CA2390696A1 - Method of stabilizing a luminescent acridinium compound and composition containing the stabilizer - Google Patents

Abstract

The object of the present invention is to provide a new means of stabilizing a luminescent acridinium compound in an aqueous medium thereby preventing a decrease in the emission yield from the compound over time in storage in the aqueous medium. This is accomplished by the presence in the aqueous medium o f 0.2 to 20 g/L of a dioxane having one bromine atom and one nitro group.</SDO AB>

Description

STABILIZING A LUMINESCENT ACRIDINIUM COMPOUND
Field of the Invention The present invention relates to a method of stabilizing a luminescent acridinium compound in an aqueous medium. The present invention also relates to a composition comprising a luminescent acridinium compound in an aqueous medium in which the luminescent acridinium compound is stabilized.
1o Background of the Invention It is known that a luminescent acridinium compound is a salt of an acridinium compound in which a nitrogen in an acridine ring forms a quaternary amine that is excited by alkaline hydrogen peroxide to emit light. Owing to this property, luminescent acridinium compounds are frequently used as labels in the qualitative or 15 quantitative analysis of analytes, e.g. in a chemiluminescent immunoassays.
The water-soluble derivatives of luminescent acridinium compounds are generally used when these compounds are employed as labels. However these water-soluble derivatives can be unstable in an aqueous medium, resulting in a decrease in their emission intensity and yield over time when stored.
2o Various attempts have been made to improve the stability and increase the emission yield of luminescent acridinium compounds in an aqueous medium.
Initially more stable acridinium compounds were synthesized, as described in JP-6357572A, JP-63112564A, JP-63101368A. Additional compounds or additives, such as cyclodextrin (see JP-7278184A), have also been added to an aqueous medium 25 containing a luminescent acridinium compound in an effort to stabilize the compound.
However, further improvements in stability and emission yield of luminescent acridinium compounds are still needed when these compounds are stored in an aqueous medium.
3o Summary of the Invention An object of the present invention is to provide a new means for stabilization of a luminescent acridinium compound in an aqueous medium thereby preventing a decrease in emission yield over time.

The inventors have found that by including a dioxane having one bromine atom and one vitro group (hereinafter referred to "bromo-vitro-dioxane") in the aqueous medium containing a luminescent acridinium compound, that the acridinium compound was more stable in the aqueous medium during storage and that the decreased emission yield typically seen over time, was prevented.
One aspect of the present invention relates to a method of stabilizing a luminescent acridinium compound by dissolving the luminescent acridinium compound in an aqueous medium containing 0.2 to 20 g/L of bromo-vitro-dioxane.
Another aspect of the present invention relates to a composition comprising a luminescent acridinium compound in an aqueous medium containing 0.2 to 20 g/L
of bromo-vitro-dioxane.
Detailed Description of the Invention As used herein, the singular forms "a", "an", and "the" include plural reference unless the context clearly dictates otherwise. In addition, all references to the published scientific and patent literature (including patent applications) are hereby incorporated by reference.
The luminescent acridinium compounds discussed herein are defined as those luminescent acridinium compounds that are water-soluble. Water-soluble luminescent acridinium compounds, as used in the present invention, include those which are soluble in an aqueous liquid to the extent that they can be used as detectable labels in a test for an analyte, or are useful as detection labels in an assay such as an immunoassay. Compounds that are weakly soluble in an aqueous medium also are included in this definition, as are compounds that are dissolved first in a polar solvent such as methanol, then introduced into an aqueous medium.
The luminescent acridinium compounds of the present invention may be conjugated, directly or indirectly via a linker, to one of the members of a specific binding pair. A "specific binding pair" refers to two different molecules where one of the molecules, through chemical or physical means, specifically binds to the second molecule. A "specific binding member," as used herein, is a member of a specific binding pair. In addition to antigen and antibody specific binding pairs of common immunoassays, other specific binding pairs can include biotin and avidin, carbohydrates and lectins, complementary nucleotide sequences, effector and receptor WO 01/35103 CA 02390696 2002-05-08 pCT/IB99/01827 molecules, cofactors and enzymes, enzyme inhibitors and enzymes, and the like.
Furthermore, specific binding pairs can include members that are analogs of the original specific binding members, for example, an analyte-analog.
Immunoreactive specific binding members include antigens, antigen fragments, antibodies and antibody fragments, both monoclonal and polyclonal, and complexes thereof, including those formed by recombinant DNA molecules.
In the present invention, the member of the binding pair, which is labeled with the acridinium compound may be used to detect the corresponding member of the specific binding pair (e.g. an analyte in a patient sample). Alternatively, the same to member of a specific binding pair that is labeled may also be the analyte being detected, when a competitive format is used.
Many different luminescent acridinium compounds may be used in the present invention. Well-known examples include those described in U.S. Patent No.
5,468,646, issued November 21, 1995, U.S. Patent No. 5,543,524, issued August 6, 1996, U.S. Patent No. 5,545,739, issued August 13, 1996, U.S. Patent No.
5,565,570, issued October 15, 1996, U.S. Patent No. 5,669,819, issued September 23, 1997, and U.S. Patent No. 5,783,699, issued July 21, 1998, all of which are incorporated herein by reference. Preferred luminescent acridinium compounds are salts of 10-substituted-9-carboxamido-acridinium, also described in JP-63112564A.
Different 2o groups may be substituted at the 10-position, including but not limited to an alkyl group, aryl group or heterocyclic group, which may be substituted or unsubstituted (e.g. 3-sulfopropyl).
The 9-carboxamide nitrogen may be bound to one group or two groups which are the same or different. Non-limiting examples of such groups include an unsubstituted alkyl, aryl, heterocyclic (e.g. 2-carboxyethyl or 3-sulfopropyl), sulfinyl (e.g. a tosyl) or sulfonyl group (e.g. a trifluoro-methanesulfonyl, halo- or nitro-benzenesulfonyl group). An alkyl group as the 10-substituent or group attached to the nitrogen of the 9-carboxamide, may contain a nitrogen, sulfur or oxygen atom, or a carbonyl, oxycarbonyl, amido or similar group in its carbon chain. Other examples of substitute groups (for example, a substitute group in a substituted alkyl) for the 10-substituent position or as the group attached to the nitrogen of the 9-carboxamide include halogens, carboxyl, sulfonyl, sulfinyl, thiol, amino, hydroxyl and succinimide groups. Among these substitute groups, at least one group may be preferred for its WO 01/35103 CA 02390696 2002-05-08 pCT/IB99/01827 ease in forming a covalent bond with one of the members of a specific binding pair through, for example, a carboxyl, amino or thiol group. The most preferred luminescent acridinium compound is 10-(3-sulfopropyl)-9-[N-(2-carboxyethyl)-N-tosyl]carboxamido-acridinium trifluoromethanesulfonate.
A water-soluble acridinium conjugate comprising a luminescent acridinium compound linked to one member of a specific binding pair is prepared by any of a number of methods known to those skilled in the art. Such conjugation methods include but are not limited to binding via a linker, or direct binding of an amino, carboxyl or sulfhydryl group of an antigen, antibody or other protein member of a specific binding pair, to a carboxyl, amino or sulfhydryl group of the luminescent acridinium compound.
The aqueous medium is preferably a buffered solution in a pH range. The pH
of the buffer is preferably 5 to 10, and more preferably pH 6 to 8. There are various conventional buffer solutions comprising inorganic or organic acids such as 15 phosphoric acid, pyrophosphoric acid, boric acid, tris-hydrochloric acid, succinic acid, citric acid, malic acid and malefic acid, all of which can be used. The aqueous medium may also contain surfactants, such as Triton or Tween, and/or non-specific proteins such as bovine serum albumin. Further, in order to increase the solubility of a luminescent acridinium compound, a polar organic solvent, such as, for example, 2o dimethylformamide (DMF) and dimethyl sulfoxide (DMSO) may be used.
The bromo-nitro-dioxane of the present invention uses 1,4-dioxane or 1,3-dioxane wherein the hydrogen atoms at any position in the dioxane are replaced with a bromine atom and a nitro group. The preferred bromo-nitro-dioxane is 5-bromo-nitro-1,3-dioxane. Bromo-nitro-dioxanes are well-known in the art and are 25 commercially available (from, e.g. SIGMA, St. Louis, MO).
The concentration of the bromo-nitro-dioxane that is useful when present in an aqueous medium is between 0.2 to 20 g/L, and preferably between 1 to 5 g/L.
The lower limit concentration of a luminescent acridinium compound in an aqueous medium is determined such that the emission from the luminescent 3o acridinium compound can be detected. There is not necessarily an upper limit on the concentration of a luminescent acridinium compound in an aqueous medium, other than that determined by the solubility of the compound, since the solution may be diluted if the concentration of the compound is determined to be too high to use.

Storage of the luminescent acridinium compound in an aqueous medium is required for being able to use the compound for various applications. It is preferable that storage conditions include cold temperatures and the absence of light.
Luminescent acridinium compounds are even more stable when stored using the method or composition of the present invention, wherein bromo-nitro-dioxane is included the aqueous medium. This increased stability prevents or lessens the amount that the emission yield of the compound decreases over time in storage.
Luminescent acridinium compounds stored according to the method or composition of the invention, can be used in the same applications and procedures as luminescent acridinium compounds stored using methods previously known in the art.
Optionally, the luminescent acridinium compound can be separated from the aqueous medium containing the bromo-nitro-dioxane after storage during use. Some examples of the use of luminescent acridinium compounds stored according to the present invention include, but are not limited to, as a label in a chemiluminescent immunoassay, as a label in a nucleic acid hybridization assay, to label cells in flow cytometry, for use in labeling tissues in histology or the like.
Examples 2o Example 1 An acridinium conjugate comprising a recombinant Hepatitis B core antigen (rHBcAg) labeled with a luminescent acridinium compound was prepared as follows:
One mg of 10-(3-sulfopropyl)-9-[N-(2-carboxyethyl)-N-tosyl]carboxamido-acridine trifluoromethanesulfonate was dissolved in 100 pL of anhydrous dimethylformamide (DMF). Fifty pL of N-hydroxysuccinimide (5.75 mg/mL) was added and the solution was stirred at 25°C for 48 hours in the dark to activate the luminescent acridinium compound. The activated luminescent acridinium compound, diluted to 160 ~g/mL, was added to a lmg/mL solution of rHBcAg in 0.018 M phosphate buffer, pH 8.0 containing 0.9 % NaCI, and the mixture was stirred at room temperature for 10 3o minutes. The mixture was then applied to a Hiprep 16/60 S-200 FPLC column (Pharmacia, Uppsala,Sweden) to separate the fraction containing the rHBcAg labeled with acridinium (i.e., the rHBcAg-acridinium conjugate) from the reaction mixture.
Alternatively, an activated luminescent acridinium compound such as 3-[9-({ {4-[(2,5-dioxo-1-pyrrolidinyl)oxy]-4-oxobutyl } [(4-methylphenyl)sulfonyl]amino }
carbonyl)-10-acridiniumyl]-1-propanesulfonate may be used and conjugated to rHBcAg directly, as described above.
The rHBcAg-acridinium conjugate was then diluted to 0.05 ~g/mL in an aqueous medium composed of 50 mM MES (2-[N-morpholino] ethanesulfonic acid) buffer, pH 6.3 containing 1.5 % Triton-X, 2.5 % bovine serum albumin and 0.15 M
NaCI and containing 0, 1, 2 or 10 g/L 5-bromo-5-nitro-1,3-dioxane (BND). This solution was stored in the dark at 45°C for 7 days. After the 7-day storage period, the amount of luminescent emission from the rHBcAg-acridinium conjugate was determined. As a control, 0.05 p,g/mL of the conjugate was diluted in the same buffer 1 o containing 2 g/L of BND, and tested immediately without being stored.
The luminescent emission amount was determined by adding 50 ~,L of 0.2 N
NaCI containing 0.03 % H202 to the aqueous medium containing the conjugate.
The amount of light emitted (RLu) was then determined using a photo-counting luminometer.
The results, shown in Table 1, indicate the luminescence of the HBcAg-acridinium conjugate decreased 43%, from a control of 251 to 143, after 7 days of storage in the absence of BND. However, if BND was present in the storage solution, no or little decrease (5.6% at 1 g/L BND) in luminescence was observed vs. the control.
Table 1 Concentration of BND Luminescence (g/L) (RLu x 103) 2 (no storage) 251 Example 2 The effectiveness of using a luminescent acridinium compound as a label in an assay was measured after storage of the acridinium conjugate in an aqueous medium with or without S-bromo-5-nitro-1,3-dioxane (BND). The assay used indirect 3 CA 02390696 2002-05-08 pCT/IB99/01827 antibody capture of IgM-HBcAb on microparticles, which was then detected using the HBcAg-acridinium conjugate labeled as described in Example 1.
Magnetic microparticles (3 ~m in diameter, Polymer Labo K.K., Japan) were coated with a solution of 30 pg/ml of anti-human IgM mouse monoclonal antibody (CosmoBio K.K., Japan) in a 0.05 M MES buffer, pH 6.2 containing 2.0 mg/mL 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDAC), for 1 hour at room temperature. The antibody solution was removed from the magnetic particles and the microparticles were washed with 0.01 M phosphate buffer, pH 7.2 containing 0.1 Tween 20 and 0.9 % NaCI.
to The anti-human IgM coated magnetic microparticles were then used to capture IgM anti-HBc from a previously titered human serum sample containing HBcAb.
This was done by incubating 50 ~L of a 0.15% solution of anti-human IgM coated magnetic particles in 50 mM Tris-HCI, pH 7.4, with 50 ~L of human anti-HBc serum diluted to contain 40, 572 or 3000 U/mL HBcAb, for 18 minutes at 37°C.
The magnetic microparticles, now containing bound IgM anti-HBc, were then separated from the mixture and washed with 4 mM phosphate buffer, pH 6.8 containing 0.9%
NaCI and 0.17% Brij 35. Next, 50 ~L of the HBcAg-acridinium conjugate, prepared as in Example 1 with or without the addition of 2 g/L BND, and which had been stored at either 2 to 8°C (the control solutions) or 45°C
(accelerated stability test solutions) for 7 days, was added to the magnetic microparticles and incubated for 4 minutes at 37°C. The magnetic microparticles were again separated from the solution and washed as before. The amount of light emitted from the HBcAg-acridinium conjugate attached to the magnetic microparticles was then determined using a photo-counting luminometer. The results of the solutions stored at 45°C were then compared to the control solutions stored at 2-8°C, and expressed as the percentage of activity present versus the control solution.
As can be seen from the results in Table 2, the activity of the acridinium conjugate after storage under accelerated stability test conditions (45°C) in the absence of BND is, on average, 10% lower than when stored in the presence of BND.
3o The activity of the conjugate after storage with BND only decreases slightly. Thus BND provides a benefit for helping to maintain the activity of the acridinium compound over time in solution.

Table 2 Concentration of BND HBcAb % Activity in Storage Solution concentration (45C vs. 2-8C

(g/L) (U/mL) stored control) . ...........572 87 ............

. ...... ... -300081 --. -. ....
..

. . . . . . . . 97 . _ . .572 .
. . . . _ .
. . . . .

. . . . . . . . 95 . . -3000 -. . . . . .
. . . .

While the invention has been described in detail and with reference to specific embodiments, it will be apparent to one skilled in the art that various changes and modifications may be made to such embodiments without departing from the spirit and scope of the invention.

Claims (8)

What is claimed is:

1. A method of stabilizing a luminescent acridinium compound comprising dissolving said luminescent acridinium compound in an aqueous medium containing 0.2 to 20 g/L of a dioxane having one bromine atom and one nitro group.

2. The method of claim 1 wherein the luminescent acridinium compound is a salt of 10-substituted-9-carboxamido-acridinium.

3. The method of claim 1 wherein the dioxane having one bromine atom and one nitro group is 5-bromo-5-nitro-1,3-dioxane.

4. The method of claim 1 wherein the luminescent acridinium compound is conjugated to an antigen, antibody or nucleic acid.

5. A composition comprising a luminescent acridinium compound in an aqueous medium containing 0.2 to 20 g/L of a dioxane having one bromine atom and one nitro group.

6. A composition as claimed in claim 5 wherein the luminescent acridinium compound is a salt of 10-substituted-9-carboxamido-acridinium.

7. A composition as claimed in claim 5 wherein the dioxane having one bromine atom and one nitro group is 5-bromo-5-nitro-1,3-dioxane.

8. A composition as claimed in claim 5 wherein the luminescent acridinium compound is conjugated to an antigen, antibody or nucleic acid.