Note: Descriptions are shown in the official language in which they were submitted.
WO 01/35103 CA 02390696 2002-05-08
PCT/IB99/01827
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.
CONF1RMATIGN COPY
WO 01/35103 CA 02390696 2002-05-08 PCT/IB99/01827
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-
5-
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.
WO 01/35103 CA 02390696 2002-05-08 PCT/IB99/01827
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)-
WO 01/35103 CA 02390696 2002-05-08 PCT/IB99/01827
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)
0 143
1 237
2 250
10 248
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
6
WO 01/35103 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.
7
WO 01/35103 CA 02390696 2002-05-08 PCT/IB99/01827
Table 2
Concentration of BND HBcAb % Activity
in
Storage Solution concentration (45C vs. 2-8C
(g/L) (U/mL) stored control)
0 40 84
. ...........572 87
............
. ...... ... -300081
--. -. ....
..
2 40 93
. . . . . . . . 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.
