FR2500165A1 - METHOD AND REAGENTS OF POLARIZATION IMMUNODETERMINATION OF FLUORESCENCE USING CARBOXYFLUORESCEINES - Google Patents

Abstract

THE INVENTION RELATES TO A METHOD AND REAGENTS FOR IMMUNODETERMINATION BY POLARIZATION OF FLUORESCENCE USING CARBOXYFLUORESCEINS; TO DETERMINE LIGANDS IN BIOLOGICAL LIQUIDS SUCH AS SERUM, PLASMA, CEPHALORACHIDIAN FLUID, AMNIOTIC FLUID AND URINE, A NEW CATEGORY OF TRACERS ARE USED ACCORDING TO A PROCEDURE OF IMMUNODETERMINATION BY POLARIZATION QUIU COMICIFICATION IMMUNODETERMINATION AT THE SPEED AND CONVENIENCE OF FLUORESCENCE POLARIZATION TECHNIQUES TO DETERMINE THE QUANTITY OF A PARTICULAR LIGAND PRESENT IN A SAMPLE.

Description

The present invention relates to a method and reagents

  Fluorescence polarization immunoassay

  using carboxyfluoresceins.

  More particularly, the invention relates to a method and reagents for determining ligands in biological fluids, such as serum, plasma, cerebrospinal fluid, amniotic fluid and urine. In particular, the invention relates to fluorescence polarization immunoassay and tracers used as reagents in this assay. The fluorescence polarization immunoassay method of the invention combines the specificity of an immunodetermination with the speed and convenience of fluorescence polarization techniques to allow the determination of the amount of a particular ligand present in

a sample.

  The competitive binding immunoassays for measuring ligands rely on the competition between a ligand contained in a sample and a labeled reagent called a tracer.

  to a limited number of receptor sites for

  specific bodies of the ligand and tracer. The concentration of the ligand

  in the sample determines the amount of tracer fixed

  only to an antibody. The amount of tracer-antibody conjugate

  produced can be measured quantitatively and is inversely

  proportional to the amount of ligand present in the sample.

  In general, fluorescence polarization techniques are

  The invention is based on the principle that a fluorescently labeled compound, when excited by linearly polarized light, emits fluorescence with a degree of polarization inversely related to its rotational speed. Thus, when a molecule, such as a tracer-antibody conjugate, having a fluorescent label, is excited by a linearly polarized light, the emitted light remains strongly polarized because the rotation of the fluorophore is limited between the moment when the light is absorbed and the one where it is emitted. When a "free" (i.e., non-antibody-bound) tracer is excited by linearly polarized light, its rotation is much faster than that of the corresponding tracer-antibody conjugate and the molecules are oriented at the same time. by chance, so that the light emitted is depolarized. Thus, the polarization of fluorescence provides a quantitative means of measuring the amount of tracer-antibody conjugate produced in an immunoassay

by competitive fixation.

  Various fluorescent labeled compounds are known in the art. U.S. Patent No. 3,998,943 describes the preparation

  of a fluorescent labeled insulin derivative using isothiol

  fluorescein cyanate (FITC) as a fluorescent marker and a fluorescently labeled morphine derivative using 4-amino fluorescein hydrochloride as a fluorescent marker. We also

  used carboxyfluorescein for analytical determinations.

  R. F. Chen, in Anal. Lett., 10, 787 (1977) describes the use of

  carboxyfluorescein to indicate the activity of phospholipase.

  However, the carboxyfluorescein is not conjugated as according to the invention, it is encapsulated in lecithin liposomes and becomes fluorescent only when it is released by hydrolysis

lecithin.

  The invention comprises a method for determining ligands in a sample which comprises mixing with said sample a biologically acceptable salt of a tracer of the formula:

O O

I R-C

\

  R is a ligand analog having a single primary or secondary reactive amino group which is attached to the carbonyl carbon of the

  carboxyfluorescein, said ligand analogue having at least one epitope

  common with said ligand so as to be specifically recognized

  born by a common antibody; and an antibody capable of specifically recognizing said ligand

  and said tracer; then to determine the amount of tracer-conjugate

  antibody according to fluorescence polarization techniques to be a measure of the concentration of said ligand in the sample. The invention further relates to certain novel tracers and their biologically acceptable salts, which are useful as reagents in the previously described method. Processes and

  tracers of the invention are particularly useful for monitoring

  launches quantitative drug concentrations in serum

and plasma.

  The invention will now be described in detail.

  In the present description, the term "ligand" refers to

  a molecule, particularly a low molecular weight hapten having a single reactive amino group, to which a receptor, normally an antibody, can bind or bind. These haptens are non-protein-containing bodies, usually of low molecular weight, that do not induce antibody formation when injected

  to an animal but react with the antibodies. We form genera-

  antibodies to a hapten by first conjugating the hapten to a protein and then injecting the conjugate

  animal. The antibodies produced are isolated according to standard techniques.

isolation of antibodies.

  The ligands that can be determined according to the process of the invention have a molecular weight that varies over a wide range. Although high molecular weight ligands can be determined, it is generally preferred for best results to employ the methods of the invention to determine low ligands.

  molecular weight generally in a range of 50 to 4000.

  It is particularly preferred to determine ligands having a weight

  molecular weight range from 100 to 2000.

  The novel tracers of the invention include compounds of formula (I) wherein the ligand analog represented by R comprises radicals having a molecular weight in the range of 50 to 4000. Preferred novel tracers include formula (I) wherein the ligand analogs represented by R include radicals having a molecular weight

  located in a range of 100 to 2,000.

  Typical examples of ligands having a single reactive amino group that can be determined according to the methods of the invention include steroids, such as estrone, estradiol,

  cortisol, testosterone, prdgesterone, chenodeoxy acid

  cholic, digoxin, cholic acid, digitoxin, deoxycholic acid, lithocholic acids and esters and amides derived therefrom; vitamins such as vitamin B-12, acid

  folic; thyroxine, triiodothyronine, histamine, serotonin

  nine, prostaglandins such as PGE, FGF and PGA; drugs

  anti-asthma drugs such as theophylline, antineoplastic

  such as doxorubicin and methotrexate, drugs

  antiarrhythmics such as disopyramide, lidocalin, procaine

  mide, propranolol, quinidine, N-acetyl procainamide; anticonvulsant drugs such as phenobarbital, phenytoin, primidone, valproic amide, cailbamazepine and ethlosuximide; antibiotics such as penicillins, cephalosporins and vancomycin; anti-arthritic drugs such as salicylate; antidepressant drugs, including tricyclic compounds, such as nortriptyline, amitriptyline, imipramine and desipramine; and the like as well as their metabolites. Other ligands that can be determined according to the methods of the invention include drugs causing addiction such as morphine, heroin, hydromorphone, oxymorphone, metapon, codeine, hydrocodone, dihydrocodeine, dihydrohydroxycodéinone, pholcodine, dextromethorphan, phenazocine and deonine, and their metabolites. The tracers of the invention generally exist in equilibrium between their acid state and their ionized state and, in the ionized state, they are effective in the method of the invention. The invention thus includes tracers in the acidic or ionized state and, for simplicity, the tracer structure of the invention is shown in the acid form. When the tracers of the invention are present in their ionized state, they exist in the form of biologically acceptable salts. The term "biologically acceptable salts" is intended here to mean salts such as the sodium, potassium, ammonium and similar salts which make it possible for the tracers of the invention to exist at

  the ionized state when employed in the process of the invention.

  Generally, the tracers of the invention exist in solution in the form of salts, the particular salt corresponding to the buffer used, that is to say that, in the presence of a sodium phosphate buffer, the tracers of the invention usually exist in the ionized state

in the form of a sodium salt.

  The tracers of the invention comprise a ligand analog represented by R bonded to a carboxyfluorescein moiety of the formula OH

-C

\ O (II)

Luke \

O OH

  The term ligand analogue as used herein refers to a mono or polyvalent radical of which a significant proportion has the same spatial and polar organization as the ligand to form one or more critical or epitopic sites capable of competing with the ligand vis-à-vis receptor binding sites. A feature of such a ligand analog is that it has sufficient structural similarity with the ligand of interest to be recognized by an antibody directed against the ligand. For the most part, the structure and distribution of loads (spatial and polar organization) of. the ligand analogue are the same or substantially the same as those of the ligand of interest in a substantial portion of the molecular surface. As frequently the hapten binding site for preparing the antigen for the production of antibodies is the same as the ligand binding site, the portion of the ligand analogue that is the target of the antibody is the same as the one

  exposed by the ligand analog in the tracer.

  In general, the category of ligand analogs represented by R are derived from the corresponding ligand by the removal of a reactive hydrogen atom, i.e. from a hydrogen atom attached to a reactive amine. (primary or secondary) or by formation of an amino derivative of the ligand o a radical 1ino

H

  replaces one or more atoms originally present in the ligand at the binding site with the carboxyfluorescein moiety. Examples of ligands which, by removal of a reactive hydrogen, can form the ligand analogs represented by R, are procalinamide, thyroxine and quinidine. Examples of ligands whose amino derivatives are useful as ligand analogues are theophylline, valproic acid, phenobarbital, phenytoin, primidone, disopyramide, digoxin, chloramphenicol, salicylate, acetaminophen, carbamazepine -, desipramine and nortriptyline. In addition, the structure of a ligand can be modified by adding or removing one or more functional groups

  to form a ligand analogue while maintaining the epitope sites

  topical agents necessary for binding with an antibody. However, these

  modified ligand analogues are attached to the carboxy

fluorescein by an imino radical.

  The tracers of the invention are generally prepared according to known techniques. For example, a compound of the formula R - X (IIi) is reacted with the same definition as above and X is a reactive hydrogen, with a compound of the formula: ## STR2 ##

/.

O OH

  Where R represents a hydroxy or an active ester, and where the carboxy radical is preferably attached to the 4 or 5 position of the benzoic acid ring,

  in the presence of an inert solvent to form a compound of formula (I).

  By "active ester" is meant herein a fragment that is easily "removed" from the carboxylic carbon in the presence of a coupling agent. These "active esters" of carboxyfluorescein are readily determined by those skilled in the art and are prepared by

  reaction of carboxyfluorescein with a compound such as N-

  hydroxysuccinimide, hydrated 1-hydroxybenzotriazole or p-nitro-

  phenol in the presence of a coupling agent such as dicyclohexyl-

  carbodiimide and a solvent. The active esters of carboxyfluorescein thus produced are then reacted with a compound of

  formula (III) to obtain a tracer of formula (I).

  If the compound of formula (III) is soluble in water, the reaction mechanism is carried out by direct reaction of carboxyfluorescein with a compound of formula (III) in aqueous solution in the presence of a water-soluble carbodiimide such as 1-ethyl (3-dimethylamino-3-propyl) carbodiimide hydrochloride

as a coupling agent.

  The temperature at which the process for preparing the tracers of the invention is practiced has no particular limitation. The temperature must be sufficient to initiate and maintain the reaction. Generally, for reasons of convenience and economy, the ordinary temperature is sufficient. To prepare the tracers of the invention, the ratio of the reagents is not critical. For each mole of a compound of formula (I), 1 mole of a compound of formula (III) must be used to obtain a reasonable yield. It is preferred to use an excess of the compound of formula (III) to facilitate reaction and recovery of the reaction products. To facilitate handling and recovery of the products, the method of preparing the tracers of the invention is practiced in the presence of an inert solvent. Suitable inert solvents include those solvents which do not react with the starting materials and which dissolve them and consist for example of water (if the compound of formula (III) is soluble in

  water), dimethylformamide, dimethyl sulfoxide and the like.

  If the compound of formula (III) is a reactive amine salt, an appropriate base is added to the reaction mixture to form the free base of the reactive amine. Suitable bases include, for example, triethylamine. The reaction products of formula (I) are generally purified by thin-layer or column chromatography.

  before use in the methods of the invention.

  According to the method of the invention, a sample containing the ligand to be determined is mixed with a biologically acceptable salt of a tracer of formula (I) and an antibody specific for the ligand and the tracer. The ligand present in the sample and the tracer compete with the limiting sites of the antibody,

  which causes the formation of ligand-antibody complexes and tracer-

  antibody. When the concentration of tracer and antibody is kept constant, the ratio of ligand-antibody complex to

  tracer-antibody complex is directly proportional to the

  ligand present in the sample. So, when we excite

  mixing with polarized light and measuring the polari-

  of the fluorescence emitted by a tracer and a tracer antibody complex, the quantity can be determined quantitatively.

  of ligand present in the sample.

  In theory, the polarization of the fluorescence of a

  tracer uncomplexed with an antibody is weak and close to zero.

  After complexing with a specific antibody, the tracer antibody complex thus formed acquires the rotation of the antibody molecule which is smaller than that of the relatively small molecule of tracer, thereby increasing the observed polarization. Thus, when a ligand competes with the tracer against the sites of an antibody, the observed polarization of the fluorescence of the tracer-antibody complex has a value between that of the tracer and that of the tracer antibody complex. If a sample contains a high concentration of the ligand, the observed value of the polarization is closer to that of the free ligand, i.e., is low. If the sample contains a low concentration of the ligand, the value of the polarization is closer to that of the attached ligand, i.e. is high. When successively excites the reaction mixture of an immunodetermination with a light

  polarized vertically then horizontally and analyzed

  the vertical component of the light emitted, it is possible

  to precisely reduce the polarization of the fluorescence of the mixture

  reaction. The precise relationship between polarization and concentration

  of the ligand to be determined is established by measuring the values of

  the polarization of standards of known concentrations. We can

  to polish the ligand concentration from a standard curve

thus prepared.

  The pH at which the process of the invention is practiced must be sufficient for the tracers of formula (I) to exist in the ionized state. The pH can be between about 3 and 12 and more

  generally in the range of about 5 to 10 and more preferably about 6 to 9.

  Various buffers can be used to obtain and maintain the pH during the determination. Examples of buffers are borate, phosphate, carbonate, tris, barbital and the like. The particular buffer used has no limitations in the invention, but for a particular determination, a buffer may be preferred because of the antibody used and the ligand to be determined. The cationic portion of the buffer generally determines the cationic portion

tracer salt in solution.

  The processes of the invention are practiced at moderate temperatures, preferably at a constant temperature. The temperature is normally between about 0 and 500C and more preferably between about 15 and 40C. The concentration of ligand that can be determined generally ranges from about 10 to 10 M and more generally from about 10 to 10 M. Higher concentrations of the ligand can be determined by dilution of the original sample. In addition to the range of concentrations of the ligand of interest, such considerations as the qualitative, semi-quantitative or quantitative nature of the determination, the apparatus

  used and the characteristics of the tracer and the antibody deter-

  Normally, the concentration of the tracer and the antibody used is mined. Although the concentration of the ligand in the sample determines the concentration range of the other reagents, i.e., the tracer and the antibody, normally, to optimize the sensitivity of the determination, the concentrations should be empirically determined. individual reagents. Tracer and antibody concentrations are easily determined by humans

art.

  As previously indicated, the preferred tracers of the invention are prepared from 5-carboxy fluorescein or 4-carboxyfluorescein or their repeats and are represented by formulas I Z-C

\ / \ OV

/

  or OH (VI) The following illustrative and nonlimiting examples are intended to demonstrate to one skilled in the art how one can

  prepare particular tracers within the scope of the invention.

  tion. The symbol (CF) which appears in the developed formulas

  illustrating the compounds prepared in the following examples

  contains a moiety of the formula: ## STR2 ## wherein the carbonyl radical is attached to the 4- or 5-position of the formula by using as a starting material a mixture of

carboxy-4 and -5 fluorescdines.

EXAMPLE 1

  5 mg of m- or p-aminophenobarbital and 5 mg of carboxyfluorescein are dissolved in 0.5 ml of pyridine. To the mixture is added N, N'-dicyclohexylcarbodiimide. The reaction is carried out for 2 hours at room temperature and then the reaction product is twice purified by silica gel bed chromatography with a 2/1 mixture of chloroform and methanol as solvent.

  development to obtain an aminophenobarbital-carboxy-

fluorescein of formula: i // 0

/ N-C \ / H2CH3

0 = C C

I H

N C,

/ N-4YCF)

H 0

EXAMPLE 2

  A solution containing 1.0 g of sodium hydroxide, 2.5 g of phenytoin and 2.0 g of bromo-2-methylamine hydrochloride in 100 ml of 100% ethanol is refluxed for 2 hours and then evaporated. dry under reduced pressure. The residue is suspended in 50 ml of water and the pH adjusted to 11 by addition of 6 N sodium hydroxide to dissolve any unreacted phenytoin. The remaining precipitate of P-aminoethyl is filtered off. Rhenytcrlo, rinsed thoroughly with water and dried, prepared an active ester of carboxyfluoresceine by

  dissolution of 5 mg of N-hydroxysuccinimide, 7.5 mg of carboxyfluores-

  cetin and 20 mg of N, N'-dicyclohexylcarbodiimide in O0.5 ml of

  pyridine. The reaction is allowed to proceed for 2 hours at room temperature.

  After the addition of 10 mg of p-aminothyl-2-phenylene is dissolved in the reaction mixture. The resulting mixture was allowed to react overnight in the dark at room temperature and the reaction product was purified twice by silica gel thin layer chromatography with a 3/1 mixture of chloroform and methanol as the developing solvent for the reaction mixture. obtain a conjugate 9-aminoethyl-2 phenyto'inecarboxyfluorescein of formula: H

X / C N-CH2CH2-N-4CF)

C

C C \ C 2

\NOT

\ I H O

EXAMPLE 3

  A solution containing 620 mg of 2-carboxymethyl phenytol is left to stand at room temperature overnight.

  248 mg of N-hydroxysuccinimide and 453 mg of N, N'-dicyclohexylcarbonyl

  diimide in 6 ml of anhydrous dimethylsulfoxide. The mixture is filtered and 0.7 ml of 95% hydrazine is added to 4.5 ml of the filtrate. After 4 hours at room temperature, 40 ml of water and 0.5 ml of 10% sodium hydroxide are added to the reaction mixture. The precipitate consisting of 2-carboxymethyl phenytoinhydrazide is filtered, rinsed &

  water, dried and used without further purification.

  15 mg of N, N'-dicyclohexylcarbodiimide are added to a solution of 5 mg of 2-carboxymethyl phenytoin hydrazide and 5 mg of carboxyfluorescein in 0.5 ml of pyridine. The reaction is allowed to proceed for 2 hours at room temperature, and then the reaction product is purified twice by silica gel thin layer chromatography with a 1: 1 mixture of chloroform and acetone as developing solvent to obtain the reaction product. a carboxymethyl-2-phenytol-hydrazide-carboxyfluorescein conjugate of formula:

C-N-CH2-C-N-N-4CF)

C / N

H O

EXAMPLE 4

  15 mg of N, N'-dicyclohexylcarbodiimide are added to a

  solution of 5 mg of 5-aminoethyl-8 theophylline and 5 mg of carboxy-

  fluorescein in 0.5 ml of pyridine. The reaction is allowed to proceed for 2 hours at room temperature and then the reaction product is purified twice by thin layer chromatography on silica gel with a 2/1 mixture of chloroform and methanol as developing solvent to obtain a conjugate. P-aminoethyl-8-thiophyllinecarboxyfluorescein of formula:

OH

0 - H

CH3 H

t / '"' CH2CH2 N" <CF) O E Nv '> N

CH3

EXAMPLE 5

  The procedure of Example 4 is repeated using 8-aminomethyl theophylline instead of 8-aminoethyl-8-theophylline to obtain an 8-amino-thdophlylline-carboxyfluorescent conjugate of formula: embedded image

OH

He I

CH C N H

A 11 tCH2-N- "CF)

CH3

EXAMPLE 6

  7.5 g of α-valerolactam are dissolved in 60 ml of anhydrous tetrahydrofuran under a dry nitrogen atmosphere and 90 ml of 1,6 M butyl lithium are added dropwise to the reactor cooled with a bath of dry ice and acetone. in hexane. After completion of the addition of the butyllithium, the reaction mixture is stirred at room temperature for 1 hour, refluxed for 30 minutes and then cooled to room temperature under a dry nitrogen atmosphere. 8.0 g of 1-bromoethane are slowly added to the reactor while cooling with an ice-bath. The mixture is then stirred for 16 hours at room temperature and then 100 ml of water are slowly added. The resulting mixture is stirred at room temperature for 30 minutes and the organic layer is separated. The organic layer is extracted with 50 ml of ethyl ether,

  the organic layers are combined and dried over sodium sulfate.

  The solvent is evaporated to give a dark oil which crystallizes on standing. The crystalline residue is recrystallized from petroleum ether to give 3.8 g of a residue. The residue (2.8 g) is refluxed in 25 ml of 6N hydrochloric acid for 6 h. The water of the mixture was evaporated to give a thick dark oil of 2-ethyl-5-amino-pentanoic acid which was used without further purification. An active ester of carboxyfluorescein is prepared by

  dissolution of 5 mg of N-hydroxysuccinimide, 7.5 mg of carboxyfluores-

  cetin and 20 mg of N, N'-dicyclohexylcarbodiimide in 0.5 ml of pyridine.

  The reaction is allowed to proceed for 2 hours at room temperature.

  then 20 mg of 2-ethyl-5-amino-pentanoic acid are dissolved in the reaction mixture. The resulting mixture was allowed to react overnight in the dark at room temperature and the reaction mixture was purified twice by thin layer chromatography on silica gel using a 3/1 mixture of chloroform and methanol as the developing solvent. to obtain a conjugate ethyl 2-amino-5-pentanoic-carboxyfluorescein of formula:

H H

!!

CH3CH 2 -CH2CH2CH2-N-CF)

C = O

OH

EXAMPLE 7

  An active ester of carboxyfluorescein is prepared

  by dissolving 5 mg of N-hydroxysuccinimide, 7.5 mg of carboxy-

  fluorescein and 20 mg of N, N'-dicyclohexylcarbodimide in 0.5 ml of pyridine. The reaction is allowed to proceed for 2 hours at room temperature and then 5-dibenzo [a, d] dihydro-10,11-cycloheptene is dissolved in the reaction mixture (5-aminopropylidene). The mixture is allowed to react overnight in the dark at room temperature and the reaction product is purified twice by silica gel thin layer chromatography using a 3/1 mixture of chloroform and methanol as the developing solvent for the reaction mixture. obtain a (γ-aminopropylidene) -5H-dibenzo [a, d] dihydro-10, 11-cycloheptenecarboxyfluorescein conjugate of formula: C = CHC22H2H2 T, = -4CF)

EXAMPLE 8

  Refluxed for 2 hours a solution containing

  1.33 g of desipramine hydrochloride and 0.8 g of chloro

  acetyl in 25 ml of chloroform. The chloroform is evaporated and the residue is dissolved in 25 ml of acetone. 0.75 g of sodium iodide is added to the acetone solution and the solution is refluxed for 30 minutes. The solution is filtered and the precipitated salt is rinsed with acetone. The acetone filtrate is evaporated and the residue is taken up in 20 ml of methanol. 20 ml of concentrated ammonium hydroxide are added to the methanol solution and the resulting solution is refluxed for 1 hour. The reaction mixture is extracted three times with 25 ml of chloroform and the combined extracts are dried over sulfate.

  of sodium, filtered and evaporated to give N-aminoacetyl-

  desipramine that is used without further purification.

  5 mg of N-aminoacetyldesipramine and 5 mg of carboxyfluorescein are dissolved in 0.5 ml of pyridine. To the mixture is added N, N'-dicyclohexylcarbodiimide. The reaction is carried out for 2 hours at room temperature and then the reaction product is purified twice by silica gel thin layer chromatography with a 1: 1 mixture of chloroform and acetone as the reaction mixture.

  developing solvent to obtain an N-aminoacetyldehyde conjugate.

  pramine-carboxyfluorescein of formula:

/ <\ CH3

-CH CH CH -N-C-CH NH- * CF)

EXAMPLE 9

  Allowed to react at room temperature for 4 hours

  a solution containing 5 mg of N-hydroxysuccinimide, 7.5 mg of carboxy-

  fluorescein and 20 mg of N, N'-dicyclohexylcarbodiimide in 1 ml of pyridine. An active ester of carboxyfluorescein is precipitated by addition of 10 ml of ethyl ether to the reaction mixture. The precipitate is filtered, rinsed thoroughly with ethyl ether and redissolved in 0.5 ml dimethylsulfoxide. 10 mg of L-thyroxine are then added to the solution and the reaction is allowed to proceed for 2 hours at room temperature and then the reaction product is purified twice by thin layer chromatography on silica gel with a 3/1 mixture. chloroform and methanol as solvent

  to obtain an L-thyroxine-carboxyfluorescent conjugate.

Formula: I! 0

II O

HO O CHC-N-4CF)

H

EXAMPLE 10

  A solution containing 0.89 g of ammonium acetate, 389 mg of 3-oxo-digoxigenin and 63 mg of sodium cyanoborohydride in 5 ml of methanol is stirred at room temperature for 48 hours. The pH of solution A 1 is adjusted by addition of acid

  concentrated hydrochloric acid and evaporated to dryness under reduced pressure.

  The residue is taken up in 10 ml of water and extracted three times with ml of chloroform. The pH of the aqueous layer is adjusted to 11 with solid potassium hydroxide. The resulting solution is extracted five times with 10 ml of methylene chloride. The organic layers are combined, dried and then evaporated to dryness under reduced pressure to give 3-amino-3-deoxy digoxigenin which is used without

other purification.

  An active ester of carboxyfluorescein is prepared by

  dissolution of 5 mg of N-hydroxysuccinimide, 7.5 mg of carboxyfluores-

  and 20 mg of N, N'-dicyclohexylcarbodiimide in 0.5 ml of

  pyridine. The reaction is allowed to proceed for 2 hours at room temperature.

  and then isolating a conjugate of 3-amino-3-deoxy-digoxin

  genin-carboxyfluorescein of formula: OO

CH3

HH C3

OH H

N-ECF)

  The following tracers are also prepared according to the procedures previously described:

EXAMPLE 11

  O-aminoacetylpropanolol-carboxyfluorescein conjugate

OH

OCH CH-O-C-CH-N- (CF)

2 2

a) H2-N-CH

N H CH3

2500 165

EXAMPLE 12

  Propyl-2-amino-5-pentanoic-carboxyfluorescein conjugate

H H

I! CH 2 CH 2 CH 2 CH 2 CH-CH (CH)

3 2 Z, 222

* C = O

OH

EXAMPLE 13

  Butyl-2-amino-5-pentanoic acid-carboxyfluorescein conjugate

H H

o I

CH 2 CH 2 CH 2 -C CH 2 CH CH-N (CF)

C = O OH

EXAMPLE 14

  Aminoprimidone-carboxyfluorescein H conjugate

H CH2 CH3 H

/ N C / 3 N4CF)

CH / C

N x \

H O

EXAMPLE 15

  Conjugate (4-nitro-phenyl) -1-hydroxy-1-amino-2-hydroxy-3-propane-carboxy

fluorescein H

502N-CH-CH-N- (CF)

OH 1

CH2OH

EXAMPLE 16

  P-aminophenol-carboxyfluorescent conjugate sine H

HO-N-ECF)

EXAMPLE 17

  N- (2-aminoethyl) -ethosuximide-carboxyfluorescein conjugate

CH2. H

H N-CH2CH2-N- (CF)

CH3CH2 -C,

CH 0

EXAMPLE 18

  N'-desethyl N-acetyl procafamide-carboxyfluoresc-4 conjugate

O \\ H O H CH CH 3

C N-C-N-CH 2 CH 2 -N-4CF)

CH 3

EXAMPLE 19

  N-desethyl N-aminoacetyl N-actyl procainainide-carboxy-conjugate

fluorescent

O0 H 0 H CHCH H

2 3,

C'-NI-CH CH-N-C-CH 2 -N- "CF)

CH3

EXAMPLE 20

  1-amino-2-phenyl-2- (2-pyridyl) -4- (diisopropylamino) butane-1-conjugate

carboxyfluorescein H

CH2-N-4CF)

-10

EXAMPLE 21

  Triiodo-3,3 ', 5 L-thyronine-carboxyfluorescein I conjugate

O% OH

-CH2-C-N-4CF)

H H

EXAMPLE 22

  Tetraiodo-3,3 ', 5,5'-D-thyronine-carboxyfluorescein conjugate

0% / OH

C I H

CH2-C-N-4CF)

H

EXAMPLE 23

  Conjugate N-aminoacetyl iminodibenzyl-carboxyfluorescein CH3 H-C CH3 I HO, I

OH

N-C-CH2-N-4CF)

EXAMPLE 24

  Carbohydrazinoiminodibenzyl-carboxyfluorescein conjugate

0 H H

N-C-N-N- 'CF)

EXAMPLE 25

  Dibenzosuberonehydrazone-carboxyfluorescein H conjugate

C = N-N-ECF)

EXAMPLE 26

  5-amino-dihydro-10,11-5H-dibenzo (ad) cycloheptene-carboxy-5-conjugated conjugate

fluorescein C

N-4CF)

  As previously indicated, the tracers of the invention are effective reagents useful in fluorescence polarization immunoassays. The following examples illustrate the usefulness of the tracers of the invention in immunoassays.

  using fluorescence polarization techniques. These deter-

  The following general procedures are carried out: 1) a measured volume of standard or serum to be studied is introduced into a test tube and diluted with a buffer; 2) then adding to each tube a known concentration of a tracer of the invention optionally containing a surfactant;

  3) a known concentration of

  serum; 4) the reaction mixture is incubated at room temperature; and 5) the amount of tracer bound to the antibody is measured by fluorescence polarization techniques to measure the

  amount of ligand in the sample.

EXAMPLE 27

  Determination of phenytoin A) Materials required: 1) GGB buffer consisting of 0.1 M sodium phosphate, pH 7.5, containing 0.01% bovine ylobulin and 0.01% sodium azide.

  2) Tracer consisting of M-aminoethyl-2 phenytolene-carboxy-

  fluorescein at a concentration of about 105 nM in GGB buffer

  with addition of 5% sodium cholate.

  3) Phenytoin antiserum diluted appropriately in GGB buffer containing 0.005% benzalkonium chloride. 4) Samples of human serum or other biological fluid containing phenytoin. ) Cuvettes, glass culture tubes are used

10 x 75 mm as bowls.

  6) Fluorometer capable of measuring the polarization of the

  fluorescence with an accuracy of - 0.001 unit.

B) Method of determination:

  1) A small volume of sample is placed in each cuvette

  Ion (0.366 / μl) by pipetting 15 μl of sample and dilution with 600 μl of GGB buffer in a dilution vessel. Then, 15 μl of diluted sample is pipetted into the cuvette and then 600 μl of

GGB buffer.

  2) The tracer is added by pipetting 40 μl of

  tracer and 1000 / μl of GGB buffer in the cuvette.

  3) The antiserum is added to start the reaction by pipetting 40 μl of antiserum into the cuvette and then 1000 μl of

GGB buffer.

  4) Carefully mix the contents of all

  cuvettes and incubated for 15 min at room temperature.

  ) The polarization of the fluorescence is read with a fluorometer and a calibration curve is established to determine

unknown concentrations.

  C) The results of a series of standard sera containing phenytoin at concentrations between 0 and / ug / ml are shown below. Each concentration was determined

  in doubles and the average has been established.

  Concentration of phenytotine polarization (/ ug / ml)

O 0.222

2.5 0.196

0.178

10.0 0.154

, 0 0.132

> 0 0.110

  It can be seen that the polarization of the fluorescence decreases steadily as the phenytoin concentration increases, which makes it possible to construct a calibration curve. Quantitative analysis of identically treated unknown samples can be performed by reference to the standard curve, illustrating the utility of 5-amino-2-phenytoincarboxyfluorescein for

the measurement of phenytoin.

EXAMPLE 28

  Determination of phenobarbital A) Materials needed

1) GGB buffer (see phenytoin).

  2) Tracer consisting of aminophenobarbital-carboxyfluores-

  at a concentration of about 110 nM in tris-HCl buffer pH 7.5 containing 0.01% sodium azide, 0.01% y-globulins

  cattle and 0.125% sodium dodecyl sulphate.

  3) Phenobarbital antiserum diluted appropriately in GGB buffer containing 0.005% benzalkonium chloride. 4) Samples of human serum or other liquid

  containing phenobarbital.

) Cuvettes (see phenytoin).

6) Fluorometer (see phenytoin).

  B) Determination protocol: 1) Place a small sample volume (0. 196 / μl) in the cuvette by pipetting 10 μl of sample and dilution with 500 μl of GGB buffer in a dilution vessel. Then, 10 μl of diluted sample is pipetted and 500 μl

GCB buffer.

  2) The tracer is added by pipetting 40 μl of tracer

  and 1000 μl of CGB buffer in each cuvette.

  3) The antiserum is added to start the reaction by

  pipetting 40 μl of antiserum and 1000 μl of GGB buffer.

  4) Carefully mix the contents of all

  cuvettes and incubated for 15 min at room temperature.

  ) Fluorescence polarization is read on a fluorometer and a calibration curve is established to determine the

unknown concentrations.

  C) The results of a series of standard sera containing phenobarbital at concentrations between 0 and 80 μg / ml are given below. Each concentration was determined in duplicate

and the average has been established.

  Phenobarbital polarization concentration (/ ul)

0 0.250

0.231

0, 0, 196

0.150

0.104

80.0 0.077

  It can be seen that the polarization of the fluorescence decreases steadily as the phenobarbital concentration increases.

  allows to build a calibration curve. It can be quantified

  tatively unknown samples processed identically by reference to the calibration curve, illustrating the utility of

  aminophenobarbital-carboxyfluorescein for the measurement of

barbital.

EXAMPLE 29

  Determination of theophylline A) Materials needed:

  1) Tracer of 8-aminoethyl-theophylline-carboxy

  2 nM fluorescein in GGB buffer (see determination of

  phenytoin) containing 0.01% sodium dodecyl sulfate.

  2) Antiserum against theophylline, diluted

  appropriately in GGB buffer.

  3) Samples of human serum or other liquid

  containing theophylline.

  4) Cuvettes (see determination of phenytoin).

  ) Fluorometer (see determination of phenytoin).

250 0 165

  B) Protocol of determination: 1) Introduce 1, O / ul of tracer in all the cuvettes. 2) Introduce 2.0 μl of sample into all cuvettes. 3) Introduce 1.0 ml of antiserum into all cuvettes. 4) Mix thoroughly and incubate for 15 min.

Room temperature.

  5) Read the polarization of the fluorescence on a fluorescent

  meter and build a calibration curve.

  C) The results of a series of standard sera containing theophylline at concentrations between 0 and 40 μg / ml are given below. Each concentration was determined in duplicate

and the average was done.

  Concentration of theophylline polarization (/ ug / ml)

O 0.158

2.5 0.118

5 0.105

0.091

0,076

0,063

  It can be seen that the polarization of the fluorescence decreases steadily as theophylline concentration increases.

  allows to build a calibration curve. It can be quantified

  tatively unknown samples treated identically with reference to the standard curve, illustrating the utility of 8-aminoethyl-theophylline-carboxyfluorescein for the measurement of

theophylline.

EXAMPLE 30

  Determination of digoxin A) Materials required: 1) GB buffer consisting of 0.1 M sodium phosphate pH 7.5 containing 0.01% bovine yglobulin and 0.01% sodium azide. 2) Tracer consisting of digoxin-carboxyfluorescein at a concentration of about 2 nM in GGB buffer. 3) Rabbit antisdrum directed against digoxin, diluted

  suitably in GGB buffer.

  4) Samples of human serum or other liquid

  containing digoxin.

  ) Precipitating reagent: 5% trichloroacetic acid in water. 6) Cuvettes, culture tubes are used as cuvettes

in glass of 10 x 75 mm.

  7) Fluorometer capable of measuring the polarization of the

  fluorescence with a precision of 0.001 unit.

B) Protocol of determination:

  (1) In a test tube, 100 μl of trichloroacetic acid is added

  5% acetic acid 7. 100 μl of standard or unknown sample. The tubes containing the sample are capped and shaken to form a

vortex.

  2) Centrifuge tubes containing the standard or

  the sample in trichloroacetic acid.

  3) In a test tube containing 1.8 ml of GGB buffer and / ul of 35 C antiserum, 150 μl of the supernatant solution is added.

trichloroacetic acid.

  4) The test tubes containing the antiserum and the supernatant are incubated for 6 min at 35 ° C. and then the fluorescence polarization of the tubes is measured. This measure constitutes the polarization of

  basic fluorescence of standard or unknown sample.

  5) 10 min after the addition of the supernatant to the antiserum,

  25 μl of tracer are added to the test tube.

  6) 6 min after the addition of the tracer, measure the polarity

  fluorescence of the standard and sample tubes and subtract the baseline value from the polarization of the previously measured fluorescence to obtain the polarization of the fluorescence

  of the antibody-tracer complex formed.

  7) The results of a series of standard sera containing digoxin at concentrations between 0 and 5 ng / ml are given below. Four samples were analyzed for each

  concentration and averaged.

  Concentration of digoxin (ng / ml) O 0.5 1.0 2.0 3.0, 0 Polarization 0, 142 0.134 0.123 0.106 0.092 0.070 It can be seen that the polarization of fluorescence decreases steadily as the concentration of digoxin is increasing, which

  allows to build a calibration curve. It can be quantified

  tatively unknown samples processed identically by reference to the calibration curve, illustrating the utility of

  digoxin-carboxyfluorescein for the measurement of digoxin.

  The following table summarizes the various fluorescence polarization determinations that were carried out according to the procedures described above with tracers prepared in the preceding examples. The tracers used are identified by the number of the example and the particular ligands determined

are indicated.

Example n

5

10

  Ligands Phenobarbital Phenytoin Phenytoin Theophylline Theophylline Valproic acid Nortriptyline; amitriptyline Imipramine; Desipramine Thyroxine Digoxin

Example n

14

19

24

  Ligands Propanolol Valproic acid Valproic acid Primidone Chloramphenicol Acetaminophen Ethosuximide N-Acetylprocainamide N-acetylprocainamide Disopyramide Triiodothyronine Thyroxine Imipramine; desipramine Imipramine; desipramine Nortriptyline; amitriptyline Nortriptyline; Amitriptyline As the results above show, tracers

  of the invention are reagents effective in immunoassays for

  by fluorescence polarization. In addition to the aforementioned properties, the tracers of the invention possess a high degree of thermal stability, a high degree of polarization action, high quantum efficiencies and are relatively easy

to produce and purify.

  Of course, various modifications can be made by those skilled in the art to the devices or processes which have just been described as non-limiting examples.

  without departing from the scope of the invention.

2500 165

Claims (42)

R E V E N D I C A T IO N S   1. A method for determining ligands in a sample   characterized in that it consists in mixing with said sample a tracer of formula OH R-C 0 \ OH \   R represents a ligand analog having an amino radical   primary or secondary, reactive, single, fixed to a carbon   nyl of a carboxyfluorescein moiety, said ligand analog having at least one epitope common with said ligand so as to be specifically recognizable by a common antibody; and an antibody capable of specifically recognizing said ligand and said tracer; then to determine the amount of tracer bound to the antibody according to fluorescence polarization techniques   to measure the amount of ligand in the sample.   2. Method according to claim 1, characterized in   said ligand is a corresponding drug or metabolite.   3. Process according to claim 2, characterized in that the carbonyl radical attached to the radical R is also attached to   position 4 or 5 of the carboxyfluorescein moiety.   4. Method according to claim 3, characterized in that said drug is a sterotope, a hormone, an antiasthmatic,   an antineoplastic agent, an antiarrhythmic agent, an anticonvulsant, an   arthritis, an antidepressant, a glucoside acting on the heart or one of their metabolites.   5. Method according to claim 4, characterized in that   that R has a molecular weight in the range of 50 to 4000.   6. Process according to claim 5, characterized in that   that R has a molecular weight in the range of 100 to 2,000.   7. Process according to claim 6, characterized in that   that said drug is an anticonvulsant.   8. Process according to claim 7, characterized in that   that said anticonvulsant drug is phenobarbital.   9. Process according to claim 7, characterized in that   that said anticonvulsant drug is phenytoin.   10. Process according to claim 7, characterized in that   that said anticonvulsant drug is primidone.   11. The method of claim 6, characterized in that that said drug is a steron.   12. Process according to claim 11, characterized in that   that said sting is digoxin.   Method according to claim 6, characterized in that   that said drug is an antiarrhythmic.   14. The method of claim 13, characterized in that   that said antiarrhythmic drug is propranolol.   15. Process according to claim 6, characterized in that   that said drug is an antiasthmatic.   16. The method of claim 15, characterized in that   that said antiasthmatic drug is theophylline.   17. A compound characterized in that it corresponds to the formula OH 1.0A wherein R is a ligand analog having a primary amino group or   secondary, reactive, single, which is attached to a carbon-carbon   of a carboxyfluorescein fragment 2500 1 65   molecular weight in the range of 100 to 2,000; said ligand analog having at least one common epitope with a ligand so   to be specifically recognizable by a common antibody.   18. Compound according to claim 17, characterized in that the carbonyl radical attached to the radical R is also attached to the   position 4 or 5 of the carboxyfluorescein moiety.   19. Compound according to claim 18, characterized in that R is derived from a ligand chosen from steroids, hormones,   anti-asthmatics, antineoplastics, antiarrhythmic drugs,   convulsants, anti-arthritics, antidepressants, and cosides acting on the heart.   20. A compound according to claim 19, characterized in that   that R derives from an anticonvulsant.   21. Compound according to claim 20, characterized in that that R derives from phenobarbital.   22. Compound according to claim 21, characterized in that R corresponds to the formula: H O \ N _ C \ / CH2CH3 C C 23. Compound according to claim that R derives from the phenytoin.   24. A compound according to claim 28, wherein R is of the following formula: -0 0 H H C N - 4CH 4- (C-N) -N- C 2n m N C H /   o n is an integer from 1 to 3 and m is 0 or 1.   Compound according to claim 24, characterized in that that n is 2 and m is 0.   26. A compound according to claim 20, characterized in that   that R is derived from valproic acid.   27. Compound according to claim 26, characterized in that R corresponds to the following formula: 0 H H C-C-CH CH CH-N- I- ,, 2 22 HO (CH2) p H   o p is an integer from 2 to 4.   28. A compound according to claim 20, characterized in that that R derives from primidone.   29. Compound according to claim 28, characterized in that R corresponds to the following formula H o N -C CII2CH3 H N -C C   30. Compound according to claim 19, characterized in that that R derives from a steroid of.   31. Compound according to claim 30, characterized in that that R derives from digoxin.   32. Compound according to claim 31, characterized in that R corresponds to the following formula: ## STR2 ## A compound according to claim 19, characterized in that   that R derives from an antiasthmatic drug.   34. A compound according to claim 33, characterized in that that R derives from theophylline.   35. Compound according to claim 34, characterized in that R corresponds to the following formula: OH CH "' 3 C N H 2n-n- N- c> "N CH3   in which n is 1 or 2.   36. The compound of claim 19, carat that R derives from an antiarrhythmic.   37. A compound according to claim 36, cara, that R is derived from propranolol.   38. The compound of claim 37, cara, that R has the following formula: OH I .. I OCH CH-O-C-CH-N- 2 2 X J CH3 CH CNH-CH 3 2 1,1CH3 N 4 H 3   39. A compound according to claim 19, cara that R derives from a hormone.   40. Compound according to claim 19, cara that R derives from an antiarthritic.   41. A compound according to claim 19, cara,   that R derives from a glucoside acting on the heart.   42. A compound according to claim 19, cara. that R derives from an antidepressant.   43. Compound according to claim 19, cara   that R derives from an antineoplastic.   44. The compound of claim 18, wherein R has the formula: R 'I O C-OH H I I HO O CH -C-- N- 2 i H RI e I I I   R 'represents a hydrogen atom or an iodo radical.   Cérérisé in this cérérisé in this cérérisé in this cérérisé in this cérérisé in this cérérisé in this cérérisé in this cérérisé in this