FR2518096A1 - METHOD AND REAGENTS FOR IMMUNOLOGICAL DETERMINATION BY POLARIZATION OF FLUORESCENCE - Google Patents

Abstract

THE INVENTION CONCERNS NEW AMINOFLUORESCEIN DERIVATIVES OF THE FORMULA: (CF DRAWING IN BOPI) IN WHICH X IS A -SO-, -CO-NH-SO- OR -CO-CO- AND R IS ANALOGUE OF LIGAND HAVING A MOLECULAR WEIGHT IN THE RANGE FROM 100 TO 2000, THE ANALOGUE OF LIGAND HAVING AT LEAST ONE EPITOPE IN COMMON WITH LEDIT LIGAND SO AS TO BE SPECIFICALLY RECOGNIZED BY A COMMON ANTIBODY AND THIS ANALOGUE OF LIGAND BEING LINKED TO THE REST - SO-NH- BY A AROMATIC CARBON, AT THE REMAINING -CONHSO- BY A HYDROXY OXYGEN OR A REACTIVE AMINE, OR AT THE REMAIN -CO-CO- BY A REACTIVE NITROGEN. APPLICATIONS: RESEARCH AND ASSAY OF LIGANDS IN SAMPLES OF BIOLOGICAL LIQUIDS BY POLARIZATION OF FLUORESCENCE.

Description

The present invention relates to a method and reagents for determining

  ligands in body fluids

  such as serum, plasma, spinal fluid, amnion

  In particular, the invention relates to a method of immunological determination by fluorescence polarization and

  markers used as reagents in these procedures.

  The fluorescence polarization immunoassay method of the invention combines the specificity of an immunological determination 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 the measurement of ligands rely on competition between a ligand in a test sample and a labeled reagent, labeled as a marker, for a limited number of binding sites.

  receptor on antibodies specific for the ligand and the marker.

  The concentration of the ligand in the sample determines the amount of label that binds specifically to an antibody. The amount of label-antibody conjugate produced can be measured

  quantitatively and is inversely proportional to the quantity

  ligand in the test sample.

  In general, fluorescence polarization techniques are

  are based on the principle that a fluorescent marker, excited by linearly polarized light, emits fluorescence light having a degree of polarization which is inversely related to its rate of rotation. Thus, when a molecule such as that a marker-antibody conjugate having a fluorescent label is excited with linearly polarized light, the emitted light remains strongly polarized because the rotation of the fluorophore is limited between the moment of absorption of the light and the instant of emission When a "free" (i.e., non-antibody bound) marker is excited by linearly polarized light,

  its rotation is much faster than the marker-anti-conjugate

  corresponding body and the molecules are more statistically oriented

  tically, so the emitted light is depolarized So the fluorescence polarization provides a quantitative means for measuring the

  amount of marker-antibody conjugate produced in a deter-

  immunological nation by competitive fixation.

  The present invention relates to a method for the determination of ligands in a sample comprising mixing with said sample a biologically acceptable salt of a marker of general formula OH

R-HX N

  Wherein X is an oxalyl group of the formula II, a sulfonyl group of the formula ## STR2 ## or a carbamidosulfonyl group of the formula ## STR2 ## wherein

-C-N-S-

  and R is an analogue of the ligand 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 marker; and then determining the amount of label-antibody conjugate by fluorescence polarization techniques

  as a measure of the concentration of said ligand in the sample.

  The invention also relates to a new class of labels of formula (I) and their biologically acceptable salts,

  which are of interest as reagents in the process described above.

  The methods and labels according to the invention are of particular interest for the quantitative control of drug concentrations.

  Therapeutic cameras in serum and plasma.

  The term "ligand" means in this description

  to denote a molecule, in particular a low molecular weight hapten which can be obtained or form a receptor, normally an antibody. The haptens are protein-free bodies, generally of low molecular weight, which do not cause antibody formation when they are injected into an animal, but are reactive towards the antibodies. The antibodies or haptens are

  usually formed by first combining haptens with a

  The resulting antibodies are isolated by standard antibody isolation techniques.

  The ligands determinable by the process of the invention

  Although high molecular weight ligands can be determined, it is generally preferable for best results to use the methods of the invention to determine low weight ligands.

  molecular weight, usually in the range of 50 to 4000

  still need to determine ligands with a molecular weight in

the range from 100 to 2,000.

  The novel markers according to the invention comprise

  compounds of formula (I) in which the ligand analogue is

  presented by R includes radicals having a molecular weight

  in the range of 50 to 4000 The new preferred markers include

  compounds of formula (I) in which the ligand analogues represented by R include radicals having a weight

  molecular weight in the range of 100 to 2,000.

  Representative examples of ligands that can be determined by the methods of the present invention include

  steroids such as estrone, estradiol, cortisol, testosterone,

  gestérone, chenodeoxycholic acid, digoxin, cholic acid, digitoxin, deoxycholic acid, lithocholic acids and their esters and amides; vitamins such as B-12, folic acid,

  thyroxine, triiodothyronine, histamine, serotonin; prostitutes

  glandins such as PGE, PGF, PGA; anti-asthmatic drugs such as theophylline; antineoplastic drugs such

  that doxorubicin and methotrexate, antiarrhythmics such as

  pyramid, lidocaine, procainamide, propranlol, quinidine, N-acetyl-

  procainamide; anticonvulsants such as phenobarbital, phenyl

  toine, primidone, valproic acid, carbamazepine and ethosuximide; antibiotics such as penicillins, cephalosporins, erythromycin, vancomycin, gentamicin, amikacin,

  chloramph 6 nicol, streptomycin and tobramycin; antiarthritis

  ticks such as salicylates; antidepressants, including

  tricyclic compounds such as nortriptyline, amitriptyline, imine

  pramine and desipramine; and analogues, as well as their metabolites.

  In addition, the drug habitants such as morphine, heroine, hydromorphoneoxymorphone, metapon, codeine, hydrocodone, dihydrocodeine, dihydrohydroxycodéinone, pholcodine, dextromethorphan, phenazocine and deonine and their metabolites,

  can be determined according to the methods of the invention.

  The markers according to the invention generally exist in equilibrium between their acidic and ionized forms and, in the Lonized state,

  they are effective in the process of the invention.

  Thus, the labels include either the acid form or the ionized form and, for convenience, the markers of the invention.

  are structurally represented in this description under

  their acid form When the markers of the invention are present

  in the ionized state, the markers exist in the form of biologically

  The term "biologically acceptable salts" refers to salts such as sodium, potassium, ammonium, etc. salts, which allow the markers of the invention to exist in the state

  ionized when used in the process of the invention

  the markers according to the invention exist in solution to

  the salt state, the specific salt results from the buffer used, ie

  that in the presence of a sodium phosphate buffer, the markers of the invention generally exist in the ionized state in the form of a

sodium salt.

  The term "ligand analogue" is understood to mean

  description to designate a mono or polyvalent radical

  a significant proportion to the same spatial and polar organization as the ligand to define one or more critical sites or epitopes capable of competing with the ligand for receptor binding sites. A characteristic of such a ligand analog is that he has a structural resemblance

  sufficient with the interesting ligand to be recognized by the anti-

  For the most part, the ligand analogue has a similar and substantially identical charge distribution structure (spatial and polar organization) to those of the ligand

  interesting for a significant portion of the molecular surface.

  Since the binding site for a hapten is frequently the same in the preparation of the antigen for antibody production as is used for ligand binding, the same portion of the ligand analog that provides the template for the antibody is

  exposed by the ligand analogue in the marker.

When X is a sulfonyl group o

-S

ll o

  the ligand analogues represented by R are derived from the ligand cor-

  by removal of an aromatic hyogen, i.e. a hydrogen attached to an aromatic carbon, preferably a carbon

  phenyl, or by the formation of a phenyl or phenyl derivative

  In addition, a ligand can be structurally modified by the addition or removal of one or more functional groups to form a ligand analog while retaining

  the epitope sites necessary for binding to an antibody.

  However, it is preferred that these modified ligand analogues be

  fixed to the sulphonylaminofluorescein residue by an aromatic carbon.

  When X is an oxalyl group -C-C-

  ligand analogues represented by R are derived from the corresponding ligand.

  by removal of a hydrogen atom bound to a reactive amine

  tive, that is to say a hyerogen atom linked to a primary amine or

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  secondary or by the formation of an amino derivative of the ligand in which a group -Nl I replaces one or more atoms initially present

  in the ligand at the site of attachment to a residue of oxalylaminofluores-

  Examples of ligands which, by removal of a reactive amine hydrogen, form an analogue of the ligand represented by R include, for example, procainamide, thyroxine, quinidine and aminoglycoside antibiotics. whose amino derivatives are interesting as analogues

  of the ligand include theophylline, valproic acid,

  barbital, phenytoin, primidone, disopyramide, digoxin,

  chloramphenicol, salicylates, acetaminophen, carbamaze

  In addition, a ligand may be structurally modified by the addition or removal of one or more functional groups to form a ligand analogue, while retaining the epitope sites necessary for binding to a ligand. However, it is preferred that these ligand analogs

  are bound to the oxalylaminofluorescein residue by an imino group.

  When X is a carbamidosulfonylamino group 0H 0

-C-N-S-

  the ligand analogues represented by R are derived from the ligand

  corresponding by removal of a reactive hydrogen atom, that is,

  a hydrogen atom bonded to a hydroxy oxygen or a reactive amine (primary or secondary) or by the formation of an amino derivative of the ligand in which an imino H -N- group replaces one or more atoms initially present in the ligand,

  at the site of attachment to a residue of carbamidosulfonylaminofluores-

  Examples of ligands which, by removal of a reactive hydrogen, can form an analogue of the ligand represented by R include, for example, procainamide, thyroxine, quinidine and aminoglycoside antibiotics. the amino derivatives are useful as analogues of

  ligand include theophylline, valpro Yque acid, phenol

  barbital, phenytoin, primidone, disopyramide, digoxin,

  chloramphenicol, salicylates, acetaminophen, carbamaze

  In addition, a ligand may be structurally modified by the addition or removal of one or more functional groups to form a ligand analogue while retaining the epitope sites necessary for binding to an antibody. However, it is preferred that these ligand analogues

  are bound to a residue of carbamidosulfonylaminofluores-

by a group imino or oxy.

  The markers of the invention may be prepared according to known techniques. When X is a sulphonyl group, the markers of the invention are prepared by reaction of a compound of formula

R-Y (II)

  wherein R is as defined above and Y is an aromatic hydrogen, preferably bonded to a phenyl ring, with the acid

  chlorosulfonic acid to produce a chlorosulfonyl derivative of the

  ligand of formula o R-S-Cl (III)

  The chlorosulfonyl derivative of the

  of the ligand with an aminofluorescein of the formula OH ## STR3 ## in which the amino group is attached to the (IV) or (V) position of the benzoic acid ring, in the presence of an inert solvent, to give a Marker of the Invention of General Formula OH It 1

R-S-N,

  (V) When X is an oxalyl group, the markers of the invention are prepared by reacting a compound of formula RZ (vi) in which R is as defined above and Z is a hydrogen bonded to a reactive nitrogen (primary or secondary amine), with methoxyoxalyl chloride to give a methoxyoxalyl ligand analogue derivative which is hydrolyzed in the presence of a base to give a hydroxyoxalyl ligand analog derivative of formula 0 OI he

R-C-C-OH (VII).

  The ligand analogue hydoxyoxyalyl derivative is reacted with an aminofluorescein of the formula (IV) in the presence of a

  condensation agent, such as 1-ethyl-3- (3'-dihydrochloride) hydrochloride

  methylaminopropyl) -carbodiimide, in an inert solvent to give a marker of the invention of formula OOH

R-C-C-N,

(VIII)

  When X is a carbamidosulfonyl group, O H O there is

-C-N-S

  The labels of the invention are prepared by reaction of a compound of formula

R-W (IX)

  wherein R is as defined above and W is hydrogen to a reactive nitrogen (primary or secondary amine) or hydroxy oxygen with chlorosulfonyl isocyanate to give a ligand analogue chlorosulfonamidocarbonyl derivative of the formula OHO RCN-SC1 (X) The chlorosulfonamidocarbonyl ligand analogue derivative is reacted with an aminofluorescein of formula (IV) to give the markers of the invention of general formula OH

0 H OH O

R-C-N-S-N

  The temperature at which the reaction proceeds for the preparation of the markers of the invention is not essential. The temperature must be sufficient to initiate and maintain the reaction. In general, for the sake of convenience and economy, the ambient temperature is sufficient In the preparation of the markers according to the invention, the ratio of the reactants is not strictly essential. For example, two moles of chlorosulfonic acid must be used per mole of compound of formula (II) for

  to obtain a reasonable return One prefers to use an excess -

  of chlorosulfonic acid for ease of reaction and

  recovery of the reaction products.

  The compounds of formula (IV, used as raw materials in the production of the markers according to the invention are either commercially available or prepared according to

known techniques.

  For ease of handling and recovery of the product, the preparation of the markers of the invention is carried out in the presence of an inert solvent Inert solvents

  appropriate solvents which do not react appreciably

  with the raw materials and are sufficient to dissolve the raw materials. They include, for example, acetone,

  chloroform, pyridine, etc. For maximum yields,

  product, the reaction preferably takes place under conditions

  Neutral or basic bases Suitable bases include, for example, triethylamine, pyridine, etc. The reaction products are generally purified by chromatography, either in a thin layer,

  either on a column, before application in the processes of the invention.

  According to the process of the invention,

  a sample containing the ligand to be determined with a biological salt

  of a marker of formula (I) and an antibody

  specific ligand and marker The ligand present in the sample

  tillon and the scorer compete for a limited number of

  sites of the antibody leading to the formation of ligand-

  antibodies and antibody-marker By keeping the concentration constant

  marker and antibody, the ratio of the ligand-anti-

  body to the marker-antibody complex that is formed is directly

  proportional to the amount of ligand present in the sample.

  Therefore, by excitation of the mixture in fluorescent light and measurement of the polarization of the fluorescence light emitted by a marker and by a marker-antibody complex, one is able to determine

  quantitatively the amount of ligand in the sample.

  In theory, the fluorescence polarization of a

  marker not complexed by an antibody is weak and close to zero.

  By complexing with a specific antibody, the marker-antibody complex thus formed undergoes rotation of the antibody molecule which is weaker than that of the relatively small marker molecule, thereby increasing the observed polarization. a ligand is competing with the marker for sites

  of the antibody, the fluorescence polarization of the

  observed antibody takes a value between that of the

  and a marker-antibody complex If a sample contains a high concentration of a ligand, the value of the polarization

  observed is closer to that of the free ligand, i.e., weak.

  If the test sample contains a low ligand concentration, the polarization value is closer to that of the bound ligand, i.e. high by successively exciting the reaction mixture.

  an immunological determination with vertically polarized light

  then horizontally and analyzing only the vertical component of the emitted light, we can determine

  accurately the fluorescence polarization of the reaction mixture.

  The precise relationship between polarization and concentration of

  ligand to be determined is established by measuring the polarization values

  of standard substances at known concentrations.

  ligand can be extrapolated from a curve of

  prepared in this way.

  The pl using the method of the invention must be sufficient for the markers of formula (I)

  can exist in the ionized state The p H can be between

  Nos. 3 and 12, more commonly in the range of 5 to 10, preferably about 6 to 9. Various buffers may be used to obtain and

  maintain the p H during the determination of buffers characterized

  ticks include borate, phosphate and carbon buffers.

  nate, sorting, barbital, etc. The particular buffer used is

  essential for the invention, but in an individual determination

  dual, one may prefer a specific buffer for the antibody used and the ligand to determine the cation portion of the buffer

  generally determines the salt cation of the marker in solution.

  The techniques of the invention are carried out at average temperatures and, preferably, at a constant temperature. The temperature normally varies between 0 and 500 ° C., more usually between approximately 15 and 400 ° C.

  The concentration of the ligand which can be determined

  Minute range generally from about 10-2 to 113 M, more commonly from about 104 to 10 M Higher concentrations of ligand

  can be determined by dilution of the original sample.

  In addition to the range of ligand concentrations of interest, other considerations normally determine the concentration of markers and antibody to be used, such as whether the determination is qualitative, semi-quantitative or quantitative, the equipment used and the characteristics of the ligand of interest. Although the concentration of the ligand in the sample determines the range of concentrations of the other reagents, i.e., label and antibody, the individual concentrations of the reagents are normally determined empirically to optimize sensitivity of determination

  Marker and antibody concentrations are easily

  finished by the man of the art of a current skill.

  As mentioned above, the preferred markers of the invention are prepared from 1-aminofluorescein or 4-aminofluorescein and preferably exist as formulas

OH

H

R-; X 4 \ N OXII)

O OH

l and H H

-N (XIII)

he "C \

O OH

  in which R and X are as defined above.

  The following non-limiting examples serve to further illustrate to one skilled in the art the preparation of specific labels within the scope of the invention. The symbol IAF, appearing in the developed formulas illustrating the compounds prepared in the following examples, represents a residue. of formula OH H -N t / \ (XIV)

0 OH 9

  in which the nitrogen of the imino residue is bound to position 4 or 5

  in the formula above, depending on the specific isomer of aminofluores-

  ceine used as raw material.

EXAMPLE 1

  To 0.71 g of lidocaine is added 2.8 g of chlorosulfonic acid and the resulting mixture is heated at 60 ° C. for 1 h. The reaction mixture is cooled and crushed ice and water are added to the mixture. to remove unreacted chlorosulfonic acid The resulting aqueous solution is neutralized with sodium hydroxide The resulting product is extracted with two 10 ml portions of methylene chloride. methylene, dried over sodium sulphate and filtered. Evaporated to dryness to obtain 100 mg of a mixture of meta and parachlorosulfonylidocaine in the form of

  of a gummy oil to a solution containing 5 mg of 4-amino

  fluorescein in 0.5 ml of pyridine, 5 mg of the above mixture of chlorosulfonylidocanes are added. After 10 min, a crude product is formed. The crude product is purified by thin layer chromatography using silica gel and chloroform. then

  a chloroform-acetone mixture (1: 1) and finally a mixture chloroform-

  methanol (1: 1) as developing solvents, to obtain a mixture of sulfonylidinocaine-aminofluorescein conjugates of the general formula

CH O

3 a N-CHI 2-CN d O

H 5 C 2 1 '

Cii 3

EXAMPLE 2

  1.2 g of phenobarbital are slowly added with 4.5 g of chlorosulphonic acid and the resulting mixture is heated at 60 ° C. for 1 hour. The reaction mixture is cooled and crushed ice is added to the mixture. The reaction mixture is then filtered to give a white precipitate which is rinsed with water and dried in a desiccant.

  vacuum to obtain 0.8 g of a mixture of meta and para-chlorosulfo

  nylphenobarbitals having a melting point of 190-195 ° C.

  solution containing 5 mg of 5-aminofluorescein in 0.5 ml of pyridine

  dine, 5 mg of the above mixture of chlorosulfonylphenol

  After 10 min, a crude product was formed and purified twice by layer chromatography techniques.

  thin using silica gel and a mixture of chloroform

  2: 1 methanol as developing solvent to obtain a sulfonylphenobarbital-aminofluorescein conjugate of formula

H O

CH H 5 O

O = C C 2 -I-AF N Cv

H O

EXAMPLE 3

  To 2 g of α-phenethyl-α-methylsuccinimide 2.5 g of chlorosulphonic acid are added dropwise and the resulting mixture is stirred at 26 ° C. for 1 hour. The mixture of crushed ice is added to the mixture. water to remove unreacted chlorosulfonic acid, if any. The reaction product is extracted with two 10 ml portions of chloroform. The chloroform extracts are combined, dried over sodium sulphate and evaporated to give viscous oil The oil is crystallized in a mixture

18096

  of toluene and petroleum ether to obtain 0.29 g of a mixture of α (meta and para chlorosulfonylphenethyl) -n-methylsuccinimide having a melting point of 145-150 ° C. a solution containing 5 mg of 4 aminofluorescein in 0 5 ml of pyridine, 5 mg of the above mixture of? -Chlorosulfonylphenethyl) -u-methylsuccinimides are added. After 10 min, a crude product was formed which was purified twice by thin layer chromatography techniques using silica gel and a chloroform-methanol (2: 1) mixture as a developing solvent to obtain a mixture of α- (sulfonylphenethyl) α-methylsuccinimide-aminofluorescein conjugates of formula

O = C CH 2 CH 3

I \ ^ C /

H-N C CH CH / O

O TO S -FAFl

EXAMPLE 4

  To a solution containing 2 g of iminostilbene and 2 ml of triethylamine in 50 ml of chloroform is added 1.5 g of methoxyoxalyl chloride. The resulting mixture is heated under reflux for 1 h and then evaporated to dryness. The residue is dissolved in 50 ml of chloroform and then extracted with 50 ml of water. The chloroform layer is evaporated to dryness. 2N sodium hydroxide is added to the residue and the resulting mixture is heated under reflux for 30 minutes. the mixture at room temperature and then extracted with 50 ml of chloroform.

  the aqueous folded layer 1 using concentrated hydrochloric acid

  The organic extract is dried over sodium sulfate and evaporated to dryness. The residue is taken up in 50 ml of methanol and triturated with water to obtain a jet. of crystals. The mixture is filtered and the crystals are cooled for 16 hours to obtain 2.4 g of an N-hydroxyoxalyl-

  iminostilbene (m.p. 162-163 ° C).

  To 5 mg of N-hydroxyoxalyl iminostilbene is added a solution containing 5 mg of 4-aminofluorescein in 0.5 ml of pyridine. The reaction is allowed to proceed for 2 hours at 26 ° C.

* to obtain a crude product The crude product is purified by chromatography

  thin-film matography using silica gel and a

  developing solvent consisting of a mixture of chloroform

  acetone (1: 1) to obtain an N-oxalyl-iminostilbeneaminofluorescein conjugate of formula

O Q

  N-C-C AFl The following markers are also prepared according to

the procedures above.

EXAMPLE 5

  Sulfonylprimidone-aminofluorescein conjugates

H "

o 2 5 O H O q It AF

N _C O

\ 60 <HS O \ F /

EXAMPLE 6

  Para-methyl-meta-sulfonylprimidone-aminofluoresceine conjugate

HN C __

H 2 C /

H /

O CH

EXAMPLE 8

  5-sulfonylphenyl-5-ethylhydantoin-aminofluorescein conjugate H il c CH CH O 2 3

S - + AF

C 1 1

N O

H

EXAMPLE 9

  U- (sulfonylphenyl) -α-methylsuccinimide-aminofluorescein conjugate CH 2

EXAMPLE 10

  O-sulfonanidocarbonylpropranolol-aminofluorescein conjugate

H O H O

1 There is 0-CH 0-C-N-S EAF1 2 he

H CH 3

-11 I, -H

CH 3 H N

O = C '1

CI C 2 H 5

1 1 C '

I-

N C,

  H AFl It 0 CH It \\ C _ 3 It -AFl H-N '

18,096

1 7

EXAMPLE 7

  Conjugate para-methyl-meta-sulfonylphobarbital-aminofluorescein is prepared in the form of a N-sulphonyl-aminofluorescein (N-sulphonyl) aminofluorescein.

N-C-N-S- + AFI

EXAMPLE 12

  N-Sulfonamidocarbonyl-procainartide-aminofluorescein Conjugate H 5 C 2 N-C-N-C N-CH CH -N-C

H 5 2 O

EXAMPLE 13

  0-sulfonamidocarbonyl-chloramphenicol-aminofluorescein conjugate

H H O H O

1 t ily he

0 N C-C-CH-O-C-N-S EAF-1

2 2 He

H O

H C-CH Cl

EXEIMPLE 14

  N-sillfonamidocarbonyl-disopyramide-aminofluorescein conjugate

H-C H 3

H O H O

CH 3 he lives 1 fi

N-CH CH -C-C-N C-N-S E-AFI

CH / 2 2 i He

\ 3 O

H-C i

CH 3 L) 1

EXAMPLE 15

  0-Sulfonamidocarbonyl-quinidine-aminofluorescein conjugate H CHCH x 2

N 0 H

H O-C-N-S-FEAF1 OC Hil

EXAMPLE 16

  O-oxalyl-propranolol-aminofluorescein conjugate

H 0 00

I il \ \ OC? -C-O-C-C AFl

21, CH

CH-N-C-H

2 y, -

H CH 3

EXAMPLE 17

  N-oxalyl-proca conjugate Tnamide-aminofluorescein

H 5 C 2 H O H

N-CH CH -N-C - '""

2 2 \ NCC AFI

H C

EXAMPLE 18

  Conjugate Ni-acetyl-N'-desethyll-N'-oxaly 11-procaine Cnamideaminofluorescene

O H H C H

C-N C N-CH CH-N

3 C-C-1-Arl i Q Oi

EXAMPLE 19

  N oxalyl-nortriptyline-aminofluorescein / A / 1 / CH 3 conjugate

C = CHCH 2 -CH -N

2 2 \ C-C- + AFl 0 \

EXAMPLE 20

  N-oxalyl-iminodibenzyl-aminofluorescein conjugate A 00 zi i | NCC FAF1 As previously mentioned, the markers according to the invention are reagents that are effective for use in fluorescence polarization immunological determinations. The following examples illustrate the advantage of the markers according to the invention in immunological determinations using polarization techniques. These determinations are carried out according to the following general procedure: 1) A measured volume of calibrated sample or test serum is placed in a test tube and diluted with the buffer; 2) then adding to each tube a known concentration of a label of the invention optionally containing a surfactant; 3) a known concentration of the antisera is added to the tubes;

  4) the reaction mixture is incubated at room temperature

  The amount of label bound to the antibody is measured by fluorescence polarization techniques, such as

  measuring the amount of ligand in the sample.

  EXAMPLE 21 Determination of Lidocaine Products: 1) Buffer: 0.1M phosphate, pH 7.5, containing 0.01% w / v sodium azide and 0.01% w / v volume -globulin of beef (hereinafter referred to as "GGB buffer")

  2) Marker: sulfonyllidocalne-amino-conjugate

  fluorescein (prepared in Example 1) at a concentration of 2.34 10-7 M in 0.1M tris hydrochloride buffer, pH 7.8, containing 0.1% by weight per volume of sodium dodecyl sulphate, 0.01% by weight per volume of y-globulin of beef and 0.01% by weight per volume of sodium azide 3) Anti-lidocaine rabbit serum antibody diluted 1 to 80 times in the GGB buffer 4) Standards and unknown samples: human serum (or other biological fluid) containing lidocaine in a concentration range of 0 to 10 μg / ml) fluorescence polarimeter: instrument capable

  to measure the fluorescence polarization of a fluorescent solution

_g

  cein 10 M to +0.001 polarization unit.

  Procedure: 1) To 20 μl of unknown standards or samples, add 200 μl of GGB buffer. 2) At 20 μl of each standard or unknown sample diluted in a culture tube, add 200 μl of buffer. GGB 3) To each culture tube containing the diluted standard or sample, add 40 ml of marker and 1000 ml of GGB buffer. 4) Then add 40 ml of antibody and 1000 ml of GGB buffer to each culture tube 5) Mix the reagents and incubate the culture tubes containing unknown standards and samples for approximately 15 min at 23 ° C. 6) Measure the fluorescence polarization of all the tubes Characteristic results of the standard samples

  are shown in Table I below.

TABLE I

  Concentration of lidocaine (, u / ml) Polarization

0 0.224

0.5 0.186

1.0 0.162

2.5 0.124

0.094

0.071

  The polarization values decrease as the concentration of lidocaine increases, allowing a calibration curve to be drawn.

  identical can be quantified by reference to the curve of

  pling. EXAMPLE 22 Determination of Phenobarbital Products: 1) GGB Buffer

  2) Marker: Conjugate (para-methyl-meta-sulfonyl) -

  phenobarbital-aminofluorescein (prepared in Example 7) at a concentration

  5.75 10-8 M in 5.75% sodium cholate 3) Antibody: anti-phenobarbital rabbit serum diluted 1 to 78.3 times in GGB buffer 4) Standards or unknown samples: human serum ( or other biological fluid) containing phenobarbital in a concentration range of 0 to 80 μg / ml 5) Fluorescence polarimeter: instrument capable

  to measure the fluorescence polarization of a fluorescent solution

  cein 10-9 M at + 0.001 polarization unit.

  Procedure 1) To 20 μl of unknown standards or samples add 200 μl of GGB buffer 2) To 20 μl of each standard or unknown sample diluted in a culture tube add 200 μl of GGB buffer 3 ) Each culture tube containing a diluted standard or unknown sample, add 40 ml of marker and 1000 ml of GGB buffer. 4) Then add 40.1 l of antibody and 1000 μl of 1 GGB buffer to each culture tube. ) Mix reagents and incubate culture tubes containing unknown standards and samples for approximately 15 min at 23 C. 6) Measure the fluorescence polarization of all tubes Characteristic results for samples

  standards are shown in Table II below.

TABLE II

  Phenobarbital concentration (μg / ml) Polarization

O 0.155

10.108

0.092

0.074

0,060

  The polarization values decrease as the concentration of phenobarbital increases, allowing a calibration curve to be drawn. Unknown samples treated identically can be quantified by reference to the

calibration curve.

  EXAMPLE 23 Determination of Carbamazepine Products: 1) GGB Buffer

  2) Marker: N-oxaliminostilbene-amino-conjugate

  fluorescein (prepared in Example 4) at a concentration of 2 nM in BGG buffer containing 0.2% by weight per volume of sodium cholate 3) Antibody: anti-carbamazepine rabbit serum diluted 1 to 3040 times in the GGB buffer 4 days Standard or unknown samples: human serum (or other bloom) containing carbamazepine in a concentration range of 0 to 20 μg / ml 5) Fluorescence polarimeter: an instrument capable of measuring the fluorescence polarization of a solution

  from fluorescein 109 M to +0.001 polarization unit.

  Procedure 1) To 10 μl of unknown standards or samples add 300 μl of GGB buffer 2) To 10/11 of each standard or unknown sample diluted in a culture tube, add 300 x 1 of GGB buffer 3) Add 1 ml of marker to each culture tube 4) Then add to each culture tube 1.0 ml of antibody 5) Mix reagents and incubate culture tubes containing unknown standards and samples for about 15 minutes at 23 DO C. 6) Measure the fluorescence polarization

  of all the tubes Typical results for the samples

  Standard samples are shown in Table III below.

TABLE III

  Carbamazepine concentration (/ μg / ml) Polarization

0 0.224

2 0.108

4 0.135

8 0.105

12 0.088

0.077

  Polarization values decrease as carbamazepine concentration increases, allowing a calibration curve to be drawn Unknown samples treated identically can be quantified by reference to

calibration curve.

  The following table summarizes the various deter-

  fluorescence polarization immunoassays which were carried out according to the procedures described above in

  using markers prepared in the previous examples.

  The markers used are identified by the example number and

  the specific ligands determined are indicated.

  Marker (example of preparation) Ligand (s) determined Lidocaine Phenobarbital Ethosuximide Carbamazepine Primidone Primidone Marker (Exempt of preparation) Ligand (s) determined 7 Phenobarbital 8 Phenytoin 9 Ethoxysuximide Propanolol 11 Carbamazepine 12 Procainamide 13 Chloramphenicol 14 Disopyramide Quinidine Propanolol 17 Procainamide 18 N-acetyl-procainamide 19 Desipramine, Imipramine Nortriptyline, Amitriptyline As is evident from the above results, the markers according to the invention are effective reagents in

  immunological determinations by fluorescence polarization.

  In addition to the properties mentioned above, the markers of the invention

  have a high thermal stability, a high degree of polarization in the fixed state, high quantum

  are easy to produce and purify.

  It is understood that the invention is not limited to

  preferred embodiments described above as illustrative

  and the person skilled in the art may make various modifications and changes to it without departing from the scope and

the spirit of the invention.

Claims (46)

R E V E N D I C A T IO N S   1 New sulfonylaminofluorescein derivatives, characterized   in that they correspond to the general formula __, OH / 0, R-S \ v È = \   wherein R is a ligand analog having a molecular weight in the range of from 100 to 2000, said ligand analog having at least one epitope common with said ligand so as to be specifically recognizable by a common antibody and said ligand having an aromatic carbon by which it is attached to the sulfonamidofluorescein residue;   and their biologically acceptable salts.   Compounds according to claim 1 characterized in that   that they answer the general formula OH OH R-S-N O - / \ OR 0 v BH A_ or OH O R-S-N, / 0 l O   and their biologically acceptable salts.   Compounds according to claim 2, characterized in that R is derived from a ligand selected from steroids, hormones, antiasthmatics, antineoplastics, antiarrhythmics, anticonvulsants, antibiotics, antiarthritics, antidepressants and cardiac glycosides. . Compound according to claim 3, characterized in that   that R is derived from an antiarrhythmic.   Compound according to claim 4, characterized in that   that R is derived from lidocaine.   Compound according to Claim 5, characterized in that R is a group of the formula CH 3 CH 3 N-CH 2 -CN CH 2   CH 3 Compound according to claim derived from an anticonvulsant. Compound according to claim derived from phenytoin.   Compound according to Claim 1, characterized in that 7, characterized in that 8, characterized in that H% II N C -1 C ci CH 3 I '/ 2 3 1 1 /   Compound according to claim 7, characterized in that R is derived from primidone.   Compound according to claim 7, characterized in that R is a group of formula LI r% LN _C 'CH 2 N C HL N O V   in which R 'is a C 1 -C 4 alkyl group   7. R is 8. Rest 9. R is 12 Compound according to claim 7, characterized in that   that R is derived from ethosuccinimide.   Compound according to claim 12, characterized in that R is a group of formula a? CH 3- H-NC -C -'CH 2   wherein N is an integer of 0 to 3.   New carbamidosulfonylaminofluorescein derivatives, characterized in that they correspond to the general formula OH OH OH / tg I There R-C-N-S-N I O H O   wherein R is a ligand analog having a molecular weight in the range of 100 to 2000, said ligand analog having at least one epitope common with said ligand so as to be specifically recognizable by a common antibody and said ligand analog being fixed the carbamidosulfonylaminofluorescein residue with a hydroxyl group oxygen or a reactive amine;   and their biologically acceptable salts.   Compounds according to claim 14, characterized in that they correspond to the general formula OH OH OH He I '1 I R-C-N-S-N or O HO O 11 l He R-C-N-S- O   and their biologically acceptable salts.   Compounds according to claim 15, characterized in that R is derived from a ligand selected from steroids,   hormones, antiasthmatics, antineoplastics, anti-   arrhythmic drugs, anticonvulsants, antibiotics, anti-   arthritis, antidepressants and cardiac glucosides.   Compound according to claim 16, characterized in that   what R is derived from an anticonvulsant.   Compound according to claim 17, characterized in that   what R is derived from carbamazepine.   Compound according to Claim 18, characterized in that R is a group of formula \ -   Compound according to claim 16, characterized in that   what R is derived from an antiarrhythmic.   Compound according to claim 20, characterized in that   what R is derived from procainamide.   Compound according to claim 20, characterized in   what R is derived from propranolol.   23 New oxalylaminofluorescein derivatives,   in that they satisfy the general formula OH O O H 'd R-C-C-N   wherein R is a deligand analog having a molecular weight in the range of 100 to 2000, said ligand analog having at least one epitope common with said ligand so as to be specifically recognizable by a common antibody and said ligand analogue   having a reactive nitrogen by which it is attached to the oxalylamino fluorescein   and their biologically acceptable salts.   Compounds according to claim 23, characterized in that they correspond to the general formula ## STR2 ## R-C-C-N <OH \ X OOH \ Co or OH O O H He He I R-C-C-N 0 'OH \ O   and their biologically acceptable salts. 2 518096   Compounds according to claim 24, characterized in that R is derived from a ligand selected from steroids,   hormones, antiasthmatics, antineoplastics, anti-   arrhythmic drugs, anticonvulsants, antibiotics, anti-   arthritis, antidepressants and cardiac glucosides. Compound according to claim 25, characterized in that   that R is derived from an antidepressant.   Compound according to claim 26, characterized in that   that R is derived from a tricyclic drug.   Compound according to claim 27, characterized in that   that R is derived from nortriptyline, amitriptyline, imi- pramine or desipramine.   Compound according to claim 28, characterized in that   that R is derived from an anticonvulsant.   Compound according to claim 29, characterized in that   that R is derived from carbamazepine.   Compound according to Claim 30, characterized in that R is a group of formula e   32 Method for the determination of ligands in an exchange   characterized in that a biologically acceptable salt of a marker of the general formula OH is mixed with said sample OH / R-S-N ol 18096   wherein R is a ligand analog having a molecular weight in the range of from 100 to 2000, said ligand analog having at least one epitope common with said ligand so as to be specifically recognizable by a common antibody and said ligand analog having a aromatic carbon by which it is attached to the sulfonamidofluorescein residue; and an antibody capable of specifically recognizing said ligand and said label and then determining the amount of marker   bound to the antibody by fluorescence polarization techniques.   as a measure of the amount of ligand in the sample.   Process according to claim 32, characterized in that the group R-linked sulfonamido group is also linked to the   position 4 or 5 of the rest fluorescein.   The method of claim 33, characterized in that said ligand is selected from steroids, hormones, antiasthmatics, antineoplastics, antiarrhythmics, anticonvulsants, antibiotics, antiarthritics,   antidepressants, cardiac glycosides and their metabolites.   Process according to claim 34, characterized in that   that R has a molecular weight in the range of 50 to 4000.   The method of claim 35, characterized in that   that R has a molecular weight in the range of 100 to 2000.   The method of claim 36, characterized in that   said ligand is an antiarrhythmic.   Method according to claim 37, characterized in that   that said antiarrhythmic is lidocaine.   39 Process according to claim 38, characterized in that   said ligand is an anticonvulsant.   Method according to claim 39, characterized in that   that said anticonvulsant is phenytoin.   The method of claim 39, characterized in that   that said anticonvulsant is primidone.   The method of claim 39, characterized in that   said anticonvulsant is ethosuccimide.   A process for the determination of ligands in a sample   characterized in that a biologically acceptable salt of a marker of the general formula OH is mixed with said sample O H O / R-C-N-S-N   wherein R is a ligand analog having a molecular weight in the range of 100 to 2000, said ligand analog having at least one epitope common with said ligand so as to be specifically recognizable by a common antibody and said ligand being attached to the carbamidosulfonylaminofluorescein moiety by a hydroxy group oxygen or a reactive amine, and an antibody capable of specifically recognizing said ligand and said label and then determining the amount of label   to the antibody by fluorescence polarization techniques.   as a measure of the amount of ligand in the sample.   44 Process according to claim 43, characterized in that the group R-bonded carbamidosulfonylamino group is also bound   at position 4 or 5 of the fluorescein residue.   The method according to claim 44, characterized in that said ligand is selected from steroids, hormones, antiasthmatics, antineoplastics, antiarrhythmics,   anticonvulsants, antibiotics, anti-arthritics, antitoxins   depressants, cardiac glycosides and their metabolites.   46 Process according to claim 45, characterized in that   R has a molecular weight in the range of 50 to 4000.   Process according to claim 46, characterized in that   R has a molecular weight in the range of 100 to 2000.   48 Process according to claim 47, characterized in that   said ligand is an anticonvulsant.   The method of claim 48, characterized in that   said anticonvulsant is carbamazepine.   Process for the determination of ligands in a sample   characterized in that a biologically acceptable salt of a marker of the general formula OH 0 0 H is mixed with said sample 1 R-C-C-N /   wherein R is a ligand analog having a molecular weight in the range of from 100 to 2000, said ligand analog having at least one epitope common with said ligand so as to be specifically recognizable by a common antibody and said ligand analog   having a reactive nitrogen by which it is attached to the oxalylamino   fluorescein; and an antibody capable of specifically recognizing said ligand and said label and then determining the amount of marker   bound to the antibody by fluorescence polarization techniques.   as a measure of the amount of ligand in the sample.   Method according to claim 50, characterized in that the oxalylamino group bound to the R group is also linked to the position 4 or 5 of the rest fluorescein.   Process according to claim 51, characterized in that   said ligand is selected from among steroids, hormones,   asthmatics, antineoplastics, antiarrhythmic drugs, anti-   convulsants, antibiotics, anti-arthritics, anti-   depressants, cardiac glycosides and their metabolites.   53 Process according to claim 52, characterized in that   R has a molecular weight in the range of 50 to 4000.   Method according to claim 53, characterized in that   R has a molecular weight in the range of 100 to 2000.   Process according to Claim 54, characterized in   said ligand is an anticonvulsant.   Method according to claim 55, characterized in that   that said anticonvulsant is carbamazepine.