Note: Descriptions are shown in the official language in which they were submitted.
2~
~ackground Of The Invention
The present invention relates to a method ~nd re-
agents for determining ligands in biological luids such as
serum, plasma, spinal fluid, amnionic rluid and urine. In
particular, the present invention relates to a fluorescence
polarization immunoassay 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 binding immunoassays for measuring
iigands are ~ased on the competition between a ligand in a
test sample anda labeled reagent, referred to as a tracer,
Cor a limited number of receptor binaing sites on antibodies
s~ecific to the ligand and tracer. The concentration of
ligand in the sample determines the amount of tracer t~at will
specifically bind to an antibody. The amount of tracer-
antibody conjugate produced may be quantitively measured andis inversely proportional to the quantity of ligand in the
test sample.
In general, fluorescence polarization techniques
are based on the principle that a fluorescent label compound
when excited by linearly polarized light w~ll emit fluorescence
having a degree of polarizaLion inversely related to $ts rate
o~ rotation. Therefore, when a molecule such as a tracer-
anti~ody conjugate having a fluorescent label is excited
with linearly polarized light, the emitted liaht 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-antibody conjugate and
the molecules are more randomly oriented, therefore, the
emitted ligh~ is depolarized. Thus, fluorescence polarization
provides a quantitive means for measuring the amount of
tracer-antibody conjugate produced in a competitive binding
immunoassay.
- 2 -
Summary Of The Invention
The present invention encompasses a method for
determining ligands in a sample comprising intermixing with
said sample a bio].ogically acceptable salt of a tracer of
S t~e formula:
OH
H ~ (I)
R-~X~-N\
)~\ ~ `
//\\~
O OH
wherein X is a group selected from the class consisting of
an oxalyl group of the formula:
O O
Il 11
--C--C-- ;
a sulfonyl group of the formula;
o
--S-- ;
o
and a carboamidosulfonyl group of the formula:
O H O
Il l 11
-C-N-~-
and
R is a ligand-analog wherein said ligand-analog
has at least one common epitope with said
ligand so as to be specifically reconizable
by 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 invention further relates to a novel class of
tracers of formula (I) and biologically acceptable salts
thereof, which are useful as reagents in the above-described
method. The methods and tracers of the present invention
are particularly useful in quantitatively monitoring
therapeutic drug concentrations in serum and plasma.
Detailed Descri tion Of The Invention
p
The term "ligand" as used herein refers to a
~olecule in particular a low molecular weight hapten, to
which a receptor, no~mally an antibody, can be obtained or
formed. Haptens are protein-free bodies, generally of lo~
molecular weight that do not induce anti~ody formation when
injected into an animal, but are reactive to anti~odies.
~ntibodies to hapten are generally raised ~y first con-
jugating the haptens to a protein and injecting the conjugateproduct into an animal~ The resulting antibodies are isolated
by conventional antibody isolation techniques.
The ligands determinable by the method of the
present invention vary over a wide mole~ular weight range~
~lthough high molecular weight ligands may be determined, for
best results, it is generally preferable to employ the methods
o~ 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.
- 4 ~ ~ 2L~ a
The novel tracer of the present invention includes
compounds of formula (I) wherein the ligand-analog represented
by ~ include radicals having a molecular weight within a
range of 50 to 4000. l'he preferred novel tracers include
compounds of formula (I) wherein the liyand-analogs
reoresented by R include radicals having a molecular weight
within a range of 100 to 2000.
Representative of ligands determinable by the
methods of the present invention include steroids such as
esterone, estradiol, cortisol/ testosterone, progesterone,
chenodeoxycholic acid, digoxin, cholic acid, digitoxin,
deoxycholic acid, lithocholic acids and the ester and amide
derivatives thereof; vitamins such as ~-12, folic acid;
thyroxine, triiodothyronine, histamine, serotonin, prosta-
glandins such as PGE, PGF, PG~; antiasthamatic drugs such astheophylline; antineoplastic drugs such as doxorubicin and
methotrexate; antiarrhythmic drugs such as disopyramide,
lidocaine, procainamide, propranolol, quinidine r ~-acetyl-
procainamide; anticonvulsant drugs such as phenobarbital,
20 phenytoin, primidone, valproic acid, carbamazepine and
ethosuximide; antibiotics such as penicillins, cephalosporins,
erythromycin, vancomycin, gentamicin, amikacin~ chloramphenicol,
streptomycin and tobramycin, antiarthritic drugs such as
salicylate; antidepressant drugs including tricyclics such
as nortriptyline, amitriptyline, imipramin~ and desipramine;
and the like as well as the metabolites thereof~ In addition,
drugs of abuse such as-morphine~ heroin, hydromophone,
oxymorphone, metapon, codeine, hydrocodone, dihydrocodiene,
dihydrohydroxy codeinone, pholcodine, dextromethorphan,
30 phenazocine and deonin and their metabolites may be determined
in accordance with the methods of the present invention.
The tracers of the present invention generally
exist in an eouilibrium between their acid and ionized
states, and in the ionized state are effective in the
35 method of the present invention. Therefore, the present
invention comprises the tracers in either the acid or
ionized state and for convenience, the tracers of the
- 5 - f~
present invention are structurally represented herein in
their acid form. When the tracers of the preaent invention
are present in their ionized state, the tracers exist in the
form of biologically acceptable salts. As 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 e~ist in their 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
employed, 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 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 capahle of
competing with the ligand for the binding sites of a receptor.
A characteristic of such ligand-analog is that it possesses
sufficient 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 have the same or sub-
stantially the same structure and charge distribution
(spatial and polar organization~ as the ligand of interest
for a significant portion or the molecular surface. Since
fre~uently, the linking site for a hapten will be the same
in preparing the antigen for production of antibodies as ~sed
for linXing to the ligand, the same portion of the ligand
analog which provides the template for the antibody will be
exposed by the ligand analog in the tracer.
- 6 -
When X is a sulfonyl group,
--S--
Il
O
the class of ligand-analogs represented by R are derived from
the corresponding ligand by removal of an aromatic hydrogen,
that is a hydrogen bonded to an aromatic carbon, preferably
a phenyl carbon, or by the formation of a phenyl or sub-
stituted phenyl derivative of the ligand. In addition, a
ligand may be structurally modifiecl by the addition or
election of one or more functional groups to form a ligand-
analog while retaining the necessary epitope sites forbinding to an antibody, However, it is preferred that such
modified ligand-analogs be bonded to the sulfonylamino
fluorescence moiety through an aromatic carbon~
When X is an oxalyl group,
O O
1! 11
--c--c--
the class of ligand analogs represented by R are derived
from the corres~onding ligand by removal of a hydrogen atom
hond to a reactive amine, i~e., a hydrogen atom honded to
a primary or seconaary amine or by the formation of an amino
dèrivative of the ligand wherein an imino group
_~_
replaces one or more atoms originally present in the ligand,
at the site of binding to an oxalylaminofluorescein moiety.
Illustrative of ligands which upon the removal of a hydrogen
hond to a reactive amine from a ligand-analog represented by
R include, for e~ample, procainamide, thyroxine, quinidine
and the aminoglycoside antibiotics. Illustrative of ligands
whose amino derivatives are useful as ligand-analogs include
theophylline, valproic acid, phenobarbital, phenytoin,
primidone, disopyramide, digoxin, chloramphenicol, salicylate,
acetaminophen, carbamazepine, desimpramine and nortriptyline.
In addition, a ligand may be structurally modified by the
addition or deletion of one or more functional groups to
form a ligand-analog, while retainlng the necessary epitope
sites for binding to an antibody. However, it is preferred
that such modified ligand-analogs be bonded to the oxalyl-
aminofluorescein moiety through an imino group.
When ~ is a carboamidosu:Lfonylamino
O ~ O
Il ~ 11
-C-N-S-
the class of ligand analogs represented by R are derived
from the corresponding ligand by removal of a reactive
hydrogen atom7 i.e., a hydrogen atom bonded to a hydroxy
oYygen or a reactive amine (primary or secondary) or by the
formation of an amino derivative of the ligand wherein an
imino group
replaces one or more atoms originally present in the ligand,
at the site of binding to a carboamidosulfonyl-amino-
fluorescein moiety. Illustrative of ligands which upon the
removal of a reactive hydrogen may form a ligand-analog
represented by R include, for example, procainamide,
thyroxine, quinidine and the aminoglycoside antibiotics.
Illustrative of ligands whose amino derivatives are useful
as ligand-analog include theophylline, valproic acid,
phenobarbital, phenytoin, primidone, disopyramide, digoxin,
chloramphenicol, salicylate, acetaminophen, carbamazepine,
desimpramine and nortriptyline. In addition, a ligand may
- 8 -
be structurally modified by the addition or deletion of one
or more functional groups to form a ligand-an~log, while
retaining the necessary epitope sites for bindin~ to an
antibody. However, it is preferred that such modified
ligand-analogs be bonded to a carb~amidosulfonyl-
aminofluorescein moiety through an imino or oxy sroup.
The tracers of the present invention are prepared
in accordance with known techniques. When X is a sulfonyl
sroup,
1l
--S-
O
the tracers of the present invention are prepared by reacting
a compound of the formula;
(II)
R-Y
wherein R is above-defined and Y is an aromatic hydrogen,
preîerably bonded to a phenyl ringj with chlorosulfonic acid
to produce a chlorosulfonyl ligand-analog of the formula:
O
R-S-Cl (III)
O
The chlorosulfonyl ligand-analog is reacted with an amino-
fluorescein of the formula:
- 9 -
OH
H ~
H--N ~ ~ ( IV)
O C)H~
wherein the amino group is bonded to the 4 or 5 position of
the benzoic acid ring; in the presence of an inert solvent
to yield a tracer of the present invention of the formula:
OH
O H
R--S-l`i ~ _
O ~o (V)
O OH6~
- 10 -
~hen X is an oxalyl group,
O O
-C~
the tracers of the present invention are prepared by reacting
a compound br the formula
(VI)
5R-Z
wherein R is above-defined and Z is a hydrogen bonded to a
reactive nit~ogen (primary or secondary amine), with
methyloxalylchloride to yield a methc~-roxalyl ligand-analog
r~nich is hydrolyzed in the presence of a base to yield
lo ~ydroxyoxalyl ligand-analog OI the formula
O O
~ (VII)
R-C-C-OH
The hydroxyoxalyl ligand-analog is reacted with an amino-
fluorescein of formula (IV) in the presence of a coupling
agent, such as, l-ethyl-3-(3'-dimethylaminopropyl)-carbodi-
imide hydrochloride, and an inert solvent to yield a traceror ~he present invention of the formula:
OH
O O H
R-C-C-N ~
O (VIII)
O OH ~
When X is a carboamidosulfonyl,
O H O
Il l 11
-C-N-S-
the tracers of the present invention are prepared by reacting
a compound of the formula
R-~ (IX)
wherein R is above-defined and ~ is a hydrogen bonded to a
reactive nitrogen (primary or secondary amine~ or to a
hydroxy oxygen; with chlorosulfonylisocyanate to yield
a chlorosulfonamidocarbonyl-ligand-analog derivative or the
formula
O H O
Il 1 11 (X)
R-C-N-S-Cl
O
The chlorosulfonamidocarbonyl-ligand-analoq derivative is
reacted with an a~inofluorescein of formula (IV)
- 12 - ~24~
to yield the tracers of the present invention of the formula:
OH
e I e I ~
R-C-N-S-~
O OH ~ ~
The temperature at which the reaction for preparing
the tracers of this invention proceeds is not critical. The
temperature should be one which is sufficient so as to
initiate and maintain the reaction< Generally, for convenience
and economy, room temperature is sufficient. ~n preparin~ the
tracers of the present invention, the ratio of reactants is
not narrowly critical. For example, for each mole of a compound
or formula (II), one should emnloy two moles of chlorosulfonic
acid to obtain a reasonable yield. It is preferred to employ
an excess of chlorosulfonic acid for ease of reaction and
recoverv of the reaction products.
The compounds of formula (IV~ employed as starting
materials in the production of the tracers of this invention
are either commercially available or prepared in accordance
with known technlques.
~ 2~
- 13 -
For ease of handling and recove~y of product,
the process for preparing the tracers of the ~resent
invention is conducted in the presence of an inert solvent.
Suitable inert solvents include those solvents which do not
react substantially with the starting materials and are
sufficient to dissolve the starting materials and include
for example, acetone, chloroform, pyridine, and the like.
In order to provide maximum product yields r the reaction
preferably proceeds under neutral or basic conditions~
Suitable ~ases include for example triethylamine, pyridine,
and the like. The reaction products are
generally purified using either thin-layer or column
chromatography prior to application in the methods of the
~resent 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
20 compete for limiting antibody sites resulting in the formation
o~ ligand-antibody and tracer-antibody complexes. By main-
taining constant the concentration of tracer and antibody,
the ratio of ligand-antibody complex to tracer~antibody
comple~ that is formed is directly proportional to the amount
25 of ligand present in the sample. There~ore, upon exciting
the mi~tu.e with fluorescent light and measuring the polariza-
tion of the 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 tracer
not complexed to an antibody is low, approaching zero. Upon
complexing with a specific antibody, the tracer-antibody
complex thus formed assumes the rotation of the antihody
molecule which is slower than that of the relatively small
35 tracer molecule, thereby increasing the polarization observed.
Therefore, when a ligand competes with the tracer for antibody
sites, the obser~red polarization of fluorescence of the
tracer-an-tibody complex becomes a value somewhere between that
OL the tracer and tracer-antibody compleY. If a sample
contains a high concentration of a ligand, thè ohserved
polarization value is closer to that of the free ligand,
i.e., low. If the test sample conrains a low concentration
of the ligand, the polarization value is closer to that of
the bound ligand, i.e., high. ~y sequentially exciting the
reaction mixture of an immunoassay with vertically and then
horizontally polarized light and analyzing only the vertical
component of the emitted light, the polarization of fluorescence
of the reaction m~Nremay ~e accurately determined. The precise
relationship between polarization and concentration of the
ligand to be determined is established by measuring the
polarization values of calibrators with known concentrations.
The 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 ~e sufficient to allow the tracers
of formula (I) to exist in their ionized state. The pH may
range ~rom about 3 to 12, more usually in the range of from
5 to 10, most preferably from about 6 to 9. Various buffers
may be used to achieve and maintain the pH during the assay
procedure. Representative buffers include ~orate, phosphate,
carbonate, tris, barbital, and the like. The particular
buffer employed is not critical to the present invention,
but in an individual assay~ a specific buffer may be preferred
in view of the antibody employed and ligand to be determined.
The cation portion of the buffer 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. The temperature will normally range from about
0 to 50 C, more usually from about 15 to 40 C.
The concentration of ligand which may be assayed
will generally vary from about lQ to 10 13M, more usually
from about 10 4 to 10 1OM~ Higher concentrations of ligand
may be assayed upon dilution of the original sample.
In addition to the concentra-tion range of ligand
of interest, considerations such as whether the assay is
~ 2~
- 15 -
qualitative, semiquantitative, or quantitative, the
equipment employed, and the characteristics of the tracer
and antibody will normally determine the concentration of the
tracer and antibody to be employed. While the concentration
of ligand in the sample will determine the range of concentra-
tion of the other reagents, i.e., tracer and antibody, normally
to optimize the sensitivity of the assay, individual reagent
concentrations will be determined empirically. Concentrations
of the tracer and antibody are readily ascertained by one of
ordinary skill in the art.
As previously mentioned the preferred tracers of
the oresent invention are prepared from 5-aminofluorescein or
4-aminofluorescein and exist preferably as isomers of the
^ormula:
OH
R-~X~-N ~ (~II)
O
OH ~
" - 16 -
or
OH
R-~Xt-N ~ ~
~ (XIII)
\\
O// ~OH ~
~0,
wherein R and X are above defined.
The following illustrative, nonlimiting examples
will serve to furt~er demonstrate to those skilled in the art
the manner in which specific tracers within the scope of this
invention may be prepared. The symbol EAF] appearing in the
structural formulas illustrating the compounds prepared in
the .ollowing examples, represents a moiety of the formula:
- 17 -
OH
H ~ ~
(XIV)
OH ~
wherein the imino nitrogen is attached to the 4 or 5 position
in the above formula depending on the specific aminofluorescein
isomer employed as the starting material.
- 18 -
EXAMPLE I
To 0.71 g of lidocaine was added 2.8 g of chloro-
sulfonic acid and the resultant mixture was heated at 60 C
for one hour. The reaction mixture was cooled and
crushed ice and water were added to the mixture to dissipate
any unreacted chlorosulfonic acid. The resultant aqueous
solution was neutralized to pH 7 using sodium hydroxide.
The resultant product was extracted twice with 10 ml portions
of methylene chloride. The methylene chloride extracts
we~e combined, dried over sodium sulfate, filtered and
1~ evaporated to dryness to yield 100 mg of a mixture of meta-
and para- chlorosulfonyl lidocaine as a ~ummy oil, To a
solution containing 5 mg of 4-aminofluorescein in 0.5 ml of
pyridine was added 5 mg of the above chlorosulfonyllidocaine
mixture. After ten minutes, a crude product formed.
The crude product was purified by thin-layer chromatography
using sil ca gel and chloroform, then a mixture of chloroform:
acetone (1:1~ and finally a mixture of chloroform:methanol
(1:1) developing solvents to yield a mixture of sulfonyl-
lidocaine-aminofluorescein conjugate of the general formula:
\3
a5C\2 H ~ -~A~
N--CH2-C-N~
CH3
EX~PLE II
To 1.2 ~ of phenobarbital was slowly added 4.5 g
of chlorosulfonic acid and the resultant mixture was heated
at 60 C for one hour. The reaction mixture was cooled
and crushed ice and water were added to the mixture to
dissipate any unreacted chlorosulfonic acid. The reaction
-- 19 --
mixture was then filtered to yield a white precipitate which
was rinsed with water and dried in a vacuum desiccator to
yield 0.8 g of a mixture of meta- and para- chlorosulfonyl-
pheno~ar~ital having a melting point of 190-1~5 C, To a
solution containing 5 mg of 5-aminofluorescein in 0.5 ml of
pyridine was added 5 mg of the above mixture of chlorosulfonyl-
phenobarbital~ After 10 minutes, a crude product
had formed and was purified twice e-mploying thin-layer
chromatography techniques employing silica gel and a mixture
of chloroform:methanol (2:11 as a developing solvent to yield
a sulfonylphenoharbital-aminofluorescein conjugate of the
formula:
= ~ / r ~ S-~AF
E ~PLE III
To 2 g of a-phenethyl-a-methylsuccinimide was
dropwise added 2.5 g of chlorosulfonic acid and the resultant
mixture was stirred at 26 C for one hour. Crushed ice and
wa~er were added to the mixture to dissipate any unreacted
chlorosulfonic acid. A reaction product which had formed
20 was extracted twice with 10 ml portions of chloroform~ The
chloroform extracts were combined, dried over sodium sulfate
and evaporated to yield a heavy oil. The oil was crystallized
from a mixture of toluene and petroleum ether to yield 0.29 g
of a mixture of a-~meta- and para- chlorosulfonylphenethyl)-
a-methylsuccinimide having a melting point of 145 - 150 C.
To a solution containing 5 mg of 4-aminofluorescein in 0.5 ml
of pyridine was added S mg of the a~ove mixture to u-~chloro-
sulfonylphenethyl)-a-methylsuccinlmide. After 10 minutes ,
- 20 - ~ 2~
a crude product had formed which was purified twice
employing thin-layer chromatographic techniqu~s utilizing
silica gel and a mixture of chloroform:methanol (2:1~ as a
developing solvent to yield a mixture of a- (sulfonylphenethyl)-
a-methylsuccinimide-aminofluorescein conjugate of the formula:
O=C CH~ / CH3
H-~ CH2C~2- ~ 1¦
o S-~AF]
EXAMPLE IV
To a solution containing 2 g of iminostilbene
and 2 ml of triethylamine in 50 ml of chloroform was added
1.5 g of methyloxalylchloride. The resultant mixture was
refluxed for one hour and then evaporated to dryness. The
residue was taken up in 50 ml of chloroform and then extracted
with 50 ml of water. The chloroform layer was evaporated to
dryness. To the residue was added 100 ml of 2N sodium
hydroxide and the resultant mixture was refluxed for 30 minutes.
The mixture was cooled to room temperature and then extracted
with 50 ml of chloroform. The aqueous layer was acidified to
pH l using concentrated hydrochloric acid and then extracted
with 100 ml of ether. The organic extract was dried over
sodium sulfate and evaporated to dryness. The residue was
taken up in 50 ml of methanol and triturated with water to
yield a crop of crystals. The mixture was filtered and the
crystals were cooled for 16 hours to yield 2.4 g of a
N-hydroxyoxalyl-iminostil~ene ~melting point 162 - 163 C~.
To 5 mg of the N-hydroxyoxalyl-iminostil~ene was
added a solution containing 5 mg of 4-aminofluorescein in
- 21 -
0.5 ml of pyridine. The reaction was allowed to proceed
for two hours at 26 C to yield a crude produ-ct, The crude
product was ,ourified using thin-layer chromatography employing
silica gel and a developing solution consisting of a mixture
of chloroform:acetone (1:11 to yield an N-oxalyl-iminostilbene-
aminofluorescein conjugate of the formula:
~ 811
-`C-C~AF]
The following tracers were also prepared in
accordance with the above procedures:
10 E~LE V - Sulfonylprimidone-aminofluorescein conjugates
H / O
c\ /CH2H5 1l
\N - C/ \/= ~So~AF]
H ~ O ~
- 22 - ~2~
EX2~IPLE VI - Para-methyl-meta-sulfonylprimidone-
aminofluorescein conjugate
,0
N C\ / C2H5
\ N C / ~ AP]
CH3
E~lPLE VII - Para-methyl-meta-sulfonylphenobarbital-
aminofluorescein conjugate
H~~0
\ /
O=C C
\N '~/ \ O
~A F ]
\ o
CH3
EX~PLE VIII - 5-Sulfonylphenyl-5-ethylhydantoin-
aminofluorescein conjugate
H~ ll
N-- \ ~ 2 C 3 1l
~C ~S ~A F ]
- 2 3 - ~.2~ 3$
EXAMPLE IX - a- CSulfonylphenyl~-a-methylsuccinimide-
aminofluorescein conjugate
o
O CH ll
--I ~& S~A F ]
~C--CH2
E$Q~PLE X O-sulfonamidocarbonylpropranolol-
aminofluorescein conjuqate
/~ .
A H O H O
~-O--CH2-C-O--C--N-S~A F]
CH2--N--C-H
CH3
- - 24 -
E.~MPLE XI - N-sulfonamidocarbonyl-iminostilbene- -
aminofluorescein conjugate
~0~0
¢ N-C-N-S-~AF]
/~
E~P~E ~II - N-sulfonamidocarbonyl-procainamide-
aminofluorescein conjugate
H_C2 H O H O H O
'\ I 11 ~ I 11 1 11
N-CH2CH2-N-C~-N--C-N--S~AF]
H5C2 o
E ~MPLE XIII - O-sulfonamidocarbonyl-chloramphenicol-
aminofluorescein conjugate
H H O H O
~ I 1 11 1 11
02N~-C'`C-CH2-0--C-N--S~AF]
H Ç-CHC12
Il
o
- 25 -
~LE XIV - N-sulfonamidocarbonyl-disopvramide-
aminofluorescein conju~ate -
~ ~ H O H O
CH3
N-CH CH -C-C-N-C~N-S-~F]
C\3 / 2 2 1
C~3
EX~`lPLE XV - O-sulfonamidocarbonyl-quinidine-
5aminofluorescein conjugate
H
~J~CH=CH2
~1 '
\ ~ O H O
~ 11 1 11
OCH3 H-C-O-C-N-S-~AF]
~3
~ - 26 ~
EX~LE .~I - O-oxalyl-propranolol-
aminofluorescein conjugate _ -
H O O
~-OCH2-C-O-C-C~.4P ]
CH2-N-C-H
I \
H CH3
E~MPLE XVII - N-oxalYl-procainamide-
5 _ aminofluorescein conjugate
H5C~2
\H O X O O
I ~ ~ 1 11 Il'
N--CH2CH2-N-C~N-C-C~AF ]
H5C2
~'~LE XVIII - N-acetyl-N'-desethyl-N~-oxalyl-
~rocainamide-aminofluorescein conjugate
-
~C-N~--C--N- CH 2 CH2--N/
CH3 C- IC~I P ]
O O
- 27 - ~2
E~A~LE XIX - N-oxalyl-nortriptyline-
aminofluorescein conjugate
=CHC~2-CH -N <
~ AF]
E.~E ~Y - N-oxalyl-iminodibenzyl-
5aminofluorescein conjugate
~00
~ 11 11
N-C-C-~A~]
- 28 - 32~
As previously mentioned, the tracers of the
oresent invenkion are effective reagents for use in
fluorescence polarization immunoassays. The following
Examples illustrate the suitability of tracers of the present
invention in immunoassays employing fluorescence polarization
techniques Such assays are conducted in accordance with
the following 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
resent invention optionally containing a surfactant is
ihen 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) The amount of tracer bound to antibody is
~easured by fluorescence polarization techniques as a
measure of the amount of ligand in the sample.
~PLE XXI - Lidocaine Assay
~aterials:
1~ Buffer: O~lM phosphate, pH 7~5, containing
0.01% (weight/volume) sodium azide and 0~01% ~weight/volume)
bovine gamma globulin (hereinafter referred to as "BGG
burfer")
2) Tracer: 5ulfonyllidocaine-aminofluorescein
conjugate (prepared in Example I) at a concentration of
2.34 ~ 10 7M in ORlM tris hydrochloride buffer, pH 7.8,
containing 0.1~ ~weight~volume) sodium dodecyl sulfate,
0.01~ (weight/volume~ bovine gamma globulin and O.Q1%
(weight/volume) sodium azide
3) Antibody: Ra~bit antiserum to lidocaine
diluted 1 to 80 :in BGG buffer.
4) Standards or unknowns: Human serum (or other
biological fluid~ containing lidocaine in a concentration
range of 0 to 10 ug/ml.
- 29 ~ 6
5) Fluorescence ~olarimeter: Instrument capahle
of measuring the polarization of fluorescence of a 1 X la M
fluorescein solution to + 0.001 polariaztion units,
Protocol:
1) To 20 ~Q of standards and unknowns add 20a ~Q
of BGG buffer.
2) To 20 yQ of each diluted standard and unknown
in a culture tube, add 2~0 ~Q of BGG buffer.
3~ To each culture tube containing diluted
10 standard and unknown add 40 ~Q of tracer and 1000 ~Q of
BGG bu~fer.
4) Then add 40 I~Q of antibody and 1000 ~Q of
BGG buffer to each culture tube.
5) ~ix the reagents and incubate the culture tubes
containing standards and unknowns for approximately 15
minutes at 23 C.
6) Measure the fluorescence polarization of all
tubes. Typical results for standard samples are presented
in Table I.
TABLE I
Lidocaine Concentration ~g/ml) Polarization
0.224
0-5 0.186
1.0 0.162
2.5 0.124
5.0 0.094
10.0 0.071
The polarization values decreases as the
concentration of lidocaine is increased, allowinq construction
of a standard curve. Unknowns treated in an identical manner
may be quantitated by reference to the standard curve.
- 30 - ~ 2~
EXAMPLE X~YII - Phenobarbital Assay
Materials:
1) BGG buffer
2) Tracer: ~para-methyl-meta-sulfonyl~phenobarbital-
aminofluorescein conjugate ~prepared in Example VII) at aconcentration of 5.75 x 10 8M in 5.75~ sodium cholate.
3) Antibody: Rabbit antiserum to phenobarbital
diluted 1 to 78.3 in BGG buffer.
4) Standards or unknowns: Human serum ~or other
biological fluid) containing phenobarbital in a concentration
}ange of 0 to 80 ~g/ml.
5) Fluorescence polarimeter: Instrument capable
or measuring the polarization of fluorescence of a 1 X 10 ~M
fluorescein solution to + 0.001 polarization units.
Protocol:
1) To 20 y~ of standards and unkno~ns add 20Q ~Q
of BGG buffer.
2) To 20 ~Q of each diluted standard and unknown
in a culture tube, add 2Q0 ~Q of BGG buffer.
3) To each culture tube containing diluted
standard and unknown add 40 ~Q of tracer and lQ00 ~IQ of
BGG buffer.
4) Then add 40 ~Q of antibody and 1000 ~Q of
BGG buffer to each culture tube.
5) Mix the reagents and incubate the culture tubes
containing standards and unknowns for approximately 15 minutes
at 23 C.
6) L~easure the fluorescence polarization of all
tubes. Typical results for standard samples are presented
in Table II.
~c~
- 31 -
TABLE II
Phenobarbital Concentration (~g/ml~ Polarization
.
0 0.155
O.lQ8
520 0.~92
0.074
0.060
The polarization values 2ecrease as
the concentration of phenobarbital is increased, allowing
10 construction of a standard curve. Unknowns treated in an
identical manner may be quantitated by reference to the
standard curve.
E.~LE ~XIII - Carbamazepine Assay
~aterials:
151) BGG buffer.
2) Tracer: N-oxalyliminostilbene-aminofluorescein
conjugate ~prepared in Example IV~ at a concentration of 2nM
in BGG buffer containing 0~2~ ~weight~volumel sodium cholate.
3~ Antibody: Rabbit antiserum to carbamazepine
20 diluted 1 to 3040 in BGG buffer.
4) Standards or unknowns: Human serum Cor other
biological fluid) containing carbamazepine in a concentration
range of 0 to 20 ug/ml.
5) Fluorescence polarimeter: Instrument capahle
25 of measuring the polarization of fluorescence of 1 X 10 ~!~
fluorescence solution to + 0.001 polarization units.
Protocol:
1~ To lO ~ of standards and unknowns add 300 ~Q
of BGG buffer.
2) To L0 ~Q of each diluted standard and unknown
in a culture tube, add 300 ~Q of BGG buffer.
3) Add 1 ml of tracer to each culture tube.
~!2~ $
- 32 -
4) Then add 1.0 ml of antihody to each culture
tube.
5) ~ the reagents and incubate the culture tubes
containing standards and unknowns for approximately 15 minutes
at 23 C.
6~ Measure the fluorescence polarization of all
tubes. Typical results for standard samples are presented
in Table III.
10TABLE III
Carbamazepine Concentration ~ug/mllPolarization
0.22~
2 0.108
4 0~135
8 0.105
12 0.088
0.077
The polarization values decrease as the
concentration of carbamazepine is increased, allowing
construction of a standard curve. Unknowns treated in an
identical manner may be quantitated by reference to the
standard curve.
The following tahle summarizes the various
}luorescence polarization immunoassays that have been carried
out in accordance with the above-described procedures employing
tracers prepared in the preceding Examples. The tracers
employed are identified by Example number and the specific
ligand~s) determined are indicated.
~ 33
Tracer Prepared
In Example Number LigandLsl A~ayed
I Lidocaine
II Phenobar~ital
III Ethosuximide
IV Carbamazepine
V Primidone
VI Primidone
VII P.heno~arbital
10VIII Phenytoin
IX Ethosuximide
X Propanolol
XI Carbamazepine
XII Procainamide
15XIII Chloramphenicol
XIV Disopyramide
XV Quinidine
XVI Propanolol
XVII Procainamide
20XVIII N-~cetyl ~rocainamide
XIX DesiPramine; Imipramine
XY Nortriptyline;Amitriptyline
_ 34 _ ~ 6
As evident from the above results, ~he tracers
of the present invention are effective reagents in
fluorescence polarization immunoassays. In addition to the
properties mentioned above, the tracers of the present
invention possess a high degree of thermal stability, a high
degree of bound polarization, high quantum yields and are
relatively easy to produce and purify.
~ lthough this invention has been described ~ith
respect to specific modifications, the details thereof are
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
t~ereof and it is understood that such equivalent embodiments
are intended to be included therein.
