger B solutions (the latter differing in the bulk and concentration of the cationic compound). After the medium-speed injection of trigger A, the mixture was incubated for 1 second during mixing in a dual-track mode at normal speed. After adding various trigger B solutions at a low injection speed, the chemiluminescent intensity in relative light units (RLU) was recorded every 50ms over a total time of 1.5 seconds. All samples were measured with four technical replicates. In the control experiment, all different trigger B solutions were injected into the empty wells of a 96-well plate in combination with trigger A. The latter experiment reflects the signal background generated by different trigger solutions.

[0212] Data Analysis:

[0213] To illustrate the observed chemiluminescence kinetics, a representative data set was selected from four technical replicates and the signal intensity in RLU was plotted against time. In addition, the total signal intensity was calculated by adding all RLU values ​​observed within a 1.5 second time frame. The background signal intensity of the different trigger B solutions was subtracted from the total signal intensity, and all background-corrected total signal intensities were set for comparison.

[0214] Example 1

[0215] Three representative enhancer compounds according to the 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 invention were tested against the commonly used enhancer substance CTAC (e.g. proposed in US 4,927,769). In contrast, CTAC does not contain an aromatic moiety adjacent to the quaternary ammonium.

[0216] Four representative analytes were selected to study the effect of the enhancer compounds according to the present invention on the chemiluminescent signals generated by these analytes compared with common enhancer substances known in the prior art ( Figure 1-Figure 4 ). The selected analytes include those in free form ( Figure 1-Figure 2 ) and in antibody-bound form ( Figure 3-Figure 4 ) were conjugated to the acridinium compounds NSP-DMAE-NHS and NSP-SA-NHS. Polyclonal anti-human IgG-Fcγ antibodies from goats were selected to be conjugated to two types of acridinium labels.

[0217] The acridinium label, conjugate solution and chemiluminescent trigger solution were obtained according to the methods disclosed above. The chemiluminescent signal intensity was measured and the data analyzed as described above.

[0218] result:

[0219] Benzyldimethylhexadecylammonium chloride (Compound 1) and analyte NSP-DMAE-NHS ( Figure 1 , Table 3) A 43% increase in signal output was observed compared to the enhancer substance CTAC for the same analyte.

[0220] Using NSP-DMAE-NHS as the analyte, benzalkonium chloride (compound 2) and alkyl chloride (C 12 -C 14 ) dimethylethylbenzylammonium chloride (Compound 3) also resulted in 19% and 15% signal increases, respectively, compared to CTAC.

[0221] Similar results were obtained when the analyte was switched to NSP-SA-NHS ( Figure 2 , Table 3).

[0222] Compound 1 (benzyldimethylhexadecylammonium chloride) resulted in a 67% increase in signal compared to the substance CTAC. In addition, for this analyte, a 58% and 40% increase in signal was observed for compounds 2 and 3, respectively, compared to CTAC.

[0223] Additionally, it was found that the enhancement in signal output observed for both examples of acridinium-based probes in free form was also observed when the probes were covalently linked to antibodies, which is typically desired for immunoassays.

[0224] In this case, compound 1 (benzyldimethylhexadecyl ammonium chloride) resulted in a 5-fold increase in the analyte anti-human IgG-Fcγ-NSP-DMAE ( Figure 3 , Table 3) and anti-human IgG-Fcγ-NSP-SA ( Figure 4 , Table 3), the signals for these analytes increased by 45% and 66%, respectively. Compound 2 (benzalkonium chloride) also increased the signals for these two antibody conjugates by 40% and 52%, respectively. In addition, the signals for compound 3 (alkyl (C 12 -C 14 In the presence of (dimethylethylbenzylammonium chloride), a 25% and 35% increase in signal was observed for both analytes.

[0225] Table 3:

[0226] Comparison of background-corrected total signal intensities of four different analytes in the presence of 0.5% (w / v) of three different cationic enhancer compounds, each with an aromatic head group (Compounds 1-3) or in the presence of 0.5% (w / v) of the cationic detergent CTAC, which lacks an aromatic head group.

[0227]

[0228] Example 2

[0229] describe:

[0230] To test the effect of the chain length within the hydrophobic tail of the substance on the chemiluminescence enhancement, further experiments were performed with compounds 1-3 (as described in Example 1), the commonly used enhancer CTAC, an additional cationic enhancer according to the present invention (compound 4; benzyldimethyldodecylammonium chloride) and an additional substance BTEAC (benzyltriethylammonium chloride) which contains a chain length of less than three carbon atoms in the hydrophobic tail ( Figure 5-Figure 8 ). These additional experiments were performed as described above and as described in Example 1, respectively.

[0231] result:

[0232] Similar to compounds 1-3, compound 4 enhanced the activity of 1 nM NSP-DMAE-NHS ( Figure 5 )、1nM NSP-SA-NHS( Figure 6 )、0.1μg / mL anti-human IgG-Fcγ-NSP-DMAE( Figure 7 ) and 0.1 μg / mL anti-human IgG-Fcγ-NSP-SA ( Figure 8 ) chemiluminescence. In this experiment, compared with the commonly used enhancer CTAC, compounds 1-3 increased the chemiluminescence of 1nM NSP-DMAE-NHS by 45-79%, increased the chemiluminescence of 1nM NSP-SA-NHS by 62-102%, increased the chemiluminescence of 0.1μg / mL anti-human IgG-Fcγ-NSP-DMAE by 46-63%, and increased the chemiluminescence of 0.1μg / mL anti-human IgG-Fcγ-NSP-SA by 47-50% (Table 4). Similarly, compared with the commonly used enhancer CTAC, compound 4 increased the chemiluminescence of 1nM NSP-DMAE-NHS by 50%, increased the chemiluminescence of 1nM NSP-SA-NHS by 47%, increased the chemiluminescence of 0.1μg / mL anti-human IgG-Fcγ-NSP-DMAE by 50%, and increased the chemiluminescence of 0.1μg / mL anti-human IgG-Fcγ-NSP-SA by 40% (Table 4).

[0233] For BTEAC, reduced chemiluminescence was observed for all four analytes compared to the commonly used enhancer CTAC ( Figure 5-Figure 8 and Table 4). Although this material contains an aromatic head group, it does not function as an equivalent chemiluminescence enhancer, most likely, and without being bound by any theory, because the chain length of the hydrophobic tail is too short to form a micellar structure.

[0234] Table 4: Comparison of background-corrected total signal intensities of four different analytes in the presence of 0.5% (w / v) of four different cationic enhancer compounds, each having an aromatic head group and an extended hydrophobic tail possessing at least 3 carbon atoms (Compounds 1-4), or in the presence of 0.5% (w / v) of benzyltriethylammonium chloride (BTEAC), which contains an aromatic head group but lacks an extended hydrophobic tail, or in the presence of 0.5% (w / v) of the cationic detergent CTAC, which lacks an aromatic head group but contains an extended hydrophobic tail.

[0235]

[0236] In summary, the data obtained in the examples show that all four tested compounds representing examples of enhancers proposed by the present invention are 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 commonly used enhancers such as CTAC.

[0237] In addition, these new chemiluminescent enhancers all share a molecular structure comprising a cationic quaternary ammonium compound attached to an aromatic portion (i.e., an aromatic head group) and a minimum chain length of at least three carbon atoms in the hydrophobic tail of the enhancer. All substances containing these molecular moieties (i.e., an aromatic head group and a minimum chain length of at least three carbon atoms in the hydrophobic tail) may similarly enhance the chemiluminescence generated by acridinium-based analytes compared to enhancer substances lacking these molecular moieties (i.e., an aromatic head group and a minimum chain length of at least three carbon atoms in the hydrophobic tail). Substances with shorter chain lengths in the hydrophobic tail do not similarly enhance chemiluminescence, most likely because they do not form micellar structures, which are believed to facilitate chemiluminescence enhancement. While not being bound by theory, it is further believed that the newly proposed cationic chemiluminescent enhancers according to the present invention facilitate the chemiluminescent reaction process by directly interacting with the acridinium ring to form so-called hydrophobic π-π stacking interactions. In this way, the acridinium label can be efficiently recruited and positioned to react with the hydrogen peroxide anion, which is attracted by the positive charge of the adjacent cationic ammonium ion.

[0238] The data, figures, instruments, reagents and steps herein should be understood to be illustrative, rather than restrictive. Although the disclosure has been described with reference to the above specific embodiments, many modifications and variations are apparent to those skilled in the art. All modifications and variations also fall within the spirit and scope of the disclosure.

Claims

1. A method for enhancing the chemiluminescent signal of a chemiluminescent compound in a chemiluminescent reaction mixture in a chemiluminescent reaction, the method comprising oxidizing the chemiluminescent compound in the presence of an effective amount of an enhancer to obtain an enhanced chemiluminescent signal, wherein the chemiluminescent compound is an acridinium compound, wherein the enhancer is a compound or a mixture of two, three or more compounds, and wherein the or each enhancer compound is selected from the group consisting of quaternary ammonium cationic substances of formula (I) Where R 1 C3-C 20 alkyl or alkenyl; and R 2 and R 3 are independently C1-C4 alkyl or alkenyl; and R 4 , R 5 , R 6 , R 7 and R 8 are independently hydrogen, alkyl, alkenyl; and X 1- It is a halide or hydroxide ion.

2. The method according to claim 1, wherein the chemiluminescent compound is an acridinium ester or an acridinium sulfonamide.

3. The method according to claim 1, wherein X 1- Chloride or bromide.

4. The method according to claim 1, wherein -R 1 C5-C 18 Alkyl or alkenyl; -R 2 and R 3 are independently methyl, ethyl, propyl or butyl; -R 4 , R 5 , R 6 , R 7 and R 8 are independently hydrogen, alkyl, alkenyl; and / or -X 1- Chloride or bromide.

5. The method of claim 1, wherein the enhancer, at least one compound of the enhancer, or each compound of the enhancer is selected from the group consisting of: -Benzyldimethyleicosylammonium chloride, -Benzyldimethylnonadecylammonium chloride, -Benzyldimethyloctadecylammonium chloride, -Benzyldimethylheptadecylammonium chloride, -Benzyldimethylhexadecylammonium chloride, -Benzyldimethylpentadecylammonium chloride, -Benzyldimethyltetradecylammonium chloride, -Benzyldimethyltridecylammonium chloride, -Benzyldimethyldodecylammonium chloride, -Benzyldimethylundecylammonium chloride, -Benzyldimethyldecylammonium chloride, -Benzyldimethylnonylammonium chloride, -Benzyl dimethyl octyl ammonium chloride, -Benzalkonium chloride, -Benzyl dimethyl heptyl ammonium chloride, -Benzyl dimethyl hexyl ammonium chloride, -Benzyl dimethyl amyl ammonium chloride, -Benzyl dimethyl butyl ammonium chloride, -Benzyl dimethyl propyl ammonium chloride, -Benzyltripropylammonium chloride, -Benzyl dipropyl ethyl ammonium chloride, -Benzyl diethyl propyl ammonium chloride, -Benzyl dipropyl methyl ammonium chloride, -Benzylpropylethylmethylammonium chloride, -Benzyltributylammonium chloride, -Benzyl dibutyl propyl ammonium chloride, -Benzyl dipropyl butyl ammonium chloride, -Benzylbutylpropylethylammonium chloride, -Benzylbutylpropylmethylammonium chloride, -Benzylbutylethylmethylammonium chloride, -Benzyl diethyl butyl ammonium chloride, -C 12-14 -Alkyl dimethyl (ethyl benzyl) ammonium chloride, -Dodecyl(ethylbenzyl)dimethylammonium chloride, -Tetradecyl dimethyl (ethylbenzyl) ammonium chloride, -octadecyldimethyl(ar-ethylbenzyl)ammonium chloride, as well as the corresponding compounds mentioned above containing the bromide instead of the chloride.

6. The method of claim 1, wherein the effective amount of the enhancer in the chemiluminescent reaction mixture is 0.001% weight / volume (w / v) or greater, in each case based on the total volume of the chemiluminescent reaction mixture.

7. The method of claim 1, wherein the enhancer is combined with the chemiluminescent compound in a chemiluminescent reaction mixture before, simultaneously with, or shortly after the chemiluminescent reaction begins.

8. The method of claim 7, wherein the enhancer is combined with the chemiluminescent compound in a chemiluminescent reaction mixture simultaneously with the initiation of the chemiluminescent reaction.

9. The method of claim 1, wherein the chemiluminescent signal generated by the chemiluminescent reaction is detected for a fixed duration, wherein the duration of the detection begins when the chemiluminescent reaction begins.

10. The method of claim 9, wherein the chemiluminescent reaction mixture is formed, the chemiluminescent reaction is initiated and the signal is detected simultaneously.

11. The method of claim 1, wherein the chemiluminescent reaction mixture further comprises hydrogen peroxide, nitric acid and / or sodium hydroxide.

12. The method of claim 2, wherein the chemiluminescent compound is an acridinium ester according to formula (II): Where R 1 is alkyl, alkenyl, alkynyl, aryl or aralkyl, sulfoalkyl, carboxyalkyl and oxoalkyl; and Where R 3 To R 15 each independently of the others is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, aryl or aralkyl, amino, amido, acyl, alkoxy, hydroxy, carboxyl, halogen, halide, nitro, cyano, sulfo, sulfoalkyl, sulfamoyl, carboxyalkyl, succinimidyl ester and oxoalkyl or any other leaving group; or an acridinium sulfonamide according to formula (III) Where R 1 is selected from the group consisting of an alkyl group, an alkenyl group, an alkynyl group, an aryl or aralkyl group, a sulfoalkyl group, a carboxyalkyl group, and an oxoalkyl group, and Where R 2 To R 15 Each is independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, aryl or aralkyl, amino, amido, acyl, alkoxy, hydroxyl, carboxyl, halogen, halide, nitro, cyano, sulfo, sulfoalkyl, sulfamoyl, carboxyalkyl, succinimidyl and oxoalkyl or any other leaving group.

13. The method according to claim 12, wherein if there is X 1- , which is the anion in formula (II) or (III).

14. Use of a compound selected from the group consisting of quaternary ammonium cationic substances of formula (I), Where R 1 C3-C 20 alkyl or alkenyl; and R 2 and R 3 are independently C1-C4 alkyl or alkenyl; and R 4 , R 5 , R 6 , R 7 and R 8 are independently hydrogen, alkyl, alkenyl; and X 1- It is a halide or hydroxide ion used to enhance acridinium compounds in chemiluminescent reactions.

15. The use according to claim 14, wherein -R 1 C5-C 18 Alkyl or alkenyl; -R 2 and R 3 are independently methyl, ethyl, propyl or butyl, -R 4 , R 5 , R 6 , R 7 and R 8 are independently hydrogen, alkyl, alkenyl; and / or -X 1- Chloride or bromide.

16. A chemiluminescent composition comprising at least one compound selected from the group consisting of quaternary ammonium cationic substances of formula (I) Where R 1 C3-C 20 alkyl or alkenyl; and R 2 and R 3 are independently C1-C4 alkyl or alkenyl; and R 4 , R 5 , R 6 , R 7 and R 8 are independently hydrogen, alkyl, alkenyl; and X 1- is a halide or hydroxide ion, and At least one chemiluminescent compound, wherein the chemiluminescent compound is an acridinium compound.

17. A kit comprising at least one chemiluminescent compound, wherein the chemiluminescent compound is an acridinium compound and One, two or more compounds selected from the group consisting of quaternary ammonium cationic substances of formula (I) Where R 1 C3-C 20 alkyl or alkenyl; and R 2 and R 3 are independently C1-C4 alkyl or alkenyl; and R 4 , R 5 , R 6 , R 7 and R 8 are independently hydrogen, alkyl, alkenyl; and X 1- It is a halide or hydroxide ion.

18. A kit according to claim 17, comprising a diagnostically useful carrier, comprising means for capturing antigens, antibodies or autoantibodies in liquid solution and means for detecting antigens, antibodies or autoantibodies bound to the carrier, wherein said means for detecting said antigen, antibody or autoantibody bound to said carrier is labeled with said at least one chemiluminescent compound.

19. The kit according to claim 18, wherein the diagnostically useful support is a solid support.

20. Use of the chemiluminescent composition according to claim 16 or the kit according to any one of claims 17 to 19 for diagnosis of a disease.

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