Note: Descriptions are shown in the official language in which they were submitted.
t~3
Baclcground O The Invention
The present invention relates to a method and
reagents for determining ligands in biological fluids such
as serum, plasmal spinal fluid, amnionic fluid and urine.
In par~icular, the present invention relates to a fluorescence
polarization i~nunoassay procedure and to tracers employed as
reagents in such procedures. The fluorescence polarization
immunoassay procedure of the present invention combines the
specificity of an immunoassay with the speed and convenience
of fluorescence polarization techniques to provide a means
for determining the amount of a specific ligand present in a
sample.
Competitive bindin~ immunoassays for measuring
ligands are based on the competition bekween a ligand in a
test sample and a labeled reagent, re~erred to as a tracer,
~or a limited number of receptor binding sites on antibodie!s
specific to the ligand ancl tracer~ The concentration o~
ligand in ~he sample determines the amount o~ tracer that
will speci~ically b~nd to an anti~ody. The amount o~ tracer-
antibody conjugate produced may be quantitively measured and
is inversely proportional to the quantity of ligand in the
test sample.
In general, fluorescence polarization techniques
are based on the principle that a fluorescent labeled compound
when excited by linearly polarized light will emit fluorescence
having a degree o~ polarization inversely related to its rate
of rotation. Therefore, when a molecule such a~ a txacer-
antibody conjugate ha~ing a fluorescent label is excited with
linearly polarized light, the emitted light remains highly
polarized because the fluorophore is constrained from rotating
between the time light is absorbed and emitted~ When a "free"
tracer compound (i.e., unbound to an antibody) is excited by
linearly polarized light, its rotation is much faster than
the corresponding tracer-anti~ody conjugate and the molecules
-- 3 --
~t~t~i~3
are more randomly oriented, therefore, the emitted light
is depolarized~ Thus, fluorescence polarization pxovides a
quantitive means for measuring the amount of tracer-antibody
conjugate produced in a competitive binding immunoassay.
Various fluorescent la~eled compounds are known
in the art. U. S. Patent ~o. 3, 9~8,943 describes the
preparation of a fluorescently labeled insulin derivative
using fluorescein isothiocyanate (FITC) as the fluorescent
label and a fluorescently labeled morphine derivative uslng
4-aminofluorescein hydrochloride as the fluorescent label.
Carboxyfluorescein has also been used for analytical determi-
nations. Ro F. Chen, AnaZ. Lett~ ~ 10, 787 (lq77~ describes
the use of carboxyfluorescein to indicate the activity of
phospholipase. However, carboxyfluorescein is not conjugated
according to the present invention~ It is encapsuLated in
lecithin liposomes, and it will fluoresce only when released
by the hydrolysis of lecithin
Summar Of The Invention
Y
The present invention encompasses ~ method for
determining ligands in a sample comprising intermixing with
said sample a biologically acceptable salt of a tracer of the
formula:
OH
V
~\
-- 4 --
1~'7~
wherein R is a ligand-analog having a single reactive
primary or secondary amino group which is
attached to the carbonyl carbon of the
carboxyfluorescein wherein said ligand-
analog has at least one common epitope with
said ligand so as to be specifically
reconizable ~y a common antibody;
and an antibody capable of specifically recognizing said
ligand and said tracer; and then determining the amount of
tracer antibody conjugate by fluorescence polarization
techniques as a measure of the concentration of said ligand
in the sample.
The inyention further relates to certain novel
tracers and biologically acceptable salts thereof, which are
useful in reagents in the above desoribed method. The methods
and tracers o the present invention are particularly useul
in quantitatively monitoring therapeutic drug concentrations
in serum and plasmaO
_ctailed Description O~ The Invention
The term l'ligand" as used herein refers to a
molecule,in particular a low molecular weight hapten having
a single react;ve amino group, to which a receptor, normally
an antibody, can be o~tained or formed. Such haptens are
protein-free bodies, generally of low molecular weight that
25 do not induce antihody formation when injected into an
animal, but are reactive to antibodies. Antibodies to hapten
are generally raised by first conjugating the haptens to a
protein and injecting the conjugate product into an animal.
The resulting antibodies are isolated by conventional
30 antibody isolation techniques.
Ligands determinable by the method of the present
invention vary over a wide molecular weight range. Although
high molecular weight ligands may ~e determined, for best
results, it is generally preferable to employ the methods
35 of the present invention to determine ligands of low molecular
weight, generally in a range of 50 to 4000. It is more
preferred to determine ligands having a molecular weight
in a range of 100 to 2000.
- 5 -
t~
The novel tracers of the Present invention include
compounds of fQrmula LI~ whereIn the ligand-analog repre.sented
~y R include r~dicals having a molecular weight within a
range o~ 50 to 4~Q0. The preferred novel tracers include
compounds of formula (I~ wherein the ligand-analogs represented
~y R include radicals havinq a molecular weight within a
range of 100 to 2000.
Representative of ligands having a single reactive
amino group determinable by the methods of the present
invention include steriods such as esterone, estradoil, cortisol,
testoestrone, progesterone, chenodeoxycholic acid, digo~in!
cholic acid, digitoxin, deoxycholic acid, lithocholic acids
and the ester and amide derivatives thereof; vitamins such as
B-12, folic acid; thyroxine, triiodothyronine~ histamine,
serotonin, prostaglandins such as PGE, PGF, PGA; antiasthmatic
drugs such as theophylline, antineoplastic drugs such as
doxorubicin and methotrexate antiarrhy~hmic drugs such as
disopyramide, lidocaine, procainamide, propranolol, quinidine,
N-acetyl-procainamide; anticonvulsant drugs such as phenobarbital,
phenytoin, primidone, valproic acid, carbamazepine and
ethosuximide; antibiotics such as penic.illins, cephalosporins
and vancomycin; antiarthritic drugs such as salicylate; anti-
depressant drugs including tricyclics such as nortriptylinet
amitriptyline, imipramine and desipramlne; and the like as
well as the metabolites thereof. Additional ligands that
may be determined by the methods of the present invention
include drugs of a~use such as morphine, heroin, hydromorphone,
oxymorphone, metapon, codeine, hydrocodone, dihydrocodeine,
dihydrohydroxy, codeinone, pholcodine, dextromethorphan,
phenazocine and deonin and their metabolites.
The tracers of the present invention generally
exist in an equili~rium ~etween their acid and ionized
states, and in the ionized state are effective in the method
of the present invention. Th.erefore, the present invention
comprises the tracers in either the acid or ionized state
and for convenience, the tracers of the present invention
are structurally represented herein in their acid form. When
the tracers of the present invention are present in their
ionized state, the tracers exist in the form of biologically
- 6 - ~7~
acceptable salts~ ~s used herein, the term "biologically
acceptable salts" refers to salts such as sodium, potassium,
ammonium and the like which will enable the tracers of the
present invention to exist in thelr ionized state when
employed in the method of the present invention. Generally,
the tracers of the present invention exist in solution as
salts, the specific salt results from the buffer ~mployed,
i.e., in the presence of a sodium phosphate buffer, the
tracers of the present invention will generally exist in
their ionized state as a sodium salt.
The tracers of the present invention comprise a
ligand-analog represented by R linked to a carboxyfluorescein
moiety of the formula:
OH
(II)
/ \OH
_ 7 ~ 8~fi~
The term ligand-analog as used herein refers to
a mono or polyvalent radical a su~stantial proportion of
which has the same spatial and polar organization as the
ligand to define one or more determinant or epitopic sites
capable fo competing with the ligand for ~he binding sites
of a receptor. A characteristic of such ligand-analog is
that it possesses su~icient structural similarity to the
ligand of interest so as to be recognized by the antibody
for the ligand. For the most part, the ligand analog will
10 have the same or substantially the same structure and charge
distribution (spatial and polar organization) as the ligand
of interest for a significant portion of the molecular
surface. Since frequently, the linking site for a hapten
will be same in preparing the antigen for production of anti-
15 bodies as used for linking to the Ligand, the same portionof the ligand analog which provides the template for the
antibody will be exposed by the ligand analog in the tracer.
In general, the class of ligand analogs represented
by R are derived ~rom the corresponding ligancl hy removal oE
20 a reactive hydrogen atom, i~e., a hydrogen atom bonded to a
reacti~re amine (primary or secondary) or by the formation
or an amino derivative of the ligand wherein an imino group
-N-
replaces one or more atoms originally present in the ligand,
25 at the site of binding to the carbo~yfluorescein moiety.
Illustrative of ligands which upon the removal of a reactive
hydrogen may form ligand-analogs represented by R include
for example, procainamide ! thyroxine and quinidine.
Illustrative of ligands whose amino derivatives are useful
30 as ligand-analog include theophylline, valproic acid,
phenobarbital, phenytoin, primidone, disopyramide ! digoxin,
chloramphenicol, sal~cylate, acetaminophen, carbamazepine,
desipramine and nortriptyline. In addition, a ligand may be
structurally modified by the addition or deletion of one or
35 more functional groups to form a ligand-analog, while retaining
- 8 ~LiL7E~2~i~
the necessary epitope sites for hinding to an anti~ody.
However, such modified ligand-analogs are bonded to the
carboæyfluorescein moiety through an imino group.
The tracers of the present invention are generally
prepared in accordance with known techniques. For example,
a compound of the formula:
(III)
R - X
wherein R is above-defined and X is a reactive hydrogen; is
treated with a compound of the formula:
' ~ ~
~ OH
~: 1l ~
(IV
O
. ~
:
,
,:~
: ~ ; ` :
: :;
: ,
- 9 - ~17~
wherein R is hydroxy or an active ester, and wherein the
carboxy group is preferably bonded to the 4 or 5 position
of the benzoic acid ring; in the presence of an inert
solvent to yield a compound of formula (I).
As used herein, the term "active ester" refers to
a moiety which is readily "removed'l rom the car~oxy carbon
in the presence of a coupling agent. Such "active esters"
of carboxyfluorescein are readily ascertained by one of
ordinary skill in the art and are prepared from the reaction
of carboxyfluorescein with a compound such as N-hydroxy-
succinimide, l-hydroxy~enzotriazole-hydrate or p-nitrophenol
in the presence of a coupling agent, such as dicyclohexyl-
car~odiimide and a solvent. The active esters of carboxy-
fluorescein thus produced are subse~uently reacted with a
compound of formula tIII) to yield a tracer of ~rmula (I~.
If the compound o formula (III) i5 water
soluble, the reaction mechanism proceeds by directly reacting
carboxyfluorescein with a compound of formula (IIIl in
aqueous solution in the presence of a water solu~le
carbodiimide, such as 1-ethyl-3~(3Ldimethylaminopropyl)-
carbodiimide hydrochloride r as a couplin~ agent.
The temperature at which the process for preparing
the tracers of this invention proceeds is not critical. The
temperature should be one wh~ch is sufficient so as to
initiate and maintain the reaction. Generally, or con-
venience and economy, ro~m temperature is sufficient. In
preparing the tracers of the present invention, the ratio
of reactants is not narrowly critical. For each mole of a
compound of formula (II~, one should employ one mole of a
compound of formula tIII) to obtain a reasona~le yield. It
is preferred to employ an excess of comp~und of formula (III)
for ease of reaction and recovery of the reaction products.
For ease in handling and recovery of product !
the process for preparing the tracers of the present
invention is conducted in the presence of an inert sol~ent,
Suitable inert solvents include those solvents which do not
react with the starting materials and are sufficient to
dissolve the starting materials, and include for example
10- ~:L'7i~
water (if the compound o formula (III) is water solublel,
dimethylformamide, dimethylsulfoxide and the like, If the
compound of formula (III~ is a reactive amine salt, a
suitable base is added to the reaction mixture to form the
free base of the reactive amine. Suitahle bases include
for example, triethylamine. The reaction products of formula
~I) are generally purified using either thin-layer or column
chromatography prior to application in the methods of the
present invention.
In accordance with the method of the present
invention, a sample containing the ligand to be determined
is intermixed with a biologically acceptable salt of a
tracer of formula (I) and an antibody specific for the ligand
and tracer. The ligand present in the sample and the tracer
compete for limiting antibody sites resulting in the
formation of ligand-antibody and tracer-antlbody complexes.
By maintaining constant the concentration of tracer and
antibody, tha ratio of ligand-antibody complex to -tracer
antibody complex that is formed is directly proportional
to the amount of ligand present in the sample. Therefore~
upon exciting the mixture with polarized light and measuring
the polarization of ~he fluorescence emitted by a tracer and
a tracer-antibody complex, one is able to quantitatively
determine the amount of ligand in the sample.
In theory, the fluorescence polarization of a
txacer not complexed to an antibody is low, approaching
zero. Upon complexing with a specific anti~ody, the tracer-
antibody complex thus formed assumes the rotation of the
antibody molecule which is slower than that of the relatively
small tracer molecule, thereby increasing the polarization
observed. Therefore, when a ligand competes with the tracer
for antibody sites, the observed polarization o~ fluorescence
of the tracer-antibody complex becomes a value somewhere
between that of the tracer and tracer-antibody complex. If
a sample contains a high concentration of the ligand, the
observed polarization value is closer to that of the free
ligand, i.e., low. If the test sample contains a low
concentration of the ligand, the polarization value is
L7~ 9
closer to that of the bound ligand, i.e., high. By
sequentially exciting the reaction mixture of an Lmmunoassay
with vertically and then horizontally polarized light and
analyzing only the vertical component of the emitted light,
the polarization of fluorescence in the reaction mix may
be accurately determined. The precise relationship between
polarization and concentration ofthe ligand to be determined
is established by measuring the polarizat:ion values of
calibrators with known concentrations. rrhe concentration
of the ligand can be extrapolated from a standard curve
prepared in this manner.
The pH at which the method of the present
invention is practiced must be sufficient to allow the tracers
of formula (I) to exist in their ionized state. The pH may
range from about 3 to 12, more usually in the range of from
about 5 to 10, most preferably from a~out 6 to 9. Various
buffers may be used to achieve and maintain the pH during
the assay procedure. R~presentative buffers include ~orate,
phosphate, carbonate, tris, barbital and the like. The
particular bu~er employed is not critical to the present
invention, ~ut in an individ~al assay, a specific ~uffer
may be preferred in view of the antibody employed and ligand
to be determined. The cation portion of the huffer will
generally determine the cation portion of the tracer salt
in solution.
The methods of the present invention are practiced
- at moderate temperatures and preferably at a constant
temperature. ~he temperature will normally range from about
0 to 50 C, more usually from a~out 15 to 40 C~
The concentration of liaand which may be assayed
will generally vary from about 10 2 to 10 13M, more usually
from a~out 10 4 to 10 QM. Hi~her concentrations of ligand
may be assayed upon dilution of the original sample.
In addition to the concentration range of ligand
of interest, considerations such as whether the assay is
qualitative, semiquantitative or quantitative, the equipment
employed, and the characteristics of the tracer and antibody
will normally determine the concentration o~ the tracer
and antibody to be employed. While the concentration of
- 12 - ~ ~ 7~
ligand in the sample will determine the range of con-
centration of the other reagents, i.e~, tracer and anti~ody,
normally to optimize the sensitivity of the assay, individual
reagent concentrations will be determinecl empirically. Con-
centrations o the tracer and antibody are readilyascertained by one of ordinary skill in the art.
As previously mentioned the preferred tracers of
the present inention are prepared from 5--carboxyfluorescein
or 4-car~oxyfluorescein or mixtures thereof and are represented
~y the formulas:
-- 1 3
OH
~_1C ~ ~ (V)
or
OH
z ~l ~
~ (VI)
~,
0/~ \0}~ ~ ,
- 14 -
The following illustrative, nonlimiting examples
will serve to further demonstrate to those skilled in the
art the manner in which specific tracers within the scope
of ~he is invention may be prepared. The symbol ~C~]
appearing in the structural formulas illustrating the com-
pounds prepared in the following examples, represents a
moiety o the formula:
OH
(VII)
~ \o~
wherein the carbonyl carbon is attached to the 4 or 5
position in the above formula in view of the fact that a
mixture of 4- and 5-carboxy1uorescein is employed as
starting material.
- 15 - ~ ~'7
EXAMPLE I
Meta- or para- aminophenobarbital ~5 mg) and
carboxyfluorescein (5 mg) were dissolved in 0.5 ml of
pyridine. To the mixture was added N,N'-dichohexylcarbodi-
imide (15 mg). The reaction proceeded for two hours atroom temperature, after which time the reaction product was
purified twice employing silica gel thin-layer chromatography
using a chloroform:methanol (2:1) mixture as developing
solvent to yield an aminophenobarbital-carboxyfluorescein
conjugate of the formula:
H O
~ _ ~ ~ ~3 ¦ ~cF~
- 16 ~
EXAMPLE II
A solution containing sodium hydroxide (1.0 g),
phenytoin ~2.5 g) and 2-bromomethylamine hydrobromide (2.0 g)
in 100 ml of 100~ ethanol was re~luxed for two hours and then
evaporated to dryness under reduced pressure. The residue
was suspended in 50 ml of water and the pH was adjusted to
pH 11 by the addition of 6N sodium hydroxide to dissolve any
unreacted phenytoin. The remaining preclpitate, 2-~-amino-
ethylphenytoin, was filtered, rinsed thoroughly with water
and dried.
An active ester o~ carboxyfluorescein was prepared
by dissolving N-hydroxysuccinimide ~5 mg), carboxyfluorescein
~7.5 mg) and N,N'-dicyclohexylcarbodiimide (20 mg) in 0.5 ml
of pyridine. The reaction was allowed to proceed for two
hours at room temperature after which time 2 ~-aminoethyl-
phenytoin (10 mg) was dissolved ln the reaction mixture.
The resultin~ mixture was allowed to react overnight in
the dark at room temperature and the reaction product was
purified twice employing silica gel thin layer chromatography
using a chloroform:methanol (3:1) mixture as developing solvent
to yield a 2-~-aminoethylphenytoin-carboxyfluorescein
conjugate of the formula;
O H
C _ N-CH2CH2-N [C~}
/=\ / 2~ C
H / O
- 17 - ~ ~78'Z~3
EXAMPLE III
A solution containing 2-carboxymethylphenytoin
~620 mg), N-hydroxysuccinimide (248 mg) and N,N'-dicyclo-
hexylcar~odiimide (453 mg~ in 6 ml of dry dimethylsulfoxide
was allowed to stand at room temperature overnight. The
mixture was filtered and 0.7 ml of 95% hydrozine was added
to 4.5 ml of the filtrate. After four hours at room
temperature, 40 ml of water and 0.5 ml of 10% sodium
hydroxide were added to the reaction mixture. The precipitate,
2-carboxymethylphenytoin hydrazide, was filtered, rinsed
with water, dried and used without further purification.
N,N'-dicyclohexylcarbodiimide (15 mg) was added
to a solution of 2-carboxymethylphenytoin hydrazide (5 mg)
and carboxyfluorescein (5 mg) in 0.5 ml of pyridine. The
reaction was allowed to proceed for two hours at room
temperature, and the reaction product was then puri~ied
twice employing silica gel thin-layer chromatography using
a chloroform:acetone (1:1) mixture as developing solvent
to yield a 2-carboxymethylphenytoin hydrazide-car~oxy-
1uorescein conjugate of the formula:
O H H
~ ~ 11 1 1
~ ~C N-C~2-C-N-N~CF]
~ H O
- 18 - ~'7~Z6~
EXAMPLE IV
N,N'-dicyclohexylcarbodiimide (15 mg) was added to
a solution of 8-~-aminoethyltheophylline (5 mg) and carboxy-
fluorescein ~5 mg~ in 0~5 ml of pyridine. The reaction was
allowed to proceed for two hours at room temperature and the
reaction product was purified twice employing silica gel
thin-layer chromato~raphy using a thin chloroform:methanol
~2:1) mixture as developing solvent to yield an 8-~-
amino ethyltheophylline-car~oxyfluoresceirl conjugate of the
formula:
CH3
- 19 - ~L7~
EXAM ~E V
The procedure of Example IV was employed utilizing
8~aminomethyltheophylline in lieu of 8-3-aminoethylthoephylline
to yield an 8-aminomethylthoephylline-carboxyfluorescein
conjugate of the formula;
J ~CH2_N~CF
C \ N
CH3
~XAMPLE ~I
~ -Valerolactam ~7 5 g~ was dissolved in 6a ml
of dry tetrahydrofuran, under a dry nitrogen atmosphere and
n-butyllithium (1.6 M, 90 mll tn hexane were added dropwise
to the reaction flask and chilled in a dry ~ce-acetone ~ath.
Upon completion of the additton of n-~utyllithium, the
reaction mixture was stirred at room temperature ~or one
hour, refluxed for thirty minutes, and cooled to room
temperature under dry nitorgen atmosphere. l-Bromoethane
(8.0 g) was slowly added to the reaction flask while the
flask was chilled in an ice ~ath. The resulting mi-xture was
then stirred for sixteen hours at room temperature after
which time 100 ml of water was slowly added. The resulting
mixture was stirred at room temperature for thirty minutes
and the organic layer separated. The aqueous layer was
extracted with 50 ml of diethyl ether and the organic layers
were com~ined and dried over sodium sulfate. The solvent
- 20 - ~ ~7~Z~
was evaporated to give a dark oil, which crystallized on
standing. The crystalline residue was recrystallized from
petroleum ether to yield 3.8 g of a residue. The residue
(2.8 g) was refluxed in 25 ml of 6N hydrochlorid acid for
six hours. The water was evaporated from the mixture to
yield a dark, thick oil--2-ethyl-5-aminopentanoic acid--
which was used without further purification.
An active ester of carboxyfluorescein was prepared
by dissolving N-hydroxysuccinimide (5 mg), carboxyfluorescein
(7.5 mg) and N,N'-dicyclohexylcarbodiimide (20 mg) in 0.5 ml
of pyridine. The reaction was allowed to proceed for two
hours at room temperature, after which time 2-ethyl-5-
ami~opentanoic acid (20 mg) was dissolved in the reaction
mixture. The resulting mixture was allowed to react over-
15 night in the dark at room temperature and the reaction
product was purified twice employing silica gel thin-layer
chromatography using a chloroform:methanol (3:1) mixture as
developing solvent to yield a 2-ethyl-5-aminopentanoic acid-
carboxyfluorescein conjugate oe the formula:
CH3CH2 f CH2CH2 2 ~~ ]
Cl=O
OH
- 21 ~
EXAMPLE ~II
An active ester of carboxyfluorescein was prepared
by dissolving N-hydroxysuccinimide tS mg), carboxyfluorescein
(7.5 mg) and ~,N'-dicyclohexylcarhodiimide (20 mg) in 0.5 ml
5 of pyridine. The reaction was allowed to proceed for two
hours at room temperature, after which time 5-(y-amino-
propylidene)- 5H -dibenzo[a,d]-10,11-dihydrocycloheptene
~20 mg) was dissolved in the reaction mixture. The resulting
mixture was allowed to react overnight in the dark at room
10 temperature and the reaction product was purified twice
employing silica gel thin-layer chromatography using a
chloroform:methanol (3:1~ mixture as developing solvent to
yield a 5-tY-aminopropylidene~-5H-dibenzo[a~d]-10~11-di-
hydrocycloheptene-carboxy1uorescein conjugate of the formula:
~/~
~'CHCE12CH2-NH ~ CF]
- 22 - ~ &~
XAMPLE VIII
A solution containing desipramine hydrochloride
(1~33 g) and chloroacetyl chloride (0.8 g) in 25 ml of chloro-
form was refluxed for two hours, The chloroform was evaporated
5 and the residue was dissolved in 25 ml of acetone. Sodium
iodide (0.75 g) was added to the acetone solution, and the
solution was refluxed for thirty minutes, The solution was
filtered and the prec;pitated salt was rinsed with acetone.
The acetone filtrate was evaporated and the residue was
10 taken up in 20 ml of methanol, Concentrated ammonium
hydroxide (20 ml) was added to the methanol solution and the
resulting solution was refluxed for one hour. The reaction
mixture was extracted three times with 25 ml of chloroform
and combined extracts were dried over sodium sulfate,
15 filtered and evaporated to yield N-aminoacetyldesipramine
which was used without further purification.
N-aminoacetyldesipramine (5 mg) and carboxy-
fluorescein ~5 mg) were dissolved in 0.5 ml of pyridine.
To the mixture was added N,N'-diclohexylcarbodiimide (15 mg).
20 ~he ,reaction proceeded for two hours at room temperature,
a~ter which time the reaction product was purified twice
employing silica gel thin-layer chromatography using a
chloro~orm:acetone tl:l) mixture as developing solving
to yield a N-aminoacetyldesipramine-carboxyfluorescein
25 conjugate of the formula:
23
CH3
~ I,`i
N-C~ CH CH -N-C-CH2NH-~CF]
E~YAMPLE IX
A solution containing N-hydroxysuccinimide (S mg~,
carboxyfluorescein (7~5 mg1 and N,N~-dicyclohexylcarbodi-
s imide (20 mg) in 1 ml of pyridine was allowed to reack at
room temperature for ~our hours. An acti~e ester of
carboxy1uorescein was precipitated by adding lO ml o~ di-
ethylether to the reaction mixture. Tha precipitate was
I filtered, rinsed well with diethylether and redissolved
10 in 0.5 ml of dimethylsulfoxide. L-thyroxine (10 mg~ was
then added to the solution and the reaction was allowed to
proceed for two hours at room temperature after which time
the reaction product was purified twice employing silica
gel thin-layer chromatography using a chloro~orm:methanol
lS (3:1) mixture as developing solvent to yield a L-thyroxine-
carboxyfluorescein conjugate of the formula:
I\ I \ ~
T-OH
H(:) I CH2 -C N~ CF ]
2 ~
EXAMPLE X
A solution containing ammonium ace~ate (0.89 g),
3-oxodigoxigenin (389 mg) and sodium cyanoborohydride (63 mg)
in 5 ml of methanol was stirred at room temperature for 48
5 hoursA The solution was adjusted to pH 1 by the addition
of concentrated hydrochloric acid and evaporated to dryness
under reduced pressure. The residue was taken up in 10 ml
of water and extracted three times with 10 ml of chloroform.
The aqueous layer was adjusted to pH 11 by using solid
10 potassium hydroxide~ The resulting solution was extracted
five times with 10 ml of methylene chloride. The organic
layers were combined, dried and then evaporated to dryness
under reduced pressure to yield 3-amino-3-deoxydigoxigenin
which was used without further puriication.
An active ester of carboxyfluorescein was prepared
by dissolvi.ng N-hydroxysuccinimide (5 mg~, carboxyfluorescein
(7.5 mg) and N,N'-dicyclohexylcarbodiimide (20 mg~ in 0.5
ml of pyridine. The reac~ion was allowed to proceed for
two hours at room temperature, after which time a 3-amino-
20 3-deoxydigoxigenin-carboxyfluorescein conjugate of the
formula was isolated:
2 5 ~ Z~3
o~
~N~CF ]
~ JLt;~ 3
- 26 -
The following tracers were also prepared in accordance
with the procedures previously dascribed:
XAMPLE XI - O-~minoacetyl-propranolol-carboxyfluor~scein
coniuqate
O H
Il I
OcH2cH-o-c-cH2-N~cF]
XAMPLE XII - 2-Propyl-5-aminopentanoic acid-
carbox~fluorescein coniuqate
H H
CH3cH2cH2~f-cH2cH2cH2-N~cF ]
C=O
I
OH
EXAMPLE XIII - 2-Butyl-5-aminopentanoic acid-
carboxyfluorescein conjugate
7 H
CH3cH2cH2cH2-c-cH2cH2cH2-N~cF]
C=O
OH
EXAMPLE XIV - Aminoprimidone-carboxy~luorescein conjugate
,~ C' '' CH 2 CH 3
CH ~ C
~ N - C~
H O
- 27 - ~'3L7~
EXAMPLE XV ~ 4'-nitrophenyl~-l-hydroxy-2-
amino-3-hydroxypropane-
_ carboxyfluorescein conjugate
02N-~-CH-fH-N~CF~
CH2 ~
5 XAMPLE XVI - p-amlnophenol-carbaxyfluorescein conjugate
Ho_e~-N~CF]
EXAMPLE XVII - N- (2-aminoethyl)-ethosuximide-
_ _ carboxy~luorescein conjugate
CH 2 C tH
~ N-CEI2CH2-N~CP3
CH3 2 1
CH3 0
10 EXAMPLE XVIII - N'-desethyl-N-acetyl-procainamide-
carboxyfluorescein conjugate
-
C - N -~ C -N- CH2 CH 2 - N{`C li ]
CH3
- 28 ~ 8~
EXAMPLE XIX - N'-desethyl-N'-aminoacetyl-N-
acetyl-procainamide-carboxy-
fluorescein coniugate
O H ~ O H CH2CH3 Hl
~-N-~-c-N-cH2cH2-N-c-cH2-N~cFJ
CH3
5 EXAMPLE XX - l-amino-2-phenyl-2-(2'-pyridyl)-4-
(diisopropylamino)-butane-
_ carbo ~ rescein conjugate
CH3 H
H-C CH2-N--~CF]
C ;
C ~ N
CH3 ~
EXAMPLE XXI - 3,3',5-Triiodo-L-thyronine-
lO _ carboxy~luorescein conjugate
~C /H
HO- ~ O- ~ _C~2_~ CF]
- 29 - ~7~9
EXAMPLE XXII - 3,3',5,5'-tetraiodo-D-thyronine-
carboxyfluorescein~c~o~n ~
.
O OH
~C H
HO-~- o~ CH2 - C-N~ C F ]
EXA~PLE XXIII ~ N-aminoacetyl-iminodibenzyl
5car~oxyfluorescein conjugate
~ O H
N-C-CH2-N~CF]
/
EXAMPLE XXIV - Carbhydrazinoimino-dibenzyl
carboxyfluorescein conjugate
O H H
N-C-N-N-~ CF ]
- 30 - ~ 9
EX~MPLE X~- Dibenzosuberonehydrazone
fluorescein conjugate
H
~=N-N-~CP]
EXAMPLE XXVI - 5-amino-10,11-dihydro-SH-dibenzo-
S~a,d3-cycloheptene
~H
N~CF]
~.~'7~
- 31 -
As previously mentioned~ the tracers of the
present in~ention are effective reagents for use in
fluorescence polari2ation immunoassays. The followlng
Examples illustrate the suitablility of tracers of the
5 present invention in immunoassays employing fluorescence
polarization techniques. Such assays are conclucted in
accordance with the Eollowing general procedure:
1) A measured volume of standard or test serum
is delivered into a test tube and diluted with. buffer,
2~ A known concentration of a tracer of the
present invention optionally CQntaining a surfactant is
then added to each tube;
3) A known concentration of antisera is added to
the tubes;
4) The reaction mixture is incubated at room
temperature; and
5) ~he amount of tracer bound to antibody is
measured by fluorescence polariæation techniques as a measure
of the amount o ligang in the sample~
- 32 - ~7~
EXAMPLEXXVqI- Phen tion assa
Y y
A~ Materials required:
1) BGG buffer consisting o~ 0.1 M sodium phosphate,
pH 7.5, containing bovine gammaglobulin, 0.01% and sodium
azide, 0.01~.
2) Tracer, consisting of 2-~-aminoethyl phenytoin-
carboxyfluorescein at a concentration of approximately 105
nM in BGG buffer with 5~ sodium cholate added.
3) Antiserum, consisting of antiserum raised
against phenytion diluted appropriately in BGG buffer contain-
ing 0.005% benzalkonium chloride.
4) Samples of human serum or other biologiGal
1uid containing phenytoin.
5) Cuvettes, lO x 75 mm glass culture tubes
used as cuvettes.
6) Fluorometer capable of measuring ~luorescence
polarization with a precl~ion of ~ 0.001 unit5.
Bl Assay Method:
1) A small volume of sample (0.366 microliters)
is placed in each cuvette by pipetting 15 ~Q of sample and
diluting with 600 ~Q BGG buffer in a dilution vessel. Next,
15 ~Q of diluted sample is pipetted into the cuvette followed
by 600 ~Q BGG buffer.
2) Tracer is added by pipetting 40 ~ tracer and
lO00 ~Q BGG buffer into the cuvette.
3~ Antiserum is added to start the reaction by
pipetting 40 ~Q antiserum into the cuvette followed by lO00 ~Q
BGG buffer.
4) The contents of all cuvettes are well mixed
and allowed to incubate for 15 minutes at ambient temperature.
5) The fluorescence polarization is read on a
fluorometer and a standard curve constructed to determine
unknowns.
- 33 - ~ ~'7~
C~ The results of a series of serum standards
containing phenytoin at concentrations between 0 and 40 ~g,/ml
are presented below. Each concentration was assayed in
duplicate and averaged.
S Concentration of
Phenytoin (~g/ml)Polarlzation
0.222
2.5 0.1~6
5.0 0.178
10,0 Q.154
20.0 0.132
40 0 0.110
The polarization of fluorescence is seen to
decrease in a regular manner as the phenytoin concentration
increases, allowing construction of a standard curve. Unknown
specimens treated in an identical manner can be quan-titated
by reerence to the standard curve, thereby illustrating the
utility of 2-~ aminoethyl phenytoin-carboxy~luorescein for
the measurement of phenytoin.
EXAMPLE XNIII -Phenobarbital assay
A) Materials required:
1) BGG buffer lsee Phenytoin)
22 Tracer, consisting of amlnophenobarbital
23 carboxyfluorescein at a concentration of approximately 110 nM
in tris HCl buffer, pH 7.5, containing 0.01~ sodium azide,
0.01% bovine gamma globulin, and 0.125% sodium dodecyl sulfate.
3) Antiserum, consiting of antiserum against
phenobarbital diluted appropriately in BGG buffer containing
0.0~5~ benzalkonium chloride.
4) Samples of human serum or other ~iological
fluid containing phenobarbital.
5) Cuvettes (see Phenytoin)
6~ Fluorometer ~see Phenytoin~
iLi7~
- 34 -
B~ Assay Protocol:
1~ A small volume of sample (0.196 micr~liter~
is placed in the cuvette by ipetting 10 ~Q o sample and
diluting with 500 ~Q BGG buffer in a dilution vessel. Next,
10 ~Q of diluted sample is pipetted into the cuvette followed
by 500 ~Q BGG buffer.
2) Tracer is added by pipettin~ 40 ~ of tracer
and 1000 ~Q BGG buffer into each cuvette.
3) Antiserum is added to start the reaction by
10 pipetting 40 ~Q antiserum followed by 1000 ~ BGG buffer.
4) The contents of all cuvettes are mixed well
and allowed to incubate for 15 minutes at ambient temperature.
5) The fluoxescence polarization is read on a
fluorometer and a standard curve ccnstructed to determine
unknowns.
C) The results of a series of senlm standards
containing phenobarbital at concentrations between 0 and 80
~g/ml are presented below. Each concentration was assayed in
duplicate and the values averaged.
Concentration of
P enobarbital (~Q)Polarization
0 0.250
5.0 0.~31
10.0 0.196
20.0 0.150
40.0 0.104
80.0 0 077
The polarization of fluorescence is seen to decrease in a
regular manner as the phenobarbital concentration increases,
30 allowing construction of a standard curve. Unknown specimens
treated in an identical manner can be quantitated by references
to the standard curve thereby illustrating the utility of
aminophenobarbital-carboxyfluorescein for the measurement
of phenobarbital.
- 35 - ~ ~7
_AMPLE XXIX _ - Theophylline assay
A) Materials required:
1) ~racer, consisting of 2 nM of 8-aminoethyl
theophylline-carboxyfluorescein in BGG buffer (see Phentoin
assay~ containing 0.01~ sodium dodecyl ~ulfate.
2) Antiserum, consisting of antiserum raised
against theophylline diluted appropriately in BGG buffer.
3) Samples of human serum or other biological
fluid containing theophylline.
4~ Cuvettes, (see Phenytoin assay)
5) Fluorometer, (see Phenytoin assay)
B) Assay protocol:
1) Place 1,0 ml tracer in all cuvettas.
2) Add 2.0 ~Q sample to all cuvettes.
3) Add 1.O ml antiserum to all cuvettes,
4) Mix well and incubate 15 minutes at ambient
temperature.
5) Read the fluorescence polarization on a ~luoro-
meter and construct a standard curve.
C~ The results of a series of serum standards
containinq theophylline at concentrations between 0 and 40
~g/ml are presented. Each concentration was assayed in
duplicate and the average is presented.
Concentration of
Theo hvlline C~q/mllPolarization
P ,~
o 0.158
2,5 0.118
0.105
la 0.091
0.076
0.063
~7~
- 36 -
The polarization of fluore~cence is seen to decrease in a
regular manner as the theophylline concentration increases,
allowing construction of a standard curve. Unknown specimens
treated in an identical manner can be ~uantita~ed by reference
to the standard curbe thereby illustrating the utility of
8-aminoethyltheophylline-carbox~tfluorescein for the measure-
ment of the theophylline.
E~L~ XXX - Digoxin assa
Y
A) Materials required:
1) BGG buffer consisting of O.lM sodium phosphate,
pH 7.5, containing bovine gammaglobulin, 0.01% and sodium
azide, 0.01~.
2) Tracer, consisting of digoxin carbo~yfluorescein
at a concentration of approximately 2nM in BGG bufer.
3) Antiserum, consisting oE rabbit antiserum raised
against digoxin diluted appropriately in BGG buffer.
4) Samples or human serum or other biological
fluid containing phenytoin.
5) Precipitation reagent --5% trichloroacetic
20 acid in water.
6) Cuvettes, 10 x 75 mm glass culture tubes used
as cuvettes.
7) Fluorometer capable of measurin~ fluorescence
polarization with a precision of ~ 0.001 units.
B) Assay protocol:
1) To 100 ~Q of 5% trichloroacetic acid in a test
tube is added 100 ~Q of a standard or unknown sample. The
tubes containing the sample are capped and vortexed.
2~ The tubes containing standard or sample in
30 trichloroacetic acid are centrifuged.
3) To a test tube 1.8 ml of BGG buffer and 25 ~Q
of antisera at 35 C is added 150 ~Q of the trichloroacetic
supernatant solution.
4) The test tubes containing antisera and supernatant
35 i5 incubated for 6 minutes at 35 C, at which time the
- 37 ~
fluorescence polarization of the tubes are measured. This
measurement is the background fluorescence polarization of
the standard or unknown.
5) Ten minutes after the addition of supernatant
5 to antisera, 25 ~Q of the tracer is added to the test tube.
62 Six minutes after the addition of tracer, the
fluorescence polarization of the standards and sample ~ubes
are measured and the previously measured background fluorèscence
polarization is substracted to yield the fluorescence
10 polarization of the antibody-tracer complex that had formed.
7) The results of a series of serum standards
containing digoxin at concentrations between 0 and 5 ng/ml
are presented below. Four samples at each concentration were
assayed and averaged.
15Digoxin Concentration (n~/mll Polarization
0 0.1~2
0.5 0.134
1.0 0.123
2.0 Q.106
3.0 0.092
5.0 0.070
The polarization of fluorescence is seen to
decrease in a regular manner as the digoxin concentration
increases, allowing construction of a standard curve.
25 Unknown ~pecimens treated in an identical manner can be
quantitated by reference to the standard curve, thereby
illustrating the utility of digoxin carboxyfluorescein for
the measurement of digoxin.
The following table summarizes the various
30 fluorescence polarization assays that have been carried out
in accordance with the above-described procedures employing
tracers prepared in the preceeding examples. The tracers
employed are identical by Example number and the specific
ligand(s) determined are indicated.
- 38 - ~ ~7
Example No. _ Ligand(s)
I Phenobarbital
II Phenytoin
III Phenytoin
5IV Theophylline
V Theophylline
VI Valproi.c acid
VII Nortriptyline; Amitriptyline
VIII Imipramine; Desipramine
10IX Thyroxine
X Digoxin
XI Propranolol
XII Valproic acid
XIII Valproic acid
15 XIV Primidone
XV Chloramphenicol
XVI Acetaminophen
XVII Ethosuximide
XVIII N-acetylprocainamide
20 XIX N-acetylprocainamide
XX Disopyramide
XXI Triiodothyronine
XXII Thyroxine
XXIII Imipramine; Desipramine
25 XXIV Imipramine; Desipramine
XX~ ~ortriptyline; Amitriptyline
XXVI Nortrip~yline; ~mitriptyline
- 39 ~
As evident from the above results, the tracers
of the present invention are effective reagents in
fluorescence polarization immunoassays. In addition to the
properties mentioned above, the tracers of the present
5 invention possess a high degree of thermal stability, a high
degree of bound polarization, high quantum yields and are
realitively easy to produce and purify.
Although this invention has been described with
respect to specific modifications, the details thereof are
10 not to be construed as limitations, for it will be apparent
that various equivalents, changes and modifications may be
resorted to without departing from the spirit and scope
thereof and it is understood that such equivalent embodiments
are intended to be included therein.
