NZ202613A - N-substituted aminofluorescein derivatives;method for determining ligands by fluorescence polarisation immunoassay - Google Patents

Description

New Zealand Paient Spedficaiion for Paient Number £02613 202613 Priority Date(s): UzJ.3,;8l Complete Specification Filed: .0$.~,U: Qass: ,£Ua.. CP. ?&3//y CQ.lP.U.QS:;... LQ.7jQA<53.

Publication Date: ... 13.£ECJ985? P.O. Journal, No: .JqL7& NO DRAWINGS NEW ZEALAND PATENTS ACT, 1953 No.: Date: COMPLETE SPECIFICATION FLUORESCENCE POLARIZATION IMMUNOASSAY k/Abbott Laboratories, a Corporation organized and existing under the Laws of the State of Illinois, and having a principal place of business at 14th Street and Sheridan Road, North Chicago, Illinois 60064, United States of. America hereby declare the invention for which K/ we pray that a patent may be granted to r^g/us, and the method by which it is to be performed, to be particularly described in and by the following statement: - " 1 " (followed by page la) 2026 Background Of The Invention The present invention relates to a method and reagents for determining ligands in biological fluids such as serum, plasma, spinal fluid, amnionic fluid 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 ligands are based on the competition between a ligand in a test sample and a labeled reagent, referred to as a tracer, for a limited number of receptor binding sites on antibodies specific to the ligand and tracer. The concentration of ligand in the sample determines the amount of tracer that will specifically bind to an antibody. The amount of tracer-antibody conjugate produced may be quantitively measured and is inversely proportional to the quantity of ligand in the test sample.

In general, fluorescence polarization techniques are based on the principle that a fluorescent label compound when excited by linearly polarized light will emit fluorescence having a degree of polarization inversely related to its rate of rotation. Therefore, when a molecule such as a tracer-antibody conjugate having a fluorescent label is excited with linearly polarized light, the emitted light remains highly polarized because the fluorophore is constrained from rotating between the time light is absorbed and emitted.

When a "free" tracer compound Ci.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 light is depolarized. Thus, fluorescence polarization provides a quantitive means for measuring the amount of tracer-antibody conjugate produced in a competitive binding immunoassay.

Summary Of The Invention 202613 The present invention encompasses a method for determining ligands in a sample comprising intermixing with said sample a biologically acceptable salt of a tracer of 5 th.e formula: wherein X is a group selected from the class consisting of an oxalyl group of the formula: 0 0 « II -C-C- ; a sulfonyl group of the formula: 0 I! T ! o and a carboamidosul-fcnyl group of'the formula: OHO II I « -C-N-S- i 0 202613 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 5 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 10 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 15 are particularly useful in quantitatively monitoring therapeutic drug concentrations in serum and plasma.

Detailed Description Of The: Invention The term "ligand" as used herein refers to a molecule in particular a low molecular weight hapten, to 2 0 which a receptor, normally an antibody, can be obtained or formed. Haptens are protein—free bodies, generally of low molecular weight that do not induce antibody formation when injected into an animal, but are reactive to antibodies. Antibodies to hapten are generally raised by first con-25 jugating the haptens to a protein and injecting the conjugate product 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 molecular weight range. 30 Although high molecular weight ligands may be determined, for best results, it is generally preferable to employ the methods of the present invention to determine ligands of low molecular weight, generally in a range of 50 to 4000. It is more preferred to determine ligands having a molecular weight in 3 5 a range of 100 to 2 000. 202613 t - 4 The novel tracer of the present invention includes compounds of formula (.1) wherein the ligand-analog represented by R include radicals having a molecular weight within a range of 50 to 4000. The preferred novel tracers include 5 compounds of formula (.1) wherein the ligand-analogs represented 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 and hormoneS'such "as 10 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 B-12, folic acid; thyroxine, triiodothyronine, histamine, serotonin, prosta-15 glandins such as PGE, PGF, PGA; antiasthmatic drugs such as theophylline; antineoplastic drugs s-uch as doxorubicin and methotrexate; antiarrhythmic drugs such as disopyramide, lidocaine, procainamide, propranolol, quinidine, N-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; antiarthxitic drugs such as salicylate; antidepressant drugs including tricyclics such 25 as nortriptyline, amitriptyline, imipramine and desipramine; cardiac glycosides; and the like as well-'as the metabolites thereof. In addition, drugs of abuse such as morphine, heroin, hydromophone, oxyraorphone, 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 equilibrium between their acid and ionized states, and in the ionized state are effective in the 3 5 method of the present invention. Therefore, th.e present invention comprises the tracers in either the acid or ionized state and for convenience, the tracers of the 202613 present invention are structurally represented herein in their acid form. When the tracers of the prelent invention are present in their ionized state, the tracers exist in the form of biologically acceptable salts. As used herein, the 5 term "biologically acceptable salts" refers to salts such as sodium, potassium,ammonium and the like which will enable the tracers of the present invention to exist in their ionized state when employed in the method of the present invention. Generally, the tracers of the present invention exist in 10 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 15 a mono- or polyvalent radical a substantial proportion of which has the same spatial and polar organization as the ligand to define one or more determinant or epitopic sites capable of competing with the ligand for the binding sites of a receptor. A characteristic of such ligand-analog is that it possesses 20 sufficient structural similarity to th.e 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 substantially the same structure and charge distribution tspatial and polar organization), as the ligand of interest 25 for a significant portion of the molecular surface. Since frequently, the linking site for a hapten will be th.e same in preparing the antigen for production of antibodies as used for linking 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. 202613 When X is a sulfonyl group, 0 II -S- II 0 the class of ligand-analogs represented by R are derived from the corresponding ligand by removal of an aromatic hydrogen, 5 that is a hydrogen bonded to an aromatic carbon, preferably a phenyl carbon, or by the formation of a phenyl or substituted phenyl derivative of the ligand. In addition, a ligand may be structurally modified by the addition or election of one or more functional groups to form a ligand-10 analog while retaining the necessary epitope sites for binding to an antibody. However, it is preferred that such modified ligand-analogs be bonded to the sulfonylamino-fluorescence moiety through an aromatic barbon.

When X is an oxalyl group, 0 0 I! II -c-o the class of ligand analogs represented by R are derived from the corresponding ligand by removal of a hydrogen atom bond to a reactive amine, i.e., a hydrogen atom bonded to a primary or secondary amine or by the formation of an amino 20 derivative of the ligand wherein an imino group H I -N- 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 25 bond to a reactive amine from a ligand-analog represented by-R include, for example, procainamide, thyroxine, guinidine and the aminoglycoside antibiotics. Illustrative of ligands 20261 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 retaining the necessary epitope sites for binding to an antibody. However, it is preferred that such modified ligand-analogs be bonded to the oxalyl-10 aminofluorescein moiety through an imino group.

When X is a carboamidosulfonylamino OHO II I II -C-N-S- II O the class of ligand analogs represented by R are derived from the corresponding ligand by removal of a reactive 15 hydrogen atom, i.e., 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 wherein an imino group H I -N-. 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, 25 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, 30 desimpramine and nortriptyline. In addition, a ligand may 202613 be structurally modified by the addition or deletion of one or more functional groups to form a ligand-analog, while retaining the necessary epitope sites for binding to an antibody. However, it is preferred that such modified ligand-analogs be bonded to a carboamidosulfonyl-aminofluorescein moiety through an imino or oxy group.

The tracers of the present invention are prepared in accordance with known techniques. When X is a sulfonyl group, 0 I! -S- II 0 the tracers of the present invention are prepared by reacting a compound of the formula; (ID R-Y wherein R is above-defined and Y is an aromatic hydrogen, preferably bonded to a phenyl ring; with chlorosulfonic acid to produce a chlorosulfonyl ligand-analog of the formula; 0 R-S-Cl 11111 II 0 The chlorosulfonyl ligand-analog is reacted with an amino-fluorescein of the formula: 2026 OH 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 2026 1 3 When X is an oxalyl group, 0 0 the tracers of the present invention are prepared by reacting a compound of the formula (VI) R-Z wherein R is above-defined and Z is a hydrogen bonded to a reactive nitcrogen (primary or secondary amine), with methyloxalylchloride to yield a methoxyoxalyl ligand-analog which is hydrolyzed in the presence of a base to yield 10 hydroxyoxalyl ligand-analog of the formula 0 0 II |l (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-15 imide hydrochloride, and an inert solvent to yield a tracer of the present invention of the formula: 202613 0 0 H II II I R-C-C-Nv \ //\ A 0 OH (VIII) When X is a carboamidosulfonyl, OHO II I II -C-N-S- II 0 the tracers of the present invention are prepared by reacting 5 a compound of the formula wherein R is above-defined and W is a hydrogen bonded to a reactive nitrogen (.primary or secondary amine) or to a hydroxy oxygen; with chlorosulfonylisocyanate to yield 10 a chlorosulfonamidocarbonyl-ligand-analog derivative of the formula OHO II I II (X) R-C-N-S-Cl II 0 The chlorosulfonamidocarbonyl-ligand-analog derivative is reacted with an aminofluorescein of formula (IV) to yield the tracers of the present invention of the formula: OH The temperature at which the reaction for preparing the tracers of this invention proceeds is not critical. The 5 temperature should be one which is sufficient so as to initiate and maintain the reaction. Generally, for convenience and economy, room temperature is sufficient, In preparing the tracers of the present invention, the ratio of reactants is not narrowly critical, For example, for each mole of a compound 10 of formula (II), one should employ 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 recovery of the reaction products.

The compounds of formula (.IV). employed as starting 15 materials in the production of the tracers of this invention are either commercially available or prepared in accordance with known techniques. 202613 For ease of handling and recovery of product, the process for preparing the tracers of the present invention is conducted in the presence of an inert solvent. Suitable inert solvents include those solvents which do not 5 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, the reaction preferably proceeds under neutral or basic conditions. 10 Suitable bases 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 present invention.

In accordance with the method of the present invention, a sample containing the ligand to be determined is intermixed with a biologically acceptable salt of a tracer of formula CI) 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 of ligand-antibody and tracer-antibody complexes. By maintaining constant the concentration of tracer and antibody, the ratio of ligand-antibody complex to tracer-antibody complex that is formed is directly proportional to the amount 25 of ligand present in the sample. Therefore, upon exciting the mixture with fluorescent light and measuring the polarization 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. 3 0 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 antibody 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 observed polarization of fluorescence of the tracer-antibody complex becomes a value somewhere between that 20261 of the tracer and tracer-antibody complex. If a sample contains a high concentration of a ligand, the observed polarization value is closer to that of the free ligand, i.e., low. If the test sample contains a low concentration of the ligand, the polarization value is closer to that of the bound ligand, i.e. , high. B: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 mixture may be 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 be sufficient to allow the tracers of formula (I) to exist in their ionized state. The pH may range from about 3 to 12, more usually in the range of from 2 0 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 borate, 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 -2 -13 will generally vary from about 10 to 10 M, more usually -4 —10 from about 10 to 10 M, Higher concentrations of ligand may be assayed upon dilution of the original sample.

In addition to the concentration range of ligand of interest, considerations such as whether the assay is 202613 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 5 of ligand in the sample will determine the range of concentration 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 0 ordinary skill in the art.

As previously mentioned the preferred tracers of the present invention are prepared from 5-aminofluorescein or 4-aminofluorescein and exist preferably as isomers of the formula; OH or 202613 OH o wherein R and X are above defined.

The following illustrative, nonlimiting examples will serve to further demonstrate to those skilled in the art the manner in which specific tracers within the scope of this invention may be prepared. The symbol [AF] appearing in the structural formulas illustrating the compounds prepared in the following examples, represents a moiety of the formula: 2026 1 3 OH f~\ A»\ \ (XIV) 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. 20261 3 EXAMPLE I To 0.71 g of lidocaine was added 2.8 g of chlorosulfonic acid and the resultant mixture was heated at 50° C for one hour. The reaction mixture was cooled and crushed ice and water were added to the mixture to dissipate 5 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 were comhined, dried over sodium sulfate, filtered and 10 evaporated to dryness to yield 100 mg of a mixture of meta-and para- chlorosulfonyl lidocaine 3s a gummy 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. 15 The crude product was purified by thin-layer chromatography using silica gel and chloroform, then a mixture of chloroform: acetone (.1:1) and finally a mixture of chlorofqrm:methanol Cl:l) developing solvents to yield a mixture of sulfonyl-lidocaine-aminofluorescein conjugate of the general formula: EXAMPLE II To 1.2 g of phenobarbital was slowly added 4.5 g of chlorosulfonic acid and the resultant mixture was heated 25 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 2026 1 3 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-phenobarbital having a melting point of 190i-195o C, To a 5 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 employing thin-layer chromatography techniques employing silica gel and a mixture 10 of chloroform:methanol (.2:1) as a developing solvent to yield a sulfonylphenobarbital-aminofluorescein conjugate of the formula: 0=C S--MF] EXAMPLE 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 water 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-Cmeta- and para- chlorosulfonylphenethyl)-25 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 5 mg of the above mixture to a-Cchloro-sulfonylphenethyl)-a-methylsuccinimide. After 10 minutes , 2026 a crude product had formed which was purified twice employing thin-layer chromatographic techniques utilizing silica gel and a -mixture of chloroformrmethanol (.2:11 as a developing solvent to yield a mixture of (sulfonylphenethyl)-5 a-methylsuccinimide-aminofluorescein conjugate of the formula: 0=C CH. CH, ^ / 3 H-N C" 1 0 CH2CH2 EXAMPLE IV To a solution containing 2 g of iminostilbene and 2 ml of triethylamine in 50 ml of chloroform was added 10 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 15 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 1 using concentrated hydrochloric acid and then extracted with 100 ml of ether. The organic extract was dried over 20 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-iminostilbene (melting point 162 - 163° C). 25 to 5 mg of the N-hydroxyoxalyl-iminostilbene was added a solution containing 5 mg of 4-aminofluorescein in 202613 0.5 ml of pyridine. The reaction was allowed to proceed for two hours at 26° C to yield a crude product. The crude product was purified using thin<-layer chromatography employing silica gel and a developing solution consisting of a mixture of chloroform:acetone (1:1) to yield an N-oxalyl-iminostilbene-aminofluorescein conjugate of the formula: The following tracers were also prepared in accordance with the above procedures: EXAMPLE V - Sulfonylprimidone-aminofluorescein conjugates H.

\ / \ 'N i—EAJ] H 202613 EXAMPLE VI - Para-methyl-meta-sulfonylprimidone- aminofluorescein conjugate ' EXAMPLE VII - Para-methyl-meta-sulfonylphenobarbital- aminofluorescein conjugate /N ^ ^ o=c c -N O h' v0 -S-f4F] 0 CH3 EXAMPLE VIII - 5-Sulfonylphenyl-5-ethylhydantoin- aminofluorescein conjugate 0 Hx II N C CE.CE, 0 \ / 2 3 II EXAMPLE IX - a-CSulfonylphenyl)-a-methylsuccinimide- aminofluorescein conjugate K-n \ / .1 \ / CH.

-C-\\ V '// -CH, EXAMPLE X - O-sulfonainidocarbonylpropranolol- aminofluorescein conjugate H OHO V, //-O-CH.-C-O-C-N-S—f-AF] \ / 2 'J H CH, I I 3 CH--N-C-H 2 I CH„ 202613 EXAMPLE XI - N-sulfonamidocarbonyl-iminostilbene- aminofluorescein conjugate " EXAMPLE XII - N-sulfonamidocarbonyl-procainamide- aminofluorescein conjugate ft 0 ft 0 EXAMPLE XIII - O-sulfonamidocarbonyl-chloramphenicol- aminofluorescein conjugate ' H H OHO 02N-<^ ^-C-C-CH^-O-C^N-jj—fcflF] / \ H C-CHC1, 202613 EXAMPLE XIV - N-sulfonamidocarbonyl-disopyramide- aminofluorescein conjugate t .CH.

CH 3 CH-, V H-C' O H 0 H O ,N-CH2CH2-C-C-N-C-N-S—f-AF] \ CH.

N EXAMPLE XV - o-sulfonamidocarbonyl-quinidine-5 aminofluorescein conjugate t^CH=CH2 OCH N OHO . II I II H-C-O-C-N-S—\AF] II O 20261 3 EXAMPLE XVI - O-oxalyl-propranolol- aminofluorescein conjugate \J \ // H 0 0 I I I -0CH--C-0-C-C—frAF] CH- / CH--N-C-H 2 I \ H CH.

EXAMPLE XVII - N-oxalyl-procainamide-5 aminofluorescein conjugate H5C,2 H 0 I I ^N-CH2CH2-N-CT \ /) H 0 0 I II II -N-C-C—MF] H5C2 EXAMPLE XVIII - N-acetyl-N' -desethyl-N' -oxalyl— procainamide-aminofluorescein conjugate 0 H V o a CH.

C-N-(v At-C-N-CH2CH2^NC -C2H5 ^C-C-WF] II » 0 0 EXAMPLE XIX - N-oxalyl-nortriptyline- aminofluorescein conjugate !=CHCH2-Ctt2-Nr ,ca3 'C-C-frAF] II II 0 0 EXAMPLE XX - N-oxalyl-iminodibenzyl- aminofluorescein conjugate 1 ■ 20261 As previously mentioned, the tracers of the present invention are effective reagents for use in fluorescence polarization immunoassays. The following Examples illustrate the suitability of tracers of the present 5 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 present invention optionally containing a surfactant is then added to each tube; 3) A known concentration of antisera is added to the tubes; 4) The reaction mixture is incubated at room temperature; and ) The amount of tracer bound to antibody is measured by fluorescence polarization techniques as a measure of the amount of ligand in the sample.

EXAMPLE XXI - Lidocaine Assay Materials: 1) Buffer: 0,lM phosphate, pH 7.5, containing 0.01% (weight/volume! sodium azide and Q.01% (weight/volume1 bovine gamma globulin (hereinafter referred to as "BGG buffer"). 2) Tracer: Sulfonyllidocaine^aminofluorescein conjugate (prepared in Example II at a concentration of 2.34 x 10 ^M in O.lM tris hydrochloride buffer, pH 7.8, containing 0.1% (weight/volume) sodium dodecyl sulfate, 3 0 0.01% (weight/volume) bovine gamma globulin and 0.01% (weight/volume) sodium azide. 3) Antibody: Rabbit 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. 2026 ) Fluorescence polarimeter; Instrument capable - _ q of measuring the polarization of fluorescence of a, 1 X 10 ,M fluorescein solution to * 0.001 polariaztion units, Protocol; 11 To 20 of standards and unknowns add 200 vl of BGG buffer. 2) TO 20 pt of each diluted standard and unknown in a culture tube, add 200 of BGG buffer. 3) To each culture tube containing diluted 10 standard and unknown add 40 pi of tracer and 1000 uS. of BGG buffer. 4) Then add 40 of antibody and 1000 yl of BGG buffer to each culture tube.

) Mix the reagents and incubate the culture tubes 15 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 The polarization values decreases as the concentration of lidocaine is increased, allowing construction 30 of a standard curve. Unknowns treated in an identical manner may be guantitated by reference to the standard curve.

Lidocaine Concentration Cug/ml) 0 Polarization 0.224 0 .186 0.162 0.124 0. 094 0.071 0.5 1.0 2.5 5.0 10, 0 20261 3 EXAMPLE XXII - Phenobarbital Assay Materials: 1). BGG buffer 2) Tracer: Cpara-methyl-meta-sulfonyl)phenoba,rbital- aminofluorescein conjugate (prepared in Example VII). at a — 8 concentration of 5.75 x 10 M 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 range of 0 to 80 ug/ml, ) Fluorescence polarimeter: Instrument capable -9 of measuring the polarization of fluorescence of a 1 X 10 M fluorescein solution to t 0.001 polarization units.

Protocol: 1) To 20 yS, °f standards and unknowns add 200- yH of BGG buffer. 2). To 20 y £ of each diluted standard and unknown in a culture tube, add 200 yJl of BGG buffer. 3) To each culture tube containing diluted standard and unknown add 40 pi of tracer and 100Q y& of BGG buffer. 4) Then add 40 y£ of antibody and 1000 y£ of BGG buffer to each culture tube.

) Mix 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 II. 2026 TABLE II Phenobarbital Concentration (pg/mll Polarization 0 10 20 40 80 0.155 0.108 0.092 0. 074 0.060 The polarization values decrease 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.

EXAMPLE XXIII - Carbamazepine Assay Materials: 1) BGG buffer. 2) Tracer: N-oxalyliminostilbene-aminofluorescein conjugate (prepared in Example IVj_ at a concentration of 2nM in BGG buffer containing 0,2% (weight/volume1 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 yg/ml, ) Fluorescence polarimeter: Instrument capable - 9 of measuring the polarization of fluorescence of 1 X 10 M fluorescence solution to - 0,001 polarization units.

Protocol; 1) To 10 pi of standards and unknowns add 300 of BGG buffer, 2) To 10 pi of each diluted standard and unknown in a culture tube, add 300 yi. of BGG buffer, 3) Add 1 ml of tracer to each culture tube. 202613 4). Then add 1.0 ml of antibody to each, culture tube. 51 Mix the reagents and incubate the culture tubes containing standards and unknowns for approximately 15 minutes 5 at 23° C. 61 Measure the fluorescence polarization of all tubes. Typical results for standard samples are presented in Table III.

TABLE XII Carbamazepine Concentration tug/ml) Polarization 0 0,224 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 20 construction of a standard curve. Unknowns treated in an identical manner may be quantitated by reference to the standard curve.

The following table summarizes the various fluorescence polarization immunoassays that haye been carried 25 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 ligandCs) determined are indicated. 202613 Tra,cer Prepared In Example Number I II III IV V VI VII VIII IX X XI XII XIII XIV XV XVI XVII XVIII XIX XX Ligand (.s). Assayed Lidocaine Phenobarbital Ethosuximide Carbamazepine Primidone Primidone Phenobarbital Phenytoin Ethosuximide Propanolol Carbamazepine Procainamide Chloramphenicol Disopyramide Quinidine Propanolol Procainamide N-Acetyl Procainamide Desipramine; Imipramine Nortriptyline;Amitriptyline