CA 02234450 1998-04-08
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SULFO BENZ[e]INDOCYANINE FLUORESCE'N'T DYES
FIELD OF THE INVENTION
The present invention relates to a new class of fluorescent dyes and their
valence tautomers belonging to the sulfo Benz[e]indocyanine family. The
instant invention also relates to the synthesis of the new class of
fluorescent
dyes belonging to the sulfo bent[e]indocyanine family. The new fluorescent
dyes can be excited using powerful yet inexpensive light emitting diodes and
diode lasers, they exhibit good water solubility, and can be attached or
conjugated to a wide variety of molecules or surfaces for labeling purposes.
The new fluorescent dyes are particularly useful in techniques such as
immunoassays, DNA probes, high pressure liquid chromato~aphy (HPLC),
capillary electrophoresis, fluorescence polarization, total internal
reflection
fluorescence (T.LR.F.), flow cytometry, DNA sequencing, and optical sensors.
BACKGROUND OF THE INVENTION
The use of fluorescence technology has become widespread in the areas
of clinical chemistry, i.e., laboratory testing and the medical diagnostic
areas.
The technology is particularly effective for making very sensitive and
specific
test determinations, competing effectively in many areas with
radioimmunoassays and enzymatic immunoassays.
The phenomenon of fluorescence occurs when a molecule or atom is
bombarded with light of given wavelengths; namely, the conversion of that
Light to an emission of light of a different wavelength. In macroscopic terms,
the conversion is instantaneous, but in real terms the finite time differences
between the absorption of the light by the molecule and the time interval
during
which the emitted light is given off is a measure of the characteristics of
the
bodies being measured.
The process of fluorescence starts with the absorption of light photons
by atoms or molecules. The frequency of light absorption varies with the atom
or molecule involved.
Fluorescent molecules in any specific environment have two
characteristic spectra. The first, the so-called excitation spectrum, is
represented by a series of wavelengths of light which are absorbed by the
molecule with~differ~ing efficiencies. That is, out of a possible number of
existing wavelengths which may be absorbed by the molecule to cause
fluorescence, usually one of these will be absorbed at a l,~reater level. Most
atoms or molecules that absorb light convert this light energy into heat, but
a
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few emit Iight or "fluoresce" at a lower light frequency. Photon absorption
occurs rapidly in about 10-IS seconds. If the light excitation is abruptly
interrupted, as with a very short pulse of light, photon light emission in the
second spectrum will decay rapidly with a time constant that depends on the
atom or molecule involved. The range of decay times is usually between IO-10
to 10-6 seconds (0.1 to 1000 nanoseconds). The intensity of the emission
spectrum is directly proportional to the intensity of the exciting light.
It happens also that the intensity of the emitted light is also directly
proportional to the concentration of the fluorescent molecules in the sample.
It
thus can be seen that a very sensitive technique for measuring the
concentration
of a fluorescent body can be evolved by controlling the intensity of the
exciting
light and other physical constants of the measuring system.
The analytical value of fluorescence decay time measurement arises
from the fact that each atom or molecule has its own distinctive rate of
decay.
Each atom or molecule is excited at a different frequency and emits light only
at a particular emission wavelength.
Analytical probes having fluorescent labels are valuable reagents for the
analysis and separation of molecules and cells. Specific applications include:
(1) identification and separation of subpopulations of cells in a mixture of
cells
by the techniques of fluorescence flow cvtometry, fluorescence-activated cell
sorting, and fluorescence microscopy; (2) determination of the concentration
of
a substance that binds to a second species (e.g., antigen-antibody reactions)
in
the technique of fluorescence immunoassay; (3) localization of substances in
gels and other insoluble supports by the techniques of fluorescence staining.
These techniques are described by Herzenberg et al., "Cellular Immunologry,"
3rd ed., chapt. 22, Blackwell Scientific Publications, 1978 (fluorescence-
activated cell sorting); and by Goldman, "Fluorescence Antibody Methods,"
Academic Press, New York, 1968 (fluorescence microscopy and fluorescence
staining).
When using fluorescers for the above purposes, there are many
constraints on the choice of the fluorescer. One constraint is the absorption
and emission characteristics of the fluorescer. Many ligands, receptors, and
materials associated with such compounds in the sample in which the
compounds are found, e.g., blood, urine, and cerebrospinal fluid, will
fluoresce
and interfere with an accurate determination of the fluorescence of the
fluorescent label. Another consideration is the ability to conjugate the
fluorescer to ligands and receptors and the effect of such conjugation on the
fluorescer. In many situations, conjugation to another molecule may result in
a
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substantial change in the fluorescent characteristics of the fluoresces and in
some cases, substantially destroy or reduce the quantum efficiency of the
fluoresces. A third consideration is the quantum effciency of the fluoresces.
Also of concern is whether the fluorescent molecules will interact with each
other when in close proximity, resulting in self quenching. An additional
concern is whether there is non-specific binding of the fluoresces to other
compounds or container walls, either by themselves or in conjunction with the
compound to which the fluoresces is conjugated.
The value of the methods indicated above are closely tied to the
availability of suitable fluorescent compounds. In recent years, the evolution
of solid state emitting diodes and solid state detectors has progressed
rapidly as
has the chemistry of fluorescent dyes. This evolution opened several
opportunities for applications in the red and near infrared region (617 to
2500
nm). The red and near infrared region appears particularly suitable for
biological analysis because of the low background fluorescence generated by
biological material or by chemical compounds. Moreover, the development of
inexpensive commercial diode lasers with emitting wavelengths of 670, 750,
780, and 810 nm, led to research into dyes that can be excited at these
wavelengths.
Earlier efforts in this field produced many dyes, most of them belonging
to the cyanine family. However, these dyes did not satisfy all the
requirements
needed for useful applications. Waggoner et al. (Bioconiu~ate Chemistm_, 4
105-I 11 (1993)) studied the chemistry of the cyanine dyes in order to develop
conjugation sites and also to enhance water solubility. They synthesized
sulfoindocyanine dyes with high hydrosolubility and reactive groups available
for conjugation with biological compounds. However, these dyes cannot be
excited by diode lasers andJor have low fluorescence quantum yields.
During the development of photo-sensitive dyes, a Kodak research
group added one more ring to the indolenine moiety and made the dye structure
more rigid by means of an additional ring in the polymethine chain. Trying to
reach the same goal, Patonay et al. (J. Ors. Chem.. 57~ 4578-4580 (1992))
studied structures bearing activated groups for conjugation purposes and a
chlorocyclohexenyl ring in the polymethine chain. The latter increases the
rigidity of the structure, thus enhancing the fluorescence quantum yield of
the
dye. It also provides a convenient site for chemical substitution at the
central
ring, useful for introducing reactive groups or electron withdrawing radicals
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capable of modifying the excitation wavelength. Some of the
drawbacks of Kodak's and Patonay's dyes are their low
hydrophilicity and/or the mismatch with commercially
available diode lasers.
. The prior art is silent regarding the sulfo
bent[e]indocyanine fluorescent dyes of the present invention
as well as their uses in immunoassays, DNA probes, DNA
sequencing, HPLC, capillary electrophoresis, fluorescence
polarization, total internal reflection fluorescence, flow
cytometry, and optical sensors. The above analytical
techniques would enjoy substantial benefits when using the
fluorescent dyes of the present invention, which have high
quantum efficiency, absorption, and emission characteristics
at the red and near infrared region, simple means for
conjugation, and are substantially free of non-specific
interference.
SUMMARY OF THE INVENTION
The present invention provides sulfo
bent[e]indocyanine fluorescent dyes.
The present invention also provides sulfo
benz[e]indocyanine fluorescent dyes of high hydrophilicity
having one or more sulfonic acid groups attached to the
benzo ring.
Further, the instant invention provides sulfo
Benz[e]indocyanine fluorescent dyes having high quantum
efficiencies.
Still further, the present invention provides
sulfo benz[e]indocyanine fluorescent dyes having reactive
sites for conjugation.
4
I i A~ il~ 1 i~ In I~iiemi,i I
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The present invention also provides sulfo
benz[e]indocyanine fluorescent dyes having a rigidity
enhancing central ring in the molecule.
Further, the instant invention provides sulfo
bent[e]indocyanine fluorescent dyes having variable
polymethine chains for tuning the fluorescent behavior of
the dyes.
The present invention also provides biologically
active molecules having attached thereon sulfo
bent[e]indocyanine fluorescent dyes.
Further, the present invention provides sulfo
bent[e]indocyanine fluorescent dyes useful in the red and
near infrared region.
The instant invention also provides for the use of
the sulfo bent[e]indocyanine fluorescent dyes in biological
assays.
4a
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The present invention is directed to fluorescent dyes and their valence
tautomers of the formula:
n( -)m
(~+m-~ ) M+
wherein Q represents a conjugated moiety that increases the fluorescent
quantum yield of the compound; Rl is a functionalized group of the formula
-(CH2)jY, wherein Y is selected from the group consisting of S03H, COON,
NH2, CHO, NCS, epoxy, phthalimido, and COOZ, wherein Z represents a
leaving group; R2 is a funcnonalized group of the formula -(CH2)kY', wherein
Y' is selected from the group consisting of SO;H, COOH, NH2, CHO, NCS,
epoxy, phthalimido, and COOZ, wherein Z represents a leaving group;1VI+ is a
counterion selected from the group consisting of ammonium, alkali metal
canons, and alkaline earth metal canons; n = 1 to 4; m = 1 to 4; j = 2 to 10;
and
k = 2 to 10.
The conjugated moiety Q is typically selected from the, group consisting
of
i ~ i .
X
/ /
(cHZ)i ; and
X
/ /
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wherein X is selected from the group consisting of hydrogen, F, C1, Br, I, and
substituted aryl, wherein said aryl substituent is selected from the group
consisting of S03H, COOH, NH2, CHO, NCS; epoxy and COOZ, and wherein
Z represents a leaving group; and r = 0 or 1.
The instant invention is also directed to the use of the above fluorescent
dyes to label biological molecules which are useful in biological assays.
Additional features and advantages of the invention are set forth in the
description which follows and in part will be apparent from the description.
These and other advantages of the invention will be realized and
attained by the fluorescent dyes of the invention and their uses as
particularly
pointed out in the written description, claims, and appended drawings.
It is to be understood that both the foregoing general description and the
following detailed description are exemplary and explanatory and are intended
to provide further explanation of the invention as claimed.
BRIEF DESCRIPTION OF THE FIGURES
Fig. 1 shows the absorption and emission spectra of dye (7).
Fig. 2 shows the absorption and emission spectra of dye (8).
Fig. 3 shows the absorption and emission spectra of dyes (9) or (11).
Fig. 4 shows the absorption and emission spectra of dye ( 10).
Fie. 5 shows the calibration curve for the determnnation of a-fetoprotein
(AFP).
DETAILED DESCRIPTION OF THE INVENTION
The present invention is directed to a fluorescent dye and its valence
tautomers of the formula:
)m
n ( ~03S
(~+m_~ ~ M+
wherein Q represents a conjugated moiety that increases the fluorescent
quantum yield of the compound; R1 is a functionalized group of the formula
-(CH2)jY, wherein Y is selected from the group consisting of S03H, COOH,
NHS, CHO, NCS, epoxy, phthalimido, and COOZ, wherein Z represents a
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leaving group; R2 is a functionalized group of the formula -(CH2)kY', wherein
Y' is selected from the group consisting of S03H, COOH, NH2, CHO, NCS,
epoxy, phthalimido, and COOZ, wherein Z represents a leaving group; M+ is a
counterion selected from the group consisting of ammonium, alkali metal
cations, and allcaline earth metal cations; n = 1 to 4; m = I to 4; j = 2 to
10; and
k = 2 to 10. The conjugate moiety Q preferably also enhances the tuning
behavior of the compound.
The conjugate moiety Q is preferably selected from the group consisting
of-.
x
i i i i
(cHZ)i ; and
X
/ i
wherein X is selected from the group consisting of hydrogen, F, C1, Br, I, and
substituted aryl, wherein said aryl substituent is selected from the brroup
2o consisting of S03H, COOH, NH2, CHO, NCS, epoxy and COOZ, and wherein
Z represents a leaving group; and i = 0 or 1.
More specifically, the fluorescent labeling dyes of the present invention
can be represented by the following general formulae:
03 )m
(n+m-I)M +
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_ \ /
n( ~3S) \CHg Cf'Ig CH3 C/-[3 (S~3 )m
/
/ / \ ~ (n+m-1)M+
Ri Rz
n( ~sS
~3 )m
~n+m_ 1 )~ r
a
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fl~ ~3S ~3 111
(n+m_ ~ ~M+
wherein R1 is a functionalized group of the formula -(CH?)jY, wherein Y is
selected from the group consisting of S03H, COOH, NH2, CHO, NCS, epoxy,
phthalimido, and COOZ, wherein Z represents a leaving group; R2 is a
functionalized group of the formula -(CH?)kY', wherein Y' is selected from
the group consisting of S03H, COOH, NH2, CHO, NCS, epoxy, phthalimido,
and COOZ, wherein Z represents a leaving group; 1~IT is a counterion selected
from the group consisting of ammonium, alkali metal cations and alkaline earth
metal cations; n = 1 to 4; m = 1 to 4; j = 2 to 10; k = 2 to 10; i = 0 or 1
and
wherein X is selected from the group consisting of hydrogen, F, Cl, Br, I, and
substituted aryl, wherein said aryl substituent is selected from the group
consisting of S03H, COOH, NH2, CHO, NCS, epoxy, and COOZ, wherein Z
represents a leaving group.
The leaving group Z is basically any organic leaving group which can be
easily displaced when the fluorescent dyes of the present invention are
attached
or conjugated to a specific molecule, e.g., an antibody. A typical leaving
group
is n-hydroxysuccimide. The fluorescent dyes of the present invention can be
symmetrical, i.e., R1 and R2 are identical. or asymmetrical, i.e., R1 and R~
are
not identical.
Valence tautomerism means the shifting of the conjugated bonds as
shown below for an asymmetric fluorescent dye:
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-03S -M+
( j H~5 t j ~?>4
COOH COOH
15
-O S ~ / CH3 CH~H3 CHg/ g0 -M+
3 3
~+
\ y \ N \
(~ H 2)4
(C H2)5
COOH
C OOH
The fluorescent dyes of the instant invention have the following
important features:
1. One or more sulfonic groups attached to the benzo ring to give
high hydrophilicity to the compound and suppress non-specific binding
to proteins and surfaces, including skin, thus allowing its use as a
biochemical label. In addition, the negative charge borne by the
sulfonic groups helps reduce the stacking between dye molecules by
means of electrostatic repulsion. Such stacking is known to reduce
the fluorescence quantum yield of the dye.
2. The presence of the ring increases the rigidity of the structure and
therefore increases the quantum yield of fluorescence.
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3. The groups bonded at the N in 1 or 1' position (generally
carboxyalkyl, sulfoallcyl or alkylamine chains) have reactive sites
for conjugation purposes or in order to increase solubility in
aqueous media.
4. The central ring increases the rigidity of the dye, thus enhancing
its fluorescence quantum yield. The central ring also hinders attack by
oxygen, therefore increasing the stability of the dye with respect
IO to oxidation. It can be used to introduce substituents with reactive
sites for conjugation purposes or in order to increase solubility in
aqueous media.
5. The polymethine chain length present in the dye provides the
main tool for tuning the fluorescent behavior of this class of dyes.
The compounds of the present invention are synthesized by starting with
the new compound 2,3,3-trimethyl Benz[e]indolenine-7-sulfonic acid (4). The
synthesis of compound (4) is outlined in Scheme I below:
Scheme I
HCI
H03S \ Na O H03S I / S
I HCI
NH2 \ I N +
2
(1) (2)
I \
CHgCOOH
H 03S
I
\ NH-NH2 CHg -C-CH -Cr~3
O CH3
(3)
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H03S / ICHg CH3
N~CH3 '
(4)
Starting with compound (4) a multiple number of intermediates such as
compounds (5) and (6) can be synthesized as shown in Scheme II below:
Scheme II
CH3
H03S ~ ~ CH3 Sulfolane
\ N~CH3 Br(CH~~COOH
25 \
CHg
-pas / ~ CH3
N "CH3
(CH?)5
COOH
(5)
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' ~ CH3
HOgS / ~ CH3 Sulfolane
\ N~CH3 O
\ ~ N -(CH~2CHZBr
O
03S / CHg CH3
t~
N - -CH
(CH?~3
O N O
30
The symmetrical or asymmetrical dyes are synthesized starting with
intermediates such as compounds (5) and (6) by condensation with triethyl
orthoformate or various dialdehyde dianilides as shown in Scheme III below:
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Scheme III
s \
CH3
HOgS / ~ CH3 pyridine
\ N~CH3 HC(OC2H5)3
l
([H2)5
C OOH
CH3 CH3CH3 CH3 ~ SO M~
is ~ / s
\ N / /
(CH2)5 (CHAS
COOH COOH
(7)
CH3
HC~3S / ( CH3 Pyridine
\ N~CHg Malonaldehyde
dianyl hydrochloride
(CH2)5
COOH '
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s -03s ~s_M+
. .
I
(i H2)5 ( j H2)s
COOH C~OH
($)
CH3
H03S ~ CH3 Pyridine
s-phenylamino-2,4-
N "CHg
2o trimethylene-2,4
pentadtenylidene
(CH2)5 phenylammonium chloride
COOH
-03S Q3-M +
35
(9)
(~%~2)s W~2)s
I I
COOH COGH
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DMF
sodium ethanethiolate
-~3S M f
20
(10)
CH3CH20H'CH3COQ-Nay
5-phenylamino-2,4-trimefhylene-
2,4-pentadienyiidene phenyl -
ammonium chloride
16
~CH2)5 ~..~ ~~s
COUH COOH
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-p3 s SQ3_M +
~H2)3 ~ ~H2)3
O N O O N O
(91 )
As noted above, the cyanine dyes were synthesized starring from a new
intermediate, the 2,3,3-trimethyl Benz[e]indolenine-7-sulfonic acid. From this
molecule different cyclammonium quaternary salts were obtained, and the
latter were condensed with compounds such as triethyl orthoformate or various
dialdehyde dianilides. The side chains attached to the 1- and I'- nitrogen
atoms of the benz[e]indolenine may be the same or different, leading to
symmetrical or asymmetrical dyes. All these compounds are usually in the salt
form and the counter-ion (M+) depends on reaction and purification conditions.
It should be noted that as with other cyanine dyes, the dyes of the present
invention may exist as monomers, H-aggregates and J-aggregates, depending
on the chemical equilibrium involved. Of course the pKa and pH will have a
substantial effect as to the form which the dye takes when dissolved in a
given
solvent. Additionally, an equilibrium also exists between monomers and
aggregates consisting of dimers, trimers, or higher polymers. Factors such as
ionic strength, dye concentration, temperature and solvent effect the
equilibrium.
The fluorescent dyes of the present invention combine all the optimum
properties that are desirable in these types of dyes. The desirable properties
of
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these dyes include absorption maxima matching light emitting diodes and laser
diodes (see Table I and Figures 1 to 4), emission maxima in the low
background red and near infrared spectral region; and chemical properties
fulfilling labeling requirements (such as presence of reactive sites,
hydrosolubility, lack of non-specific binding to proteins and surfaces,
including
skin, and stability).
Table I
DYE ABSORPTION EMISSION COMMERCIAL
(~ MAX) (~.1~~ LIGHT EMITTING
DIODES/LASER
DIODES: EMITTING
WAVELENGTH
7 580 599 585
8 672 694 670
9) or 741, 810 847 750, 810
(11
670, 780 805 670, 780
10 As mentioned in the background of the present invention, the fluorescent
dyes of the present invention are particularly useful in clinical chemistry,
immunochemistry and analytical chemistry. The compounds can be used in
fluorometric immunoassays, DNA probes, high pressure liquid chromatography
(HPLC), capillary electrophoresis, fluorescence polarization, tota.I internal
reflection fluorescence (T.LR.F.), flow cytometry, DNA sequencing, and
optical sensors. When the fluorescent compounds of the present invention are
used in immunochemical determinations, it is typically necessary to conjugate
the fluorescent label to one of the binding partners, e.g., an antibody or an
antigen or a biologically active protein molecule.
Depending upon the molecule being labeled, a wide variety of linking
groups may be employed for conjugating the protein to the other molecule. For
the most part, with small molecules the functional group of interest for
linking
will be carbonyl, either an aldehyde to provide for reductive amination or a
carboxyl, which in conjunction with carbodiimide or as an activated ester,
e.g.,
N-hydroxy succinimide, will form a covalent bond with the amino groups
present in the protein. Another useful functional group is a thio ether or
disulfide, where the protein may be modified with an activated olefin and a
mercapto group added or activated mercapto groups joined, e.g., Ellman's
reagent, isothiocyanate, diazonium, nitrene, or carbene. There is ample
18
r n. .~ l. l ~~ i.e ~~ . l
CA 02234450 2004-11-10
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literature for conjug$ting a wide variety of compounds to proteins. See, for
example, A.N. Grazer, The Proteins. Vol. IIA, 3rd Ed, N. Neurath and R.L.
Hill, eds., Academic Press, pp. 1-103 (1976); A.N: Grazer et al., "Chemical
Modification of Proteins," Laborator3r Techniques in Biochemistry
Molecular Biology, Vol. 4, PRT I, T.S. Work and E. Work, eds., North-
Holland Publishing Co. (1975); and K. Peters et al., Ann. Rev. Biochem.. 46,
423-51 (1977). Examples of commercially available
cross-linking reagents are disclosed in the
Pierce 1981-82 Handbook arid General Catalog, pp. 161-166, Pierce Chemical
1 o Co., Rockford, Ill.
As indicated above, antigens or antibodies are labeled with the
fluorescent dyes of the invention by ordinary chemical reaction. Thus, a
fluorescent dye can be reacted with an antigen or antibody to form a labeled
reaction product by covalent linkage; specifically, the reaction occurs
between
the functional group (i.e., mercapto, amino, hydroxy, or carboxy) of the dye
and an amino, imino, mercapto, carboxy, carboxylic acid amide, or hydroxy
groups) of an antigen or antibody. The reaction between the two can be
carved nut by any of the following procedures:
(1) Fluorescent dyes are directly reacted with the functional ~,~roups;
(2) Fluorescent dyes and the functional groups are reacted using an
activating agent; or
(3) Fluorescent dyes and the functional groups are reacted through at
least one compound having a bifunctional group.
Groups which are reactive with the functional groups of antigens or
antibodies and methods for reacting the same are described in detail, in,
e.g.,
Lectures on Experimental Biochemistry, vol. 1 subtitled "Chemistry of
Proteins", vol. 2, subtitled "Chemistry of Nucleic Acids", vol. 3, subtitled-
"Chemistry of Lipids" and vol. 4, subtitled "Chemistry of Sugars", all edited
by
the Biochemical Association, Japan, published by Tokyo Kagaku Dojin ( 1977);
Izumiya, Peptide Gosei (Peptide Synthesis); and Greenstein et al., Chemistry
of
the Amino Acids. vols. I-III (1961), John Wiley & Sons Inc., New York. One
skilled in the art can easily perform such reactions for forming the linking
from
knowledge in the art and these publications.
Examples of compounds containing groups which react with the
functional groups described above further include, e.g., activated esters,
activated halogens, aldehydes, activated vinyl esters, activated halogens,
aldehydes, activated vinyl compounds, acid anhydrides, acid halides,
thioisocyanates, isocyanates, carboxylic acids, amides, alkyl halides,
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nitrophenyl halides, etc. Accordingly, these functional groups can originally
be present in the fluorescent dyes or can be introduced as a result of the
reaction of a compound having a bifunctional group and the fluorescent dye.
Reaction conditions for labeling vary depending upon the kind of the
antigen or antibody, the kind of fluorescent dye etc., and conditions are
selected so as to not damage the biological activity of the antigen or
antibody to
be labeled. Accordingly, the reaction temperature is generally chosen from the
range of from 40 to 60C, preferably -20 to 40C; and the reaction time from the
range of from 10 minutes to 16 hours. The reaction pressure is preferably
1 o atmospheric pressure, but can suitably be chosen from the range of 1 to 20
atmospheres. It is advantageous that water or a pH buffer solution be used as
a
solvent for the labeling. Organic solvents such as DMF (dimethylformamide),
methylene chloride, etc., can also be employed. These reaction conditions are
common to reaction conditions which are generally applicable to modification
of proteins or enzymes and details are described in the publications referred
to
above.
The amount of fluorescent dyes used for labeling varies depending upon
the kind of substances to be labeled, but is generally in a molar ratio of
1/100
to 100 moles per 1 mole of the antigen or antibody, preferably 1/20 to 20
times
per 1 mole of the antigen or antibody, more preferably '/ to ? times per 1
mole
of the antigen or antibody.
Useful methods for confirming completion of labeling include methods
for measuring spectra such as UV, visible rays, IR, mass, and NMR spectra,
etc., and a method confirming labeling via disappearance of the terminal group
at which the labeling substance is to be introduced. Simple tests will be
enough to confirm completion of labeling. Where it is confirmed utilizing
absorption spectrum, following completion of the labeling reaction, an
absorption spectrum of a separated and purified product is measured; if the
resulting absorption spectrum is consistent with the intrinsic absorption
spectrum which a fluorescent dye possesses, it is confirmed that the labeling
reaction was effected. A further method for confirming the labeling being
effected is to analyze for the presence or absence of the specific terminal
groups, e.g., an amino or carboxy group(s).
The terminal carboxy groups) are analyzed to check completion of the
labeling reaction, details of which are given in e.g., S. Akabori, K. Ohno and
K. Narita, Bull. Chem. Soc. Japan 25 214 ( 1952) (generally referred to as a
hydrazine decomposition method in the art); H. Matuo, U. Fujimoto and T.
Tatuno, Biochem. Biophys. Res. Communication 22 69 ( 1966) ( a tritium
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marking method); etc. Further, details of these terminal determination
methods are also given as a review in S.B. Needleman, Protein Se4uence
Determia~ation. published by Springer Verlag (Berlin), 1975.
The protein molecules such as the antibodies or antigens can be used in
an immunochemical determination according to one of the prefeiTed aspects of
the invention. A typical fluorometric immunoassay of an antigen in a liquid
sample comprises incubating a mixture of
(a) the liquid sample;
(b) labelled antibodies to the antigen under assay;
(c) a reagent comprising antibodies to the antigen under assay; and
(d) a reagent capable of binding to component(c) by non-covalent
bonding, but which is not directly bindable to either component (a) or
component (b). The reagent (d) is bound to a solid phase support. At least one
of components (b), (c) and (d) comprises monoclonal antibodies. The solids
fraction is separated from the liquid fraction and the amount of label in one
of
the fractions is determined. From the amount of the label, the amount of
antigen present in the sample is determined. The antibody reagents may also be
used in the form of fragments.
Another aspect of the invention is a competitive assay for determining
2o the amount of an antigen in a sample suspected of containing the antigen
comprising: (a) incubating the sample with a solution of a fluorescent-labeled
antigen, anti-antigen antibody, and an antibody against the anti-antigen
antibody; (b) adding a nonfluorescent nonlight-scattering immunoprecipitant to
the incubation mixture to form an immunoprecipitate; (c) separating the
immunoprecipitate and dissolving the immunoprecipitate in a nonfluorescent
solvent that has a Low ionic strength and maintains the pH of the resulting
solution substantially constant; and (d) measuring the fluorescence intensity
of
the solution of step (c) and comparing the fluorescence intensity to a
standard
curve.
Typically the sample that is analyzed is a body fluid such as blood,
blood serum, blood plasma, urine, Lymph fluid, bile, spinal fluid, or the
like.
The particular body fluid analyzed may vary with the antigen being assayed. In
most instances blood serum will be used. About 0.1 to about 500 N.l of fluid
is
typically used per assay.
Substances that may be assayed by the use of the fluorescent labeling
reagents of the present invention include antigens (molecules that elicit an
immune response when introduced into the bloodstream of a vertebrate host)
and haptens that are not immunogenic per se but may be conjugated to a
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protein carrier to form a conjugate that is immunogenic and capable of raising
antibodies against the hapten. The term "antigen" is used herein to
generically
denote both antigenic and haptenic compositions. Such substances include
drugs, hormones, pesticides, toxins, vitamins, human bacterial and viral
proteins, and the like. Examples of antigens that may be assayed by the
invention include thyroxine (Tq.), triodothyronine (T3), digoxin, gentamycin,
amikacin, tobramycin, kanamycin, cortisol, luteinizing hormone, human
chorionic gonadotropin, theophylline, angiotensin, human growth hormone,
HIV infection, cc fetoprotein, and the like.
The reagents that are incubated with the sample suspected of containing
antigen to form immune complexes typically are (1) fluorescent-labeled
antigen, (2) anti-antigen antibody, (3) antibody against the anti-antigen
antibody, and (4) a nonfluorescent non-Iight-scattering imrnunoprecipitant
(e.g., polyethylene glycol). A stated above, the fluorescent-labeled antigen
may
be made by coupling the antigen with a reactive derivative of the fluorescent
dye using multifunctional coupling agents such as aldehydes, carbodiimides,
dimaleimides, imidates, and succinimides.
The dyes of the present invention also have utility in any current
application for detection of nucleic acids that requires a sensitive detection
reagent. In particular, the dyes are useful for the detection of cell-free
isolated
nucleic acids, nucleic acids in solution, and nucleic acid bands in gels.
Additionally, the present dyes greatly increase the sensitivity of detection
of
nucleic acids in a variety of cells and tissues, both living and dead, plant
and
animal, eukaryotic and prokaryotic. This family of dyes displays unusually
good photostability and appears to be relatively non-toxic to cells.
Furthermore, many of the dyes rapidly penetrate cell membranes of a variety of
cells. The dyes of the invention exhibit superior properties compared to the
known cyanine dyes.
The use of the fluorescent dyes of the present invention in the detection
of nucleic acids typically comprises combining a dye of the present invention
with a sample that contains a nucleic acid, incubating the sample for a time
sufficient to obtain a detectable fluorescent response, and observing the
fluorescent response.
Typically, the dye is present as a staining solution, which is prepared by
addition of the dye to an aqueous solution that is biologically compatible
with
the sample. The staining solution is made by dissolving the dye directly in an
aqueous solvent such as water; a buffer solution such as phosphate buffered
saline; an organic water-miscible solvent such as dimethylsulfoxide (DMSO),
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70571-61
dimethylformamide (DMF), or acetonitrile; or a lower alcohol such as
methanol or ethanol. Typically the dye is preliminarily dissolved in an
organic
solvent (preferably DMSO) at a concentration of greater than about 100 times
that used in the staining solution, then diluted one or more times with an
S aqueous solvent such as water or buffer. Preferably, the dye is dissolved in
about 100% DMSO and then diluted one or more times in water or buffer such
that the dye is present in an effective amount. An effective amount of dye is
an
amount sufficient to give a detectable fluorescent response when in the
presence of nucleic acids. Typically staining solutions for cellular samples
have a dye concent<ation greater than about 0.1 nM and less than about 100
ItM, more typically greater than about 1 nM. Staining solutions for
electrophoretic gels typically have a dye concentration of greater than about
1
E.tM and less than about 10 p.M, more typically about ~ to 5 E,tM. It is
generally
understood in the art that the specific concentration of the staining solution
is
determined by the physical nature of the sample, and the nature of the
analysis
being performed.
The dye is combined with a sample that contains a nucleic acid. The
nucleic acid in the sample may be either RNA or DNA, or a mixture thereof.
When the nucleic acid present is DNA, the DNA may optionally be single-,
double-, triple-, or quadruple-stranded DNA. The nucleic acid may be either
natural (biological in origin) or synthetic (prepared artificially). The
nucleic
acid may be present as nucleic acid fragments, oligonucleotides, or nucleic
acid
polymers. The nucleic acid may be present in a condensed phase, such as a
chromosome. The presence of the nucleic acid in the sample may be due to a
successful or unsuccessful experimental methodology, undesirable
contamination, or a disease state. Nucleic acid may be present in all, or only
part, of a sample, and the presence of nucleic acids may be used to
distinguish
between individual samples, or to differentiate a portion or region within a
single sample.
The fluorescent dyes of the present invention can also be .used for DNA
sequencing methods. The method is based on Sanger's chain terminating
sequencing method as described in Sanger et al., Proc. ~latl. Acad. Sci.. 74
5464 (1977). Typically, a fluorophore-labeled probe
specific to the sequence is hybridized
with the target DNA and the sequence ladders are identified by laser induced
fluorescence or other appropriate means for detecting fluorescent labeled DNA.
The fluorescent dyes of the invention are also useful in assay
methodologies that employ DNA probes for the determination of the presence
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WO 97/13810 PCT/EP96/04377
and/or quantity of analyzes. More particularly, an assay method for an analyte
in a sample utilizing a DNA probe labeled with a dye of the invention
comprises contacting the DNA probe thus labeled under suitable conditions for
binding with the analyte, where the binding is representative of the presence
or
amount of the analyte in the sample, and determining the extent of the binding
by measuring the fluorescence of the fluorescently labeled DNA probe. As an
embodiment of this method, the analyte to be determined can be separated from
the sample in which it is present prior to being bound by the DNA probe. It is
also possible to employ first and second DNA probes in a single assay, where
the first of such probes is bound with a fluorescent dye according to the
invention, and the second probe is not labeled and is bound to a solid phase.
In this way the analyte can be separated from the sample by contacting the
sample with the second DNA probe under conditions suitable for binding the
analyte therewith, and the remaining sample is removed prior to contacting the
analyte with the first (labeled) DNA probe.
It is also within the scope of the assay methods utilizing the fluorescent
dyes of the present invention that the step of separating the analyte from the
sample in the manner just described can also be practiced with labeled binding
reagents other than DNA probes. For example, instead of utilizing first
(labeled) and second (unlabeled and bound to a solid phase) DNA probes,
plural immunologically binding reagents specific for a given analyze can be
used.
Additionally, the fluorescent dyes of the present invention can be used
in flow cytometry. The method is characterized by the use of a flow cytometer
that comprises a light source, a flow cell that permits the blood cells in a
sample to flow one by one through a constricted channel, a photometric unit
that detects light issuing from each blood cell, and an analyzer for analyzing
the detected signal. In this method, the corpuscles in the sample which are
stained are illuminated under Light and the fluorescence emitted from the
irradiated corpuscles is detected, optionally together with scattered light,
with
leukocyte classificarion being made in accordance with the intensity of the
detected signal. The examples below are given to illustrate the practice of
this
invention. They are not intended to limit or define the entire scope of this
invention.
Examples
For the synthesis of the common intermediate, 2,3,3-trimethyl
bent[eJindolenine-7-sulfonic acid, we started with Dah1's acid, 6-amino-1-
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naphthalensulfonic acid, and through diazotization and reduction we converted
it to 6-hydrazino-1-naphthalensulfonic acid; then, by classical Fischer indole
synthesis with 3-methyl-2-butanone we obtained 2,3,3-trimethyl
Benz[e]indolenine-7-sulfonic acid. It is remarkable that the cyclization takes
S place yielding just the desired isomer.
Example 1
Diazotization of Dahl's Acid.
A suspension of 13.34 g (64.2 mmol) of Dahl's acid in 45 ml of
concentrated hydrochloric acid was cooled to -5C by an ice-salt bath in a 500
ml three-necked flask equipped with mechanical stirrer, thermometer and reflex
condenser. To the slurry was added dropwise, at OC or below, an ice-cold
sodium nitrite solution (4.43 g, or 64.2 mmol, in 30 ml of water) and the
mixture was stirred for 40 minutes at the same temperature and then used
I5 without delay in the following step.
Example 2
Reduction of Diazotized Dahl's acid to 6-hvdrazino-1-naptalenesulfonic
acid 3.
A solution of stannous chloride dihydrate (43.6 g or 193.2 mmol) in 46
ml of cold concentrated hydrochloric acid was added dropwise at about OC,
with stirring, to the slurry obtained by adding 50 g of chopped ice to the
diazotized Dahl's acid. The addition requires almost 1 hour. The mixture was
stirred for another I5 minutes. The slurry was refrigerated overnight,
filtered,
and washed with brine. The final product was recrystallized from hot water to
yield 9.9 g (~64%). The 1H-NMR spectrum of the product was consistent with
the structure of 6-hydrazino-1-naphtalenesulfonic acid.
Example 3
Fischer synthesis of 2.3.3-trimethvl benzfelindolenine-7-sulfonic acid
To a 250 ml round-bottomed flask, equipped with reflex condenser and
magnetic stirrer were added acetic acid (25 ml), 3-methyl-2-butanone ( 13 .4
ml
or 124 mmol) and 6-hydrazino-1-naphthalenesulfonic acid (9.9 g or 41.6
mmol). The mixture was dissolved and heated to reflex for 3 hours. Then the
solution was evaporated and the residue washed with three n-hexane portions.
The solid residue was dried, redissolved in methanol, and precipitated in
ether
to yield 2,3,3-trimethyl benz[e]indolenine-7-sulfonic acid (80%). As
CA 02234450 1998-04-08
WO 97/13810 PCT/EP96/04377
confirmed by the 1H-NMR spectrum, the cyclization occurred only at the 5-
position of the naphthalene ring.
For the quaternarization we used methods described previously in the
literature for similar compounds. All structures were confirmed by 1H-NMR.
Example 4
Synthesis of 1-(5'-carboxypentyl)-2 3.3-trimethvl benzfelindoleninium-
7-sulfonate (5).
In a 100 ml round-bottomed flask, 2.1 g (7.26 mmol) of 2,3,3-trimethyl
benz[e]indolenine-7-sulfonate were dissolved in 27 ml of hot sulfolane under
argon. To this solution was added 6-bromohexanoic acid ( 1.76 g or 9.0 mmol).
The resulting mixture was then heated at 130C for 12 hours. After cooling, the
brown solution was mixed with toluene (~ 50 ml) and a dark solid separated;
the solid was filtered and washed with two more portions of toluene. Excess
toluene was distilled off as azeotropic mixture with ethanol. The product,
dissolved in few milliliters of methanol, was precipitated by dropwise
addition
to a large excess of ether (yield ~35%).
Example 5
Synthesis of 1-f3'-(N-nhthalimidoprogvl)1-2.3.3-trimethvl
benzfelindoleninium-7-sulfonate (6).
In a round-bottomed flask were dissolved 2,3,3-trimethyl
bent[e]indolenine-7-sulfonate (3 g or 10.4 mmol) and N-(3-bromopropyl)-
phthalimide (2.78 g or 10.4 mmol) in sulfolane (~20 ml) and suspended 0.58 g
(10.4 mmol) of KOH. The mixture was heated to 120C for 6 hours. A solid
was then precipitated from the solution by adding 50 ml of toluene. The solid
was collected on a sintered glass filter and washed with toluene. The gummy
residue was dissolved in ethanol and reprecipitated into ether to obtain a
solid
that was then thoroughly triturated with ethanol.
To obtain the free amine group it is possible to follow classical cleavage
pathways such as treatment in ethanol with hydrazine at 25C for 12 hours or
hydrolysis in concentrated hydrochloric acid. Finally, the cyanine dyes can be
obtained by condensing the benz[e]indoleninium-7-sulfonate intermediates
with triethyl orthoformate or various dialdehyde dianilides.
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Example 6
Synthesis of 2-~3'-(I"-(E-carboxypentyl)-3" 3"-dimeth
sulfobenzfe~jindolin-2"-ylidenel-I=propen'-1'-yll-1-(s-carboxwentyl)-3 3-
dimethyl benz(el indoleninium-7-sulfonate (7~
In a round-bottomed flask equipped with a reflux condenser, 306 mg
(0.76 mmol) of I-(5'-carboxypentyl)-2,3,3-trimethyl bent[e~indoleninium-7-
sulfonate were dissolved in 1 ml of pyridine. The solution was then heated to
reflux. An excess of triethyl orthoformate (225 mg or I.52 mmol) was added
and the solution was refluxed for 2 hours. The mixture was cooled and the
product was separated by addition of ether. The solid was dissolved in
methanol and reprecipitated with ether (yield 56%).
Example 7
Synthesis of 2-~5'-f 1"-($-carboxvnentyl)-3".3"-dimethvl-7"-
sulfobenz(eiindolin-2"-vlidenel-1' 3"-pentadien-I'-vll-I-(~-carboxynentyl)-3 3-
dimethyl benz[el indoleninium-7-sulfonate (8).
Malonaldehyde dianil hydrochloride (160 mg or O.G20 mmoI) was
dissolved in a hot mixture of 5 ml acetic anhydride and 1.2 ml pyridine, in a
50
ml round-bottomed flask equipped with a reflux condenser. 1-(5'-
carboxypeniyl)-2,3,3-trimethyl Benz[eJindoleninium-7-sulfonate in an amount
of 0.5 g ( 1.24 mmol) was added to the mixture. After 1 ~ minutes, the
solution
was cooled and diluted with ether; a solid separated and was redissolved in a
minimum volume of methanol and precipitated with a 2:1 2-propanol/ether
mixture (yield 46%).
Example 8
Synthesis of 2-~5'-chloro-T-(I"-_ (s-carboxypentvl)-3" 3"-dimethyl-7"-
sulfobenzfeiindolin-2"-vlidenel-3' S'-(propane-I"'3"'-divll-1' 3' S'-
heptatrien-I'-
~~-1-(s-carboxvpentyl)-3.3-dimethvl benzLl indoleninium-7-sulfonate (9)
A solution of (5-phenylamino-2,4-trimethylene-2,4-pentadienylydene)
phenylammonium chloride (0.29 g or 0.80 mmol), 1-(5'-carboxypentyl)-2,3,3-
trimethyl bent[elindoleninium-7-sulfonate (0.65 g or 1.61 mmol) and sodium
acetate (0.16 g or 1.93 mmol) in ethanol (30 mI) was heated to reflux for 1
hour. The solvent was evaporated and the residue dissolved in the minimum
volume of methanol and precipitated with ether (yield 83%).
From this molecule it is possible to synthesize cyanine dyes with
different substituents at the chloro position. These substituents can
introduce
new conjugation sites and/or shift the excitation and emission wavelengths to
a
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WO 97/13810 PCT/EP96/04377
more useful area of the spectrum. For example, the following dye can be
excited by diode lasers at 670 and 780 nm.
Example 9
Synthesis of 2-1T-f 1"-(s-carboxypeniyIl-3".3"-dimethyl-7"-
sulfobenz(elindolin-2"-ylidenel-3'.5'-propane-1"'.3"'-diyl)-I'.3' S'-
heptatrien-I'-
yll-1-(E-carboxyuentyl)-3.3-dimethyl benzj~ indoleninium-7-sulfonate (10~
To a round-bottomed flask under argon were added 15 ml of anhydrous
N,N-dimethylformamide, 300 mg (0.31 mmol) of cyanine dye (9) from
Example 9 and 0.5 g (6.1 mmol) of sodium ethanethiolate. The mixture was
refluxed for 1 hour and, after cooling, a solid was precipitated with ether
(yield
70%}.
Example 10
Synthesis oft-f5'-chloro-T-(1"-(v-(N-nhthalimidopropvl))-3".3"-
dimethyl-7"-sulfobenz(elindolin-2"-vlidene]-3'.5'-(propane-1 "'3"'-divl)-
1'.3'.5'-
heptatrien-1'-yl ~-1--(y-(N-phthalimidopropyl))-3.3-dimethvlbenz~el
indoleninium-7-sulfonate ( I I ).
1-[3'-(N-phthalimidopropyl)]-2,3,3-trimethyl Benz[e]indoleninium-7-
sulfonate (330 mg or 773 mmol), (5-phenylamino-2,4-trimethylene-2,4-
pentadienylydene) phenylammonium chloride (139 mg or 0.39 mmol) and
sodium acetate (76 mg or 0.93 mmol) were dissolved in ethanol ( 10 ml). After
20 hours of refluxing the solution was evaporated and the residue redissolved
in methanol and precipitated with ether (yield 67%).
The purification of the dyes was performed on a Millipore Waters Delta
Prep 4000 HPLC unit equipped with a Vydac C 18 R.P-column, using a gradient
elution (for example: H20/CH3CN - from 80:20 to 5:95). Because of the
numerous ionic sites present in these dyes. to obtain a well-defined
composition it was necessary to perform an ion-exchange step by passing an
aqueous solution of the dye through a strongly acidic cation-exchanger, such
as
Dowex SOW in the hydrogen form.
Example 11
General Procedure for the Preparation of Succinimidvl Esters of
Carboxyalkyl Sulfo benz('elindocvanine Dves.
The following procedure describes the preparation of succinimidyl
esters of carboxyalkyl sulfo Benz[e] indocyanine dyes. The resulting
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WO 97/13810 PCT/EP96/04377
succinimidyl esters were used as intermediates for labeling biomolecules such
as antibodies and antigens.
In a solvent mixture containing anhydrous di-N,N-dimethylformamide
(1 mI) and anhydrous pyridine (50 u,1) was dissolved 50 mg of dye containing
carboxyalkyl groups (e.g., dyes 7, 8, or 9). Disuccinimidyl carbonate ( 1.5
equivalents per carboxy group) was added and the mixture was heated at 55 to
60C for 90 minutes under an atmosphere of argon. The product was isolated
by adding ethyl acetate (anhydrous), passing the mixture through a sintered
glass filter, and washing the collected product with ethyl acetate. The raw
l0 product was dissolved in anhydrous dimethylformamide and was reprecipitated
with anhydrous ethyl acetate or ether. The yield was practically quantitative.
Example 12
General Procedure for the Labeling of Protein.
A stock solution of the succinimidyl esters of a carboxyalkyl sulfo
benzje]indocyanine dye was prepared by dissolving 1 mg of active ester in 100
u1 of anhydrous DMF. These solutions were stable for a few days when kept
in a dessicator at 4C. Aqueous solutions of the active esters were stable for
a
few hours at pH _< 7.
One can use aqueous solutions of the active ester--containing dyes in the
case where it is not possible to use DMF as a solvent when labeling the
protein.
One determines the concentration of reactive dye in the stock solution by
measuring the absorbance of an aliquot of stock solution properly diluted in
phosphate buffer saline, using the color extinction coefficient. In general,
the
labeling of protein was carried out in carbonate-bicarbonate buffer (0.1M, pH
9.4) for 15 minutes at ambient temperature.
Example 13
Labeling of Anti-ec-Fetoprotein Monoclonal Antibody.
To one milligram of anti-cc-fetoprotein monoclonal antibody (MAb anti-
AFP) dissolved in 1 ml of carbonate-bicarbonate buffer (0.1 M, pH 9.4) was
added with vortex mixing, 20 ml of active ester dye (withdrawn from the stock
solution containing 1 mg/100 1,t,1 of active ester dye). The mixture was
incubated for 15 minutes at ambient temperature. Dye that did not conjugate
with protein was separated from the labeled antibody using bel permeation
chromatography on a Sephadex G-25 column (0.7 x 70 cm) and eluted with
phosphate buffer at pH 7. Two colored bands were formed, and the first to
elute contained the labeled antibody.
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Example 14
Application: Fluorescent Immunoassay ~FIA~
As an example of FIA in heterogeneous phase of the sandwich-type,
between the analyte to be determined and two monoclonal antibodies specific
towards two epitopes different from the analyte, one can make the
determination of a-fetoprotein in serum. The analytical scheme of the assay is
as follows: the first antibody MAb, anti-AFP is used as capture antibody and
is
conjugated with biotin. The second antibody Mab2 anti-AFP is conjugated
with the indicator (for example, Dye 8 with maximum deabsorption at 670 nm.)
The immunochemical reaction between the analyte and the monoclonal
antibodies takes place in the
homogeneous phase, in the presence of a solid phase, for example, plates with
wells, sensitized with streptavidin, which constitutes half of the
unbound/bound
separation.
Schematic of Assay
( - StA.V + STET-Mab land-AFP + AFP + Mab~anti-AFP-dye
?0 ,(.
f - StAV + STET-Mab 1 anti-AFP-AFP-Mab?anti-AFP-dye
~~ incubate 1 h R.T.
f-StAV STET-iVlablanti-AFP-AFP-'Vlab?anti-AFP-dye
In the above schematic:
f -StAV is the solid phase sensitized with streptavidin;
STET-Mablanti-AFP is the capture antibody conjugated with biotin:
AFP is cc-fetoprotein; and
Mab2anti-AFP-dye is the antibody conjugated with the indicator.
After washing the plate with an appropriate washing buffer, a number of
molecules of Mab2anti-AFP-dye, equal to those of the analyte remain anchored
to the solid phase. By means of the construction of a calibration curve (as
shown in Fig. 5), one can determine the concentrations of analyte AFP in a
withdrawn serum sample. The result can be calculated from the fluorescence
CA 02234450 1998-04-08
WO 97/13810 PCT/EP96/04377
light intensity from a constant volume for every sample or reference sample,
of
a solution of guanidine, by means of which the immunocomplex remains in the
solid phase. A typical procedure for this type of determination is as follows:
~ place in each well 50 N.L of sample or reference sample;
100 p,L of solution of the STET-Mab lanti-AFP conjugate;
100 uL of the solution of Mab2anti-AFP-dye conjugate;
~ Incubate for 1 hour at room temperature.
~ a series of 5 washes with appropriate buffer is carried out
automatically using equipment known in the immunochemical art;
~ place in each well 300 uL of a solution (6 M) of guanidine dichloride;
~ transfer 300 N.L of solution to a cuvette;
~ subject sample to excitation at 670 nm and the resulting spectral
emission is integrated after correction and normalization.
The above description and accompanying drawings are provided for the
purpose of describing embodiments of the invention and are not intended to
Limit the scope of the invention in any way. It will be apparent to those
skilled
in the art that various modifications and variations can be made in the
fluorescent dyes and their uses and in the methods for their synthesis without
departing from the spirit or scope of the invention. Thus. it is intended that
the
present invention cover the modifications and variations of this invention
provided they come within the scope of the appended claims and their
equivalents.
31
