A METHOD AND REAGENTS FOR ENHANCING THE CHEMILUMINESCENT SIGNAL

Abstract

A method for enhancing the chemiluminescent signal of a chemiluminogenic compound in a chemiluminescent reaction mixture in a chemiluminescent reaction which has a step of oxidizing the chemiluminogenic compound in the presence of an effective amount of an enhancer can be performed. To obtain an enhanced chemiluminescent signal, a compound selected from the group of quaternary amine cationic substances of formula (I) for enhancing the chemiluminescent signal of an acridinium compound is used, preferably an acridinium ester or an acridinium sulfonamide. The signal is obtained in a chemiluminescent reaction, preferably in a chemiluminescent immunoassay, a related chemiluminescent composition and a related kit.

Claims

1. A method for enhancing a chemiluminescent signal of a chemiluminogenic compound in a chemiluminescent reaction mixture in a chemiluminescent reaction, the method comprising: oxidizing the chemiluminogenic compound in the presence of an effective amount of an enhancer to obtain an enhanced chemiluminescent signal, wherein the chemiluminogenic compound is an acridinium compound; wherein said enhancer is a compound or a mixture of two, three, or more compounds, and wherein said enhancer compound or each of the enhancer compounds is at least one selected from the group consisting of quaternary amine cationic substances of formula (I) ##STR00055## wherein R.sup.1 is C.sub.3-C.sub.20 alkyl or alkenyl; and R.sup.2 and R.sup.3 are independently from each other C.sub.1-C.sub.4 alkyl or alkenyl; and R.sup.4, R.sup.5, R.sup.6, R.sup.7 and R.sup.8 are independently from each other hydrogen, alkyl, or alkenyl; and X.sup.1- is halogenide or a hydroxyl-ion.

2. The method according to claim 1, wherein the enhancer compound is a quaternary amine cationic substance of formula (I), and wherein R.sup.1 is C.sub.5-C.sub.15 alkyl or alkenyl; R.sup.2 and R.sup.3 are independently from each other methyl, ethyl, propyl, or butyl; R.sup.4, R.sup.5, R.sup.6, R.sup.7 and R.sup.8 are independently from each other hydrogen, alkyl, or alkenyl; and/or X.sup.1- is chloride or bromide.

3. The method according to claim 1, wherein said enhancer, at least one compound of said enhancer, or each compound of said enhancer is at least one selected from the group consisting of 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, Benzyldibutylpropylammonium chloride, Benzyldipropylbutylammonium chloride, Benzylbutylpropylethylammonium chloride, Benzylbutylpropylmethylammonium chloride, Benzylbutylethylmethylammonium chloride, Benzyldiethylbutylammonium chloride, C.sub.12-14-alkyldimethyl (ethylbenzyl) ammonium chloride, Dodecyl (ethylbenzyl) dimethylammonium chloride, Tetradecyldimethyl (ethylbenzyl) ammonium chloride, Octadecyldimethyl (ar-ethylbenzyl) ammonium chloride and the respective aforementioned compounds containing bromide instead of chloride.

4. The method according to claim 1, wherein the effective amount of said enhancer in the chemiluminescent reaction mixture is 0.001% by weight per volume (w/v) or greater based on a total volume of the chemiluminescent reaction mixture.

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

6. The method according to claim 1, wherein the chemiluminescent signal generated by the chemiluminescent reaction is detected for a fixed duration of time, and wherein a duration of the detection starts with the start of the chemiluminescent reaction.

7. The method according to claim 1, wherein the chemiluminescent reaction mixture additionally includes hydrogen peroxide, nitric acid, and/or sodium hydroxide.

8. The method according to claim 1, wherein the chemiluminogenic compound is an acridinium ester according to formula (II) ##STR00056## wherein R.sup.1 is an alkyl, alkenyl, alkynyl, aryl or aralkyl, sulfoalkyl, carboxyalkyl, or and oxoalkyl; and wherein R.sup.3 through R.sup.15 are each independently from each other at least one selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, aryl or aralkyl, amino, amido, acyl, alkoxyl, hydroxyl, carboxyl, halogen, halogenide, nitro, cyano, sulfo, sulfoalkyl, sulfamoyl, carboxyalkyl, succinimidyl ester, oxoalkyl, and any other leaving group; and optionally, if present, X.sup.1- is an anion; or an acridinium sulfonamide according to formula (III) ##STR00057## wherein R.sup.1 is selected from the group consisting of alkyl, alkenyl, alkynyl, aryl, aralkyl, sulfoalkyl, carboxyalkyl, and oxoalkyl, and wherein R.sup.2 through R.sup.15 are each independently from each other at least one selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, aryl or aralkyl, amino, amido, acyl, alkoxyl, hydroxyl, carboxyl, halogen, halogenide, nitro, cyano, sulfo, sulfoalkyl, sulfamoyl, carboxyalkyl, succinimidyl, oxoalkyl, and any other leaving group; and optionally, if present, X.sup.1- is an anion.

9. (canceled)

10. (canceled)

11. A chemiluminescent composition, comprising: at least one compound selected from the group consisting of quaternary amine cationic substances of formula (I) ##STR00058## wherein R.sup.1 is C.sub.3-C.sub.20 alkyl or alkenyl; and R.sup.2 and R.sup.3 are independently from each other C.sub.1-C.sub.4 alkyl or alkenyl; and R.sup.4, R.sup.5, R.sup.6, R.sup.7 and R.sup.8 are independently from each other hydrogen, alkyl, or alkenyl; and X.sup.1- is halogenide or hydroxyl-ion, and at least one chemiluminogenic compound, wherein the chemiluminogenic compound is an acridinium compound.

12. A kit, comprising: at least one chemiluminogenic compound, wherein the chemiluminogenic compound is an acridinium compound, and one, two, or more compounds selected from the group consisting of quaternary amine cationic substances of formula (I) ##STR00059## wherein R.sup.1 is C.sub.3-C.sub.20 alkyl or alkenyl; and R.sup.2 and R.sup.3 are independently from each other C.sub.1-C.sub.4 alkyl or alkenyl; and R.sup.4, R.sup.5, R.sup.6, R.sup.7 and R.sup.8 are independently from each other hydrogen, alkyl, or alkenyl; and X.sup.1- is halogenide or hydroxyl-ion.

13. The kit according to claim 12, further comprising: a diagnostically useful carrier, comprising a capturer for an antigen, antibody or autoantibody in a liquid solution and a detector for the antigen, the antibody or the autoantibody bound to the carrier, wherein the detector for the antigen, antibody or autoantibody bound to the carrier is labeled with the at least one chemiluminogenic compound.

14. The chemiluminescent composition according to claim 11, for use in the diagnosis of a disease.

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

16. The method according to claim 1, wherein X.sup.1- is chloride or bromide.

17. The method according to claim 1, wherein the effective amount of said enhancer in the chemiluminescent reaction mixture is 0.2% by w/v or greater, based on a total volume of the chemiluminescent reaction mixture.

18. The method according to claim 1, wherein the enhancer is combined with the chemiluminogenic compound in the chemiluminescent reaction mixture simultaneously to the start of the chemiluminescent reaction.

19. The method according to claim 1, wherein the chemiluminescent signal generated by the chemiluminescent reaction is detected for a fixed duration of time, and wherein the duration of time is from 0.5 to 10 seconds and wherein a duration of the detection starts with the start of the chemiluminescent reaction.

20. The kit according to claim 12, for use in diagnosis of a disease.

Description

[0172] FIG. 1 shows the 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 (C.sub.12-C.sub.14) dimethylethylbenzyl-ammonium chloride) or in the presence of 0.5% (w/v) of the cationic compound CTAC lacking an aromatic head group.

[0173] FIG. 2 shows the 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 (C.sub.12-C.sub.14) dimethylethylbenzyl-ammonium chloride) or in the presence of 0.5% (w/v) of the cationic detergent CTAC lacking an aromatic head group.

[0174] FIG. 3 shows the chemiluminescence kinetics of 0.1 g/mL Anti-human IgG-Fcy-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 (C.sub.12-C.sub.14) dimethylethylbenzyl-ammonium chloride) or in the presence of 0.5% (w/v) of the cationic detergent CTAC lacking an aromatic head group.

[0175] FIG. 4 shows chemiluminescence kinetics of 0.1 g/mL Anti-human IgG-Fcy-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 (C.sub.12-C.sub.14) dimethylethylbenzyl-ammonium chloride) or in the presence of 0.5% (w/v) of the cationic detergent CTAC lacking an aromatic head group.

[0176] FIG. 5 shows the 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 prolonged hydrophobic tail with at least three carbon atoms (compound 1: Benzyldimethylhexadecylammonium chloride; compound 2: Benzalkonium chloride; compound 3: Alkyl (C.sub.12-C.sub.14) dimethylethylbenzyl-ammonium chloride; compound 4: Benzyldimethyldodecylammonium chloride) or in the presence of 0.5% (w/v) Benzyltriethylammonium chloride (BTEAC) containing an aromatic head group but lacking a prolonged hydrophobic tail or in the presence of 0.5% (w/v) CTAC lacking an aromatic head group but containing a prolonged hydrophobic tail.

[0177] FIG. 6 shows the 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 prolonged hydrophobic tail with at three least carbon atoms (compound 1: Benzyldimethylhexadecylammonium chloride; compound 2: Benzalkonium chloride; compound 3: Alkyl (C.sub.12-C.sub.14) dimethylethylbenzyl-ammonium chloride; compound 4: Benzyldimethyldodecylammonium chloride) or in the presence of 0.5% (w/v) BTEAC containing an aromatic head group but lacking a prolonged hydrophobic tail or in the presence of 0.5% (w/v) CTAC lacking an aromatic head group but containing a prolonged hydrophobic tail.

[0178] FIG. 7 shows the chemiluminescence kinetics of 0.1 g/mL Anti-human IgG-Fcy-NSP-DMAE in the presence of 0.5% (w/v) of four different cationic compounds, each with an aromatic head group and a prolonged hydrophobic tail with at least three carbon atoms (compound 1: Benzyldimethylhexadecylammonium chloride; compound 2: Benzalkonium chloride; compound 3: Alkyl (C.sub.12-C.sub.14) dimethylethylbenzyl-ammonium chloride; compound 4: Benzyldimethyldodecylammonium chloride) or in the presence of 0.5% (w/v) BTEAC containing an aromatic head group but lacking a prolonged hydrophobic tail or in the presence of 0.5% (w/v) CTAC lacking an aromatic head group but containing a prolonged hydrophobic tail

[0179] FIG. 8 shows chemiluminescence kinetics of 0.1 g/mL Anti-human IgG-Fcy-NSP-SA in the presence of 0.5% (w/v) of four different cationic compounds, each with an aromatic head group and a prolonged hydrophobic tail with at least three carbon atoms (compound 1: Benzyldimethylhexadecylammonium chloride; compound 2: Benzalkonium chloride; compound 3: Alkyl (C.sub.12-C.sub.14) dimethylethylbenzyl-ammonium chloride; compound 4: Benzyldimethyldodecylammonium chloride) or in the presence of 0.5% (w/v) BTEAC containing an aromatic head group but lacking a prolonged hydrophobic tail or in the presence of 0.5% (w/v) CTAC lacking an aromatic head group but containing a prolonged hydrophobic tail.

[0180] 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 taken.

EXAMPLES

Preparation of Acridinium Label and Conjugate Solutions:

[0181] 1 mg/mL label stock solutions of N-Sulfopropyl-dimethyl-acridinium-N-hydroxysuccinimide (NSP-DMAE-NHS; PerkinElmer Inc., Finnland) 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, Fcy fragment specific (Anti-human IgG-Fcy; Jackson ImmunoResearch Europe Ltd., UK) was diluted to a final concentration of 0.33 mg/mL in phoshate buffered saline, pH 7.6 and was subsequently 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, model RM-2M (Hassa GmbH, Germany) at 10 rpm. The labeling reactions were stopped with addition of 17.4 mM L-lysine (VWR International GmbH, Germany) for 30 min at room temperature during rotation at 10 rpm. Excess of free label was removed from the antibody conjugates during ultrafiltration at 3.800 g in a Heraeus Megafuge 40R centrifuge (Fisher Scientific GmbH, Germany) at 4 C. using a Vivaspin 20, molecular weight cutoff 10 kDa concentrator (VWR International GmbH, Germany) and the buffer was exchanged for phosphate buffered saline, pH 7.4 containing 0.045% (w/v) sodium azide (Merck KgaA, Gemany). The purified antibody conjugates were 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).

Preparation of Chemiluminescence Trigger Solutions:

[0182] 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 either used 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, Gemany), Hexadecyltrimethylammonium bromide (Merck KgaA, Gemany) or Tetradecyltrimethylammonium chloride (Merck KgaA, Gemany) all of which lack an aromatic head group. 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 (C.sub.12-C.sub.14) 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 with Benzyltriethylammonium chloride (Merck KgaA, Gemany) which contains an aromatic head group but lacks a prolonged hydrophobic tail of at least three carbon atoms.

Measurement of Chemiluminescence Signal Intensities:

[0183] NSP-DMAE-NHS and NSP-SA-NHS label stock solutions were diluted to a final concentration of 1 nM and antibody conjugates were diluted to a final concentration of 0.1 g/mL in phoshate buffered saline, pH 7.4. 10 L each of these dilutions were transferred to a well of a 96-well LumiNunc plate (VWR International GmbH, Germany) and chemiluminescence signal intensities were measured on a LB 960 Centro Microplate Luminometer (Berthold Technologies Gmbh & Co. KG, Germany) in the presence of 100 L each of chemiluminescence trigger A and of different trigger B solutions, the latter varying in the identity and concentration of the cationic compound. After injection of trigger A with middle speed, the mixtures were incubated for 1 sec during mixing in double orbit mode with normal speed. Chemiluminescence intensities in relative light units (RLU) were recorded every 50 ms over a total time of 1.5 sec upon addition of the various trigger B solutions with low injection speed. All of the samples were measured in four technical replicates. In control experiments all different trigger B solutions were injected in combination with trigger A into empty wells of the 96-well plate. The latter experiments reflect the signal background generated by the different trigger solutions.

Data Analysis:

[0184] For graphic presentation of the observed chemiluminescence kinetics a representative data set was chosen from the four technical replicates and the signal intensities in RLU were plotted against time. Additionally, total signal intensities were calculated by addition of all RLU values observed in the 1.5 sec time frame. The background signal intensities of the different trigger B solutions were subtracted from the total signal intensities and all background-corrected total signal intensities were set in comparison.

Example 1

[0185] Three representative enhancer compounds (compounds 1-3) according to the present invention were chosen for the analysis. These molecules are all based on a cationic, quarternary ammonium compound containing an aromatic moiety adjacent to the quarternary ammonium. The enhancer compounds according to the present invention were tested against a common enhancer substance CTAC (as for example proposed in U.S. Pat. No. 4,927,769). In contrast, CTAC does not contain an aromatic moiety adjacent to the quarternary ammonium.

[0186] Four representative analytes were chosen to investigate the effect of the enhancer compounds according to the present invention in comparison to the common enhancer substance already known in the prior art on the chemiluminescence signal generated by these analytes (FIG. 1-4). The chosen analytes include the acridinium compounds NSP-DMAE-NHS and NSP-SA-NHS in free-(FIG. 1-2) and in antibody-bound-form (FIG. 3-4). A polyclonal Anti-human IgG-Fcy antibody from goat was chosen for conjugation with both types of acridinium label.

[0187] Acridinium label, conjugate solutions and chemiluminescence trigger solutions were obtained according to the methods disclosed above. Measurement of chemiluminescence signal intensities was performed and data was analysed as described above.

Results:

[0188] A 43% increase in signal output using Benzyldimethylhexadecylammonium chloride (compound 1) with analyte NSP-DMAE-NHS (FIG. 1, Table 3) was observed in comparison with the enhancer substance CTAC for the same analyte.

[0189] Benzalkonium chloride (compound 2) and Alkyl (C.sub.12-C.sub.14) dimethylethylbenzyl-ammonium chloride (compound 3) also resulted in a 19% and 15% signal increase in comparison with CTAC with NSP-DMAE-NHS as analyte, respectively.

[0190] A similar result was obtained when the analyte was switched to NSP-SA-NHS (FIG. 2, Table 3).

[0191] Compound 1 (Benzyldimethylhexadecylammonium chloride) led to a signal increased by 67% compared to the substance CTAC. Moreover, a 58% and 40% signal increase with compounds 2 and 3 were observed for this analyte in comparison to CTAC, respectively.

[0192] Additionally, it was found that the enhancement of the signal output observed for both examples of acridinium-based probes in the free form could also be observed when the probes were covalently attached to an antibody, the latter typically required for immunoassays.

[0193] In this scenario, compound 1 (Benzyldimethylhexadecylammonium chloride) led to a 45% and a 66% signal increase for the analytes Anti-human IgG-Fcy-NSP-DMAE (FIG. 3, Table 3) and Anti-human IgG-Fcy-NSP-SA (FIG. 4, Table 3), respectively in comparison with signal generation for those analytes in the presence of CTAC. Compound 2 (Benzalkonium chloride) also increased the signal of these two antibody conjugates by 40% and by 52%, respectively. Moreover, a 25% and a 35% signal increase in the presence of compound 3 (Alkyl (C.sub.12-C.sub.14) dimethylethylbenzylammonium chloride) for the two analytes were observed in comparison with the signal generation in the presence of CTAC.

TABLE-US-00003 TABLE 3 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 having an aromatic head group (compounds 1-3) or in the presence of 0.5% (w/v) of the cationic detergent CTAC lacking an aromatic head group. Analyte Anti-human Anti-human IgG-Fc- IgG-Fc- Compound NSP-DMAE-NHS NSP-SA-NHS NSP-DMAE NSP-SA Benzyldimethylhexadecyl- 4712949 RLU 3631369 RLU 2707416 RLU 1449182 RLU ammonium chloride (+43%) (+67%) (+45%) (+66%) (compound 1) Benzalkonium chloride 3913778 RLU 3432574 RLU 2621080 RLU 1325083 RLU (compound 2) (+19%) (+58%) (+40%) (+52%) Alkyl(C.sub.12- 3809207 RLU 3042201 RLU 2339403 RLU 1175726 RLU C.sub.14)dimethylethyl- (+15%) (+40%) (+25%) (+35%) benzylammonium chloride (compound 3) CTAC 3299926 RLU 2169527 RLU 1871550 RLU 872358 RLU

Example 2

Description

[0194] In order to test the effect of the chain length within the hydrophobic tail of the substances on chemiluminescence enhancement further experiments were performed with compounds 1-3 (as described in Example 1), with the common enhancer CTAC, with an additional cationic enhancer according to the present invention (compound 4; Benzyldimethyldodecylammonium chloride) and with the additional substance BTEAC (Benzyltriethylammoniumchloride), which contains a chain length of less than three carbon atoms in the hydrophobic tail (FIGS. 5-8). These additional experiments were performed as described above and as described in Example 1, respectively.

Results:

[0195] Similarly to the compounds 1-3, compound 4 enhanced the chemiluminescence of 1 nM NSP-DMAE-NHS (FIG. 5), of 1 nM NSP-SA-NHS (FIG. 6), of 0.1 g/mL Anti-human IgG-Fcy-NSP-DMAE (FIG. 7) and of 0.1 g/mL Anti-human IgG-Fcy-NSP-SA (FIG. 8) compared with the common enhancer CTAC. In this experiment, compounds 1-3 increased the chemiluminescence of 1 nM NSP-DMAE-NHS by 45-79%, of 1 nM NSP-SA-NHS by 62-102%, of 0.1 g/mL Anti-human IgG-Fcy-NSP-DMAE by 46-63% and of 0.1 g/mL Anti-human lgG-Fcy-NSP-SA by 47-50% compared with the common enhancer CTAC (Tab. 4). Similarly, compound 4 increased the chemiluminescence of 1 nM NSP-DMAE-NHS by 50%, of 1 nM NSP-SA-NHS by 47%, of 0.1 g/mL Anti-human IgG-Fcy-NSP-DMAE by 50% and of 0.1 g/mL Anti-human IgG-Fcy-NSP-SA by 40% compared with the common enhancer CTAC (Tab. 4).

[0196] For BTEAC, a reduced chemiluminescence was observed with all four analytes compared with the common enhancer CTAC (FIGS. 5-8 and Tab. 4). Although this substance contains an aromatic head group, this substance does not function as an equivalent chemiluminescence enhancer, most likely and not being bound to any theory, because the chain length of the hydrophobic tail is too short to form micellular structures.

TABLE-US-00004 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 a prolonged hydrophobic tail with at least three carbon atoms (compounds 1-4) or in the presence of 0.5% (w/v) Benzyltriethylammonium chloride (BTEAC) containing an aromatic head group but lacking a prolonged hydrophobic tail or in the presence of 0.5% (w/v) of the cationic detergent CTAC lacking an aromatic head group but containing a prolonged hydrophobic tail. Analyte Anti-human Anti-human IgG-Fc- IgG-Fc- Compound NSP-DMAE-NHS NSP-SA-NHS NSP-DMAE NSP-SA Benzyldimethylhexadecyl- 9285851 RLU 7687351 RLU 2545936 RLU 13400308 RLU ammonium chloride (+74%) (+86%) (+55%) (+48%) (compound 1) Benzalkonium chloride 9554986 RLU 8351873 RLU 2667518 RLU 13568679 RLU (compound 2) (+79%) (+102%) (+63%) (+50%) Alkyl(C.sub.12- 7746405 RLU 6669609 RLU 2389234 RLU 13333146 RLU C.sub.14)dimethylethyl- (+45%) (+62%) (+46%) (+47%) benzylammonium chloride (compound 3) Benzyldimethyldodecyl- 8015307 RLU 6074325 RLU 2450210 RLU 12677099 RLU ammonium chloride (+50%) (+47%) (+50%) (+40%) (compound 4) BTEAC 3663184 RLU 1939935 RLU 676605 RLU 5143731 RLU (31%) (53%) (59%) (43%) CTAC 5333619 RLU 4125130 RLU 1637564 RLU 9041121 RLU

[0197] Together, 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 in comparison to the signal generation by the same acridinium-based analytes in the presence of a common enhancer, as CTAC.

[0198] Moreover, these new chemiluminescence enhancers all share a molecular structure containing a cationic, quarternary ammonium compound linked to an aromatic moiety, i.e. aromatic head group, and a minimum chain length of at least three carbon atoms in the hydrophobic tail of the enhancer. It is likely that all substances which contain these molecular moieties, i.e. the aromatic head group and the minimum chain length of at least three carbon atoms in the hydrophobic tail, would similarly enhance the chemiluminescence generated by acridinium-based analytes in comparison to enhancer substances lacking these molecular moieties, i.e. the aromatic head group and the minimum chain length of at least three carbon atoms in the hydrophobic tail. Substances with a shorter chain length in the hydrophobic tail do not similarly enhance the chemiluminescence, most likely because they do not form micellular structures, which are supposed to favor the chemiluminescence enhancement. While not being bound by theory, it is further believed that the newly presented cationic chemiluminescence enhancers according to the present invention favors the chemiluminescence reaction process by direct interaction with the acridinium ring forming so-called hydrophobic TT-TT stacking interactions. In this fashion, the acridinium label may be efficiently recruited and positioned for reaction with the hydrogen peroxide anion, the latter attracted by the positive charge of the adjacent cationic ammonium ion.

[0199] The data, figures, instruments, reagents and steps herein should be understood to be illustrative, but not restrictive. Although the present disclosure was described with reference to the above concrete embodiments, many modifications and variances will be apparent to skilled persons in the art. All the modifications and variances also fall within the spirit and scope of the disclosure.