GB2111476A - Substituted carboxy- fluoresceins and their use in fluorescence polarization immunoassay - Google Patents

Substituted carboxy- fluoresceins and their use in fluorescence polarization immunoassay Download PDF

Info

Publication number
GB2111476A
GB2111476A GB8203971A GB8203971A GB2111476A GB 2111476 A GB2111476 A GB 2111476A GB 8203971 A GB8203971 A GB 8203971A GB 8203971 A GB8203971 A GB 8203971A GB 2111476 A GB2111476 A GB 2111476A
Authority
GB
Grant status
Application
Patent type
Prior art keywords
compound according
ligand
derived
gt
lt
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
GB8203971A
Other versions
GB2111476B (en )
Inventor
Michael Ernest Jolley
Stephen Denham Stroupe
Chao-Huei Jeffrey Wang
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Abbott Laboratories
Original Assignee
Abbott Laboratories
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date

Links

Classifications

    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07DHETEROCYCLIC COMPOUNDS
    • C07D405/00Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
    • C07D405/02Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings
    • C07D405/12Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings linked by a chain containing hetero atoms as chain links
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07DHETEROCYCLIC COMPOUNDS
    • C07D311/00Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
    • C07D311/02Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D311/78Ring systems having three or more relevant rings
    • C07D311/80Dibenzopyrans; Hydrogenated dibenzopyrans
    • C07D311/82Xanthenes
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07DHETEROCYCLIC COMPOUNDS
    • C07D473/00Heterocyclic compounds containing purine ring systems
    • C07D473/02Heterocyclic compounds containing purine ring systems with oxygen, sulfur or nitrogen atoms directly attached in positions 2 and 6
    • C07D473/04Heterocyclic compounds containing purine ring systems with oxygen, sulfur or nitrogen atoms directly attached in positions 2 and 6 two oxygen atoms
    • C07D473/06Heterocyclic compounds containing purine ring systems with oxygen, sulfur or nitrogen atoms directly attached in positions 2 and 6 two oxygen atoms with radicals containing only hydrogen and carbon atoms, attached in position 1 or 3
    • C07D473/08Heterocyclic compounds containing purine ring systems with oxygen, sulfur or nitrogen atoms directly attached in positions 2 and 6 two oxygen atoms with radicals containing only hydrogen and carbon atoms, attached in position 1 or 3 with methyl radicals in positions 1 and 3, e.g. theophylline
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07JSTEROIDS
    • C07J41/00Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring
    • C07J41/0005Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring the nitrogen atom being directly linked to the cyclopenta(a)hydro phenanthrene skeleton
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by the preceding groups
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531Production of immunochemical test materials
    • G01N33/532Production of labelled immunochemicals
    • G01N33/533Production of labelled immunochemicals with fluorescent label

Abstract

Novel compounds of the formula: <IMAGE> wherein R is a ligand-analog having a single reactive primary or secondary amino group which is attached to a carbonyl carbon atom of a carboxy- fluorescein moiety and having a molecular weight within the range 100 to 2000; wherein said ligand- analog has at least one common epitope with a ligand so as to be specifically recognisable by a common antibody, are useful as tracer compounds in a fluorescence polarization immunoassay procedure. <IMAGE>

Description

SPECIFICATION Fluorescence polarization im munoassay utilizing substituted carboxyfluoresceins 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 tracerantibody conjugate produced may be quantitively measured and is inversely proportional to the quantity of ligand in the test sample.

In general, fluorescence polarization techniques are based on the principle that a fluorescent labeled compound when excited by linearly polarized light will emit fluorescence having a degree of polarization inversely related to its rate of rotation. Therefore, when a molecule such as a tracerantibody 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 (i.e., unbound to an antibody) is excited by linearly polarized light, its rotation is much faster than the corresponding tracer-antibody conjugate and the molecules are more randomly oriented, therefore, the emitted light is depolarized.Thus, fluorescence polarization provides a quantitive means for measuring the amount of tracer-antibody conjugate produced in a competitive binding immunoassay.

Various fluorescent labeled compounds are known in the art. U.S. Patent No. 3,998,943 describes the preparation of a fluorescently labeled insulin derivative using fluorescein isothiocyanate (FITC) as the fluorescent label and a fluorescently labeled morphine derivative using 4aminofluorescein hydrochloride as the fluorescent label. Carboxyfluorescein has also been used for analytical determinations. R. F. Chen, Anal. Lett., 10, 787 (1977) describes the use of carboxyfluorescein to indicate the activity of phospholipase. However, carboxyfluorescein is not conjugated according to the present invention. It is encapsulated in lecithin liposomes, and it will fluoresce only when released by the hydrolysis of lecithin.

The present invention encompasses a method for determining ligands in a sample comprising intermixing with said sample of biologically acceptable salt of a tracer of the formula: <img class="EMIRef" id="027228596-00010001" />

wherein R is a ligand-analog having a single reactive primary or secondary amino group which is attached to the carbonyl carbon of the carboxyfluorescein wherein said ligand-analog has at least one common epitope with said ligand so as to be specifically recognizable by a common antibody; and an antibody capable of specifically recognizing said ligand and said tracer; and then determining the amount of tracer antibody conjugate by fluorescence polarization techniques as a measure of the concentration of said ligand in the sample.

The invention further relates to certain novel tracers and biologically acceptable salts thereof, which are useful in reagents in the above-described method. The methods and tracers of the present invention are particularly useful in quantitatively monitoring therapeutic drug concentrations in serum and plasma.

The term "ligand" as used herein refers to a molecule, in particular a low molecular weight hapten having a single reactive amino group, to which a receptor, normally an antibody, can be obtained or formed. Such 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 conjugating the haptens to a protein and injecting the conjugate product into an animal. The resulting antibodies are isolated by conventional antibody isolation techniques.

Ligands determinable by the method of the present invention vary over a wide molecular weight range. Although high molecular weight ligands may 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 a range of 100 to 2000.

The novel tracers of the present invention include compounds of formula (I) wherein the ligandanalog represented by R include radicals having a molecular weight within a range of 50 to 4000. The preferred novel tracers include compounds of formula (I) wherein the ligand-analogs represented by R include radicals having a molecular weight within a range of 100 to 2000.

Representative of ligands having a single reactive amino group determinable by the methods of the present invention include steroids such as esterone, estradoil, cortisol, testoestrone, 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, prostaglandins such as PGE, PGF, PGA; antiasthmatic drugs such as theophylline, antineoplastic drugs such as doxorubicin and methotrexate antiarrhythmic drugs such as disopyramide, lidocaine, procainamide, propranolol, quinidine, N-acetyl-procainamide; anticonvulsant drugs such as phenobarbital, phenytoin, primidone, valproic acid, carbamazepine and ethbsuximide; antibiotics such as penicillins, cephalosporins and vancomycin; antiarthritic drugs such as salicylate; antidepressant drugs including tricyclics such as nortriptyline, amitriptyline, imipramine and desipramine; and the like as well as the metabolites thereof. Additional ligands that may be determined by the methods of the present invention include drugs of abuse such as morphine, heroin, hydromorphone, oxymorphone, metapon, codeine, hydrocodone, dihydrocodeine, dihydrohydroxy, codeinone, pholcodine, dextromethorphan, phenazocine and deonin and their metabolites.

The tracers of the present invention generally exist in an equilibrium between their acid and ionized states, and in the ionized state are effective in the method of the present invention. Therefore, the present invention comprises the tracers in either the acid or ionized state and for convenience. the tracers of the present invention are structurally represented herein in their acid form. When the tracers of the present invention are present in their ionized state, the tracers exist in the form of biologically acceptable salts. As used herein, the term "biologically acceptable salts" refers to salts such as sodium, potassium, ammonium and the like which will enable the tracers of the present invention to exist in their ionized state when employed in the method of the present invention.Generally, the tracers of the present invention exist in solution as salts, the specific salt results from the buffer employed, i.e., in the presence of a sodium phosphate buffer, the tracers of the present invention will generally exist in their ionized state as a sodium salt.

The tracers of the present invention comprise a ligand-analog represented by R linked to a carboxyfluorescein moiety of the formula: <img class="EMIRef" id="027228596-00020001" />

The term ligand-analog as used herein refers to 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 sufficient structural similarity to the ligand of interest so as to be recognized by the antibody for the ligand. For the most part, the ligand analog will have the same or substantially the same structure and charge distribution (spatial and polar organization) as the ligand of interest for a significant portion of the molecular surface. Since frequently, the linking site for a hapten will be the 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.

In general, the class of ligand analogs represented by R are derived from the corresponding ligand by removal of a reactive hydrogen atom, i.e., a hydrogen atom bonded to a reactive amine (primary or secondary) or by the formation of an amino derivative of the ligand wherein an imino group <img class="EMIRef" id="027228596-00030001" />

replaces one or more atoms originally present in the ligand, at the site of binding to the carboxyfluorescein moiety. Illustrative of ligands which upon the removal of a reactive hydrogen may form ligand-analogs represented by R include for example, procainamide, thyroxine and quinidine.

Illustrative of ligands whose amino derivatives are useful as ligand-analog include theophylline, valproic acid, phenobarbital, phenytoin, primidone, disopyramide, digoxin, chloramphenicol, salicylate, acetaminophen, carbamazepine, desipramine 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, such modified ligandanalogs are bonded to the carboxyfluorescein moiety through an imino group.

The tracers of the present invention are generally prepared in accordance with known techniques.

For example, a compound of the formula: R-X (III) wherein R is above-defined and X is a reactive hydrogen; is treated with a compound of the formula: <img class="EMIRef" id="027228596-00030002" />

wherein R is hydroxy or an active ester, and wherein the carboxy group is preferably bonded to the 4 or 5 position of the benzoic acid ring; in the presence of an inert solvent to yield a compound of formula (I).

As used herein, the term "active ester" refers to a moiety which is readily "removed" from the carboxy carbon in the presence of a coupling agent. Such "active esters" of carboxyfluorescein are readily ascertained by one of ordinary skill in the art and are prepared from the reaction of carboxyfluorescein with a compound such as N-hydroxy-succinimide, 1 -hydroxybenzotriazole hydrate or p-nitrophenol in the presence of a coupling agent, such as dicyclohexyicarbodiimide and a solvent.

The active esters of carboxyfluorescein thus produced are subsequently reacted with a compound of formula (III) to yield a tracer of formula (I).

If the compound of formula (III) is water soluble, the reaction mechanism proceeds by directly reacting carboxyfluorescein with a compound of formula (III) in aqueous solution in the presence of a water soluble carbodiimide, such as 1 -ethyl-3-(3'-dimethylaminopropyl)-carbodiimide hydrochloride, as a coupling agent.

The temperature at which the process for preparing the tracers of this invention proceeds is not critical. The temperature should be one which is sufficient so as to initiate and maintain the reaction.

Generally, for convenience and economy, room temperature is sufficient. In preparing the tracers of the present invention, the ratio of reactants is not narrowly critical. For each mole of a compound of formula (II), one should employ one mole of a compound of formula (III) to obtain a reasonable yield. It is preferred to employ an excess of compound of formula (III) for ease of reaction and recovery of the reaction products.

For ease in handling and recovery of product, the process for preparing the tracers of the present invention is conducted in the presence of an inert solvent. Suitable inert solvents include those solvents which do not react with the starting materials and are sufficient to dissolve the starting materials, and include for example water (if the compound of formula (III) is water soluble), dimethylformamide, dimethylsulfoxide and the like. If the compound of formula (III) is a reactive amine salt, a suitable base is added to the reaction mixture to form the free base of the reactive amine. Suitable bases include for example, triethylamine. The reaction products of formula (I) are generally purified using either thinlayer or column chromatography prior to application in the methods of the present invention.

In accordance with the method of the present invention, a sample containing the ligand to be determined is intermixed with a biologically acceptable salt of a tracer of formula (I) and an antibody specific for the ligand and tracer. The ligand present in the sample and the tracer compete for limiting antibody sites resulting in the formation of ligand-antibody and tracer-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 of ligand present in the sample. Therefore, upon exciting the mixture with polarized 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.

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 smaller 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 tracerantibody complex becomes a value somewhere between that of the tracer and tracer-antibody complex. If a sample contains a high concentration of the ligand, the observed polarization value is closer to that of the free ligand, i.e., iow. 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.By sequentially exciting the reaction mixture of an immunoassay with vertically and then horizontally polarized light and analyzing only the verticai component of the emitted light, the polarization of fluorescence in the reaction mix 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 about 5 to 1 0, 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 500 C, more usually from about 1 50 to 400C.

The concentration of ligand which may be assayed will generally vary from about 1 0-2 to 1 0-13M, more usually from about 10-4 to 1 O-10M. Higher concentrations of ligand may be assayed upon dilution of the original sample.

The addition to the concentration range of ligand of interest, considerations such as whether the assay is qualitative, semiquantitative or quantitative, the equipment employed, and the characteristics of the tracer and antibody will normally determine the concentration of the tracer and antibody to be employed. While the concentration 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 ordinary skill in the art.

As previously mentioned the preferred tracers of the present invention are prepared from 5carboxyfluorescein or 4-carboxyfluorescein or mixtures thereof and are represented by the formulas: <img class="EMIRef" id="027228596-00040001" />

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 the invention may be prepared.

The symbol [CF] appearing in the structural formulas illustrating the compounds prepared in the following examples, represents a moiety of the formula: <img class="EMIRef" id="027228596-00050001" />

wherein the carbonyl carbon is attached to the 4 or 5 position in the above formula in view of the fact that a mixture of 4- and 5-carboxyfluorescein is employed as starting material.

Example I Meta- or para- aminophenobarbital (5 mg) and carboxyfluorescein (5 mg) were dissolved in 0.5 ml of pyridine. To the mixture was added N,N'-dichohexylcarbodiimide (15 mg). The reaction proceeded for two hours at room temperature, after which time the reaction product was purified twice employing silica gel thin-layer chromatography using a chloroform:methanol (2:1) mixture as developing solvent to yield an aminophenobarbital-carboxyfluorescein conjugate of the formula: <img class="EMIRef" id="027228596-00050002" />

Example II A solution containing sodium hydroxide (1.0 g), phenytoin (2.5 g) and 2-bromomethylamine hydrobromide (2.0 g) in 100 ml of 100% ethanol was refluxed for two hours and then evaporated to dryness under reduced pressure.The residue was suspended in 50 ml of water and the pH was adjusted to pH 11 by the addition of 6N sodium hydroxide to dissolve any unreacted phenytoin. The remaining precipitate, 2-p-aminoethylphenytoin, was filtered, rinsed thoroughly with water and dried.

An active ester of carboxyfiuorescein was prepared by dissolving N-hydroxysuccinimide (5 mg), carboxyfluorescein (7.5 mg) and N,N'-dicyclohexylcarbodiimide (20 mg) in 0.5 ml of pyridine. The reaction was allowed to proceed for two hours at room temperature after which time 2-p- aminoethylphenytoin (10 mg) was 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 employing silica gel thin-layer chromatography using a chloroform:methanol (3:1) mixture as developing solvent to yield a 2-B-aminoethylphenytoin-carboxyfluorescein conjugate of the formula: <img class="EMIRef" id="027228596-00050003" />

Example III A solution containing 2-carboxymethylphenytoin (620 mg), N-hydroxysuccinimide (248 mg) and N,N'-dicyclohexylcarbodiimide (453 mg) in 6 ml of dry dimethylsulfoxide was allowed to stand at room temperature overnight. The mixture was filtered and 0.7 ml of 95% hydrozine was added to 4.5 ml of the filtrate. After four hours at room temperature, 40 ml of water and 0.5 ml of 10% sodium hydroxide were added to the reaction mixture.The precipitate, 2-carboxymethylphenytoin hydrazide, was filtered, rinsed with water, dried and used without further purification.

N,N'-dicyclohexylcarbodiimide (15 mg) was added to a solution of 2-carboxymethylphenytoin hydrazide (5 mg) and carboxyfluorescein (5 mg) in 0.5 ml of pyridine. The reaction was allowed to proceed for two hours at room temperature, and the reaction product was then purified twice employing silica gel thin-layer chromatography using a chloroform:acetone (1:1) mixture as developing solvent to yield a 2-carboxymethylphenytoin hydrazide-carboxyfluorescein conjugate of the formula: <img class="EMIRef" id="027228596-00060001" />

Example IV N,N'-dicyclohexylcarbodiimide (1 5 mg) was added to a solution of 8-ss-aminoethyltheophylline (5 mg) and carboxyfluorescein (5 mg) in 0.5 ml of pyridine.The reaction was allowed to proceed for two hours at room temperature and the reaction product was purified twice employing silica gel thin-layer chromatography using a thin chloroform:methanol (2:1) mixture as developing solvent to yield an 8-ss- amino ethyltheophylline-carboxyfluornscein conjugate of the formula: <img class="EMIRef" id="027228596-00060002" />

Example V The procedure of Example IV was employed utilizing 8-aminomethyltheophylline in lieu of 8-P- aminoethyltheophylline to yield an 8-aminomethyltheophylline-carboxyfluorescein conjugate of the formula: <img class="EMIRef" id="027228596-00060003" />

Example VI 8-Valerolactam (7.5 g) was dissolved in 60 ml of dry tetrahydrofuran, under a dry nitrogen atmosphere and n-butyllithium (1.6 M, 90 ml) in hexane were added dropwise to the reaction flask and chilled in a dry ice-acetone bath.Upon completion of the addition of n-butyllithium, the reaction mixture was stirred at room temperature for one hour, refluxed for thirty minutes, and cooled to room temperature under dry nitrogen atmosphere. 1 -Bromoethane (8.0 g) was slowly added to the reaction flask while the flask was chilied in an ice bath. The resulting mixture was then stirred for sixteen hours at room temperature after which time 100 ml of water was slowly added. The resulting mixture was stirred at room temperature for thirty minutes and the organic layer separated. The aqueous layer was extracted with 50 ml of diethyl ether and the organic layers were combined and dried over sodium sulfate. The solvent was evaporated to give a dark oil, which crystallized on standing. The crystalline residue was recrystallized from petroleum ether to yield 3.8 g of a residue.The residue (2.8 g) was refluxed in 25 ml of 6N hydrochlorid acid for six hours. The water was evaporated from the mixture to yield a dark, thick oil-2-ethyl-5-aminopentanoic acid-which was used without further purification.

An active ester of carboxyfluorescein was prepared by dissolving N-hydroxysuccinimide (5 mg), carboxyfiuorescein (7.5 mg) and N,N'-dicyclohexylcarbodiimide (20 mg) in 0.5 ml of pyridine. The reaction was allowed to proceed for two hours at room temperature, after which time 2-ethyl-5aminopentanoic acid (20 mg) was 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 employing silica gel thin-layer chromatography using a chloroform:methanol (3:1) mixture as developing solvent to yield a 2-ethyl-5-aminopentanoic acid-carboxyfluorescein conjugate of the formula: <img class="EMIRef" id="027228596-00070001" />

Example VII An active ester of carboxyfluorescein was prepared by dissolving N-hydroxysuccinimide (5 mg), carboxyfluorescein (7.5 mg) and N,N'-dicyclohexylcarbodiimide (20 mg) in 0.5 ml of pyridine.The reaction was allowed to proceed for two hours at room temperature, after which time 5-(y-amino propylidene)-5H-dibenzo[a,d]-1 0,11 -dihydrocycloheptene (20 mg) was 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 employing silica gel thin-layer chromatography using a chloroform:methanol (3:1) mixture as developing solvent to yield a 5-(y-aminopropylidene)-5H dibenzo[a,d]-l 0,1 1-dihydrocycloheptene-carboxyfluorescein conjugate of the formula: <img class="EMIRef" id="027228596-00070002" />

Example VIII A solution containing desipramine hydrochloride (1.33 g) and chloroacetyl chloride (0.8 g) in 25 ml of chloroform was refluxed for two hours. The chloroform was evaporated and the residue was dissolved in 25 ml of acetone.Sodium iodide (0.75 g) was added to the acetone solution, and the solution was refluxed for thirty minutes. The solution was filtered and the precipitated salt was rinsed with acetone. The acetone filtrate was evaporated and the residue was taken up in 20 ml of methanol.

Concentrated ammonium hydroxide (20 ml) was added to the methanol solution and the resulting solution was refluxed for one hour. The reaction mixture was extracted three times with 25 ml of chloroform and combined extracts were dried over sodium sulfate, filtered and evaporated to yield Naminoacetyldesipramine which was used without further purification.

N-aminoacetyldesipramine (5 mg) and carboxyfluorescein (5 mg) were dissolved in 0.5 ml of pyridine. To the mixture was added N,N'-diclohexylcarbodiimide (15 mg). The reaction proceeded for two hours at room temperature, after which time the reaction product was purified twice employing silica gel thin-layer chromatography using a chloroform:acetone (1:1) mixture as developing solving to yield a N-aminoacetyidesipramine-carboxyfluorescein conjugate of the formula: <img class="EMIRef" id="027228596-00070003" />

Example IX A solution containing N-hydroxysuccinimide (5 mg), carboxyfluorescein (7.5 mg) and N,N'dicyclohexylcarbodiimide (20 mg) in 1 ml of pyridine was allowed to react at room temperature for four hours. An active ester of carboxyfluorescein was precipitated by adding 10 ml of diethylether to the reaction mixture.The precipitate was filtered, rinsed well with diethylether and redissolved in 0.5 ml of dimethylsulfoxide. L-thyroxine (10 mg) was then added to the solution and the reaction was allowed to proceed for two hours at room temperature after which time the reaction product was purified twice employing silica gel thin-layer chromatography using a chloroform:methanol (3:1) mixture as developing solvent to yield a L-thyroxine-carboxyfluorescein conjugate of the formula: <img class="EMIRef" id="027228596-00080001" />

Example X A solution containing ammonium acetate (0.89 g), 3-oxodigoxigenin (389 mg) and sodium cyanoborohydride (63 mg) in 5 ml of methanol was stirred at room temperature for 48 hours. The solution was adjusted to pH 1 by the addition of concentrated hydrochloric acid and evaporated to dryness under reduced pressure. The residue was taken up in 10 ml of water and extracted three times with 10 ml of chloroform.The aqueous layer was adjusted to pH 11 by using solid potassium hydroxide. The resulting solution was extracted five times with 10 ml of methylene chloride. The organic layers were combined, dried and then evaporated to dryness under reduced pressure to yield 3amino-3-deoxydigoxigenin which was used without further purification.

An active ester of carboxyfluorescein was prepared by dissolving N-hydroxysuccinimide (5 mg), carboxyfluorescein (7.5 mg) and N,N'-dicyclohexylcarbodiimide (20 mg) in 0.5 ml of pyridine. The reaction was allowed to proceed for two hours at room temperature, after which time a 3-amino-3deoxydigoxigenin-carboxyfluorescein conjugate of the formula was isolated: <img class="EMIRef" id="027228596-00080002" />

The following tracers were also prepared in accordance with the procedures previously described:: Example XlO-Aminoacetyl-propranolol-carboxyfluorescein conjugate <img class="EMIRef" id="027228596-00080003" />

Example XlI-2-Propyl-5-aminopentanoic acid-carboxyfluorescein conjugate <img class="EMIRef" id="027228596-00090001" />

Example XIll-2-Butyl-5-aminopentanoic acid-carboxyfluorescein conjugate <img class="EMIRef" id="027228596-00090002" />

Example XIV-Aminoprimidone-carboxyfluorescein conjugate <img class="EMIRef" id="027228596-00090003" />

Example XV-1 -(4'-nitrophenyl)-1 -hydroxy-2-amino-3-hydroxypropane-carboxyfluorescein conjugate <img class="EMIRef" id="027228596-00090004" />

Example XVI-p-aminophenol-carboxyfluorescein conjugate <img class="EMIRef" id="027228596-00090005" />

Example XVll-N-(2-a minoethyl)-ethosuxi mide-carboxyfluorescein conjugate <img class="EMIRef" id="027228596-00090006" />

Example XVlIl-N'-desethyl-N-acetylprocainamide-carboxyfluorescein conjugate <img class="EMIRef" id="027228596-00090007" />

Example XlX-N'desethyl-N'-aminoacetyl-N-acetylrocainamide-carboxyfluorescein conjugate <img class="EMIRef" id="027228596-00100001" />

Example XX-1-amino-2-phenyl-2-(2'-pyridyl)-4-(diisopropylamino)-butane-carboxyfluorescein conjugate <img class="EMIRef" id="027228596-00100002" />

Example XXl-3,3',5-Triiodo-L-thyronine-carboxyfluorescein conjugate <img class="EMIRef" id="027228596-00100003" />

Example XXII3,3',5,5'-tetraiodo-D-thyronine-carboxyfluorescein conjugate <img class="EMIRef" id="027228596-00100004" />

Example XXIII-N-aminoacetyl-iminodibenzyl carboxyfluorescein conjugate <img class="EMIRef" id="027228596-00100005" />

Example XXIV-Carbhydrazinoimino-dibenzyl carboxyfluorescein conjugate <img class="EMIRef" id="027228596-00110001" />

Example XXV-Dibenzosuberonehydrazone fluorescein conjugate <img class="EMIRef" id="027228596-00110002" />

Example XXVI-5-amino-1 1 -dihydro-5H-dibenzo-[a,d]-cycloheptene <img class="EMIRef" id="027228596-00110003" />

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 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 5) 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 XXVIl-Phenytion assay A) Materials required: 1) BGG buffer consisting of 0.1 M sodium phosphate, pH 7.5, containing bovine gammaglobulin, 0.01% and sodium azide, 0.01%.

2) Tracer, consisting of 2-P-aminoethyl phenytoin-carboxyfluorescein at a concentration of approximately 105 nM in BGG buffer with 5% sodium cholate added.

3) Antiserum, consisting of antiserum raised against phenytion diluted appropriately in BGG buffer containing 0.005% benzalkonium chloride.

4) Samples of human serum or other biological fluid containing phenytoin.

5) Cuvettes, 1 0x75 mm glass culture tubes used as cuvettes.

6) Fluorometer capable of measuring fluorescence polarization with a precision of 10.001 units.

B) Assay Method: 1) A small volume of sample (0.366 microliters) is placed in each cuvette by pipetting 1 5 yl of sample and diluting with 600 ,al BGG buffer in a dilution vessel. Next, 1 5 ,ul of diluted sample is pipetted into the cuvette followed by 600 yI BGG buffer.

2) Tracer is added by pipetting 40 yI tracer and 1000 ,ul BGG buffer into the cuvette.

3) Antiserum is added to start the reaction by pipetting 40 yl antiserum into the cuvette followed by 1000 yI BGG buffer.

4) The contents of all cuvettes are well mixed and allowed to incubate for 1 5 minutes at ambient temperature.

5) The fluorescence polarization is read on a fluorometer and a standard curve constructed to determine unknowns.

C) The results of a series of serum standards containing phenytoin at concentrations between 0 and 40 ,ug/ml are presented below. Each concentration was assayed in duplicate and averaged.

Concentration of phenytoin {/lg/mlJ Polarization 0 0.222 2.5 0.196 5.0 0.178 10.0 0.154 20.0 0.132 40.0 0.110 The polarization of fluorescence is seen to decrease in a regular manner as the phenytoin concentration increases, allowing construction of a standard curve. Unknown specimens treated in an identical manner can be quantitated by reference to the standard curve, thereby illustrating the utility of 2-P-a minoethyl phenytoin-carboxyfluorescein for the measurement of phenytoin.

Example XXVIII-Phenobarbital assay A) Materials required: 1)BGG buffer (see Phenytoin) 2) Tracer, consisting of aminophenobarbital carboxyfluorescein at a concentration of approximately 110 nM in tris HCI buffer, pH 7.5, containing 0.01% sodium azide, 0.019/0 bovine gamma globulin, and 0.125% sodium dodecyl sulfate.

3) Antiserum, consisting of antiserum against phenobarbital diluted appropriately in BGG buffer containing 0.005% benzalkonium chloride.

4) Samples of human serum or other biological fluid containing phenobarbital.

5) Cuvettes (see Phenytoin) 6) Fluorometer (see Phenytoin) B) Assay Protocol: 1) A small volume of sample (0.196 microliter) is placed in the cuvette by pipetting 10 4i of sample and diluting with 500 jul BGG buffer in a dilution vessel. Next, 10 yl of diluted sample is pipetted into the cuvette followed by 500 ul BGG buffer.

2) Tracer is added by pipetting 40 yl of tracer and 1000 yI BGG buffer into each cuvette.

3) Antiserum is added to start the reaction by pipetting 40 Ml antiserum followed by 1000 jil BGG buffer.

4) The contents of all cuvettes are mixed well and allowed to incubate for 1 5 minutes at ambient temperature.

5) The fluorescence polarization is read on a fluorometer and a standard curve constructed to determine unknowns.

C) The results of a series of serum standards containing phenobarbital at concentrations between 0 and 80 yg/ml are presented below. Each concentration was assayed in duplicate and the values averaged.

Concentrations of Phenobarbftal (i) Polarization 0 0.250 5.0 0.231 10.0 0.196 20.0 0.150 40.0 0.104 80.0 0.077 The polarization of fluorescence is seen to decrease in a regular manner as the phenobarbital concentration increases, allowing construction of a standard curve. Unknown specimens treated in an identical manner can be quantitated by references to the standard curve thereby illustrating the utility of aminophenobarbital-carboxyfluorescein for the measurement of phenobarbital.

Example XXIX--Theophylline assay A) Materials required: 1) Tracer, consisting of 2 nM of 8-aminoethyl theophylline-carboxyfluorescein in BGG buffer (see Phenytoin assay) containing 0.01% sodium dodecyl sulfate.

2) Antiserum, consisting of antiserum raised against theophylline diluted appropriately in BGG buffer.

3) Samples of human serum or other biological fluid containing theophylline.

4) Cuvettes, (see Phenytoin assay) 5) Fluorometer, (see Phenytoin assay) B) Assay protocol: 1) Place 1.0 ml of tracer in all cuvettes.

2) Add 2.0 ,ul sample to all cuvettes.

3) Add 1.0 ml antiserum to all cuvettes.

4) Mix well and incubate 1 5 minutes at ambient temperature.

5) Read the fluorescence polarization on a fluorometer and construct a standard curve.

C) The results of a series of serum standards containing theophylline at concentrations between 0 and 40,ug/ml are presented. Each concentration was assayed in duplicate and the average is presented.

Concentration of Theophylline {yg/ml) Polarization 0 0.158 2.5 0.118 5 0.105 10 0.091 20 0.076 40 0.063 The polarization of fluorescence is seen to decrease in a regular manner as the theophylline concentration increases, allowing construction of a standard curve. Unknown specimens treated in an identical manner can be quantitated by reference to the standard curve thereby illustrating the utility of 8-aminoethyltheophylline-carboxyfluorescein for the measurement of the theophylline.

Example XXX-Digoxin assay A) Materials required: 1) BGG buffer consisting of 0.1 M sodium phosphate, pH 7.5, containing bovine gammaglobulin, 0.01% and sodium azide, 0.01%.

2) Tracer, consisting of digoxin carboxyfluorescein at a concentration of approximately 2 nM in BGG buffer.

3) Antiserum, consisting of rabbit antiserum raised against digoxin diluted appropriately in BGG buffer.

4) Samples of human serum or other biological fluid containing phenytoin.

5) Precipitation reagent -5% trichloroacetic acid in water.

6) Cuvettes, 10x75 mm glass culture tubes used as cuvettes.

7) Fluorometer capable of measuring fluorescence polarization with a precision of +0.001 units.

B) Assay protocol: 1) To 100 ,ul of 5% trichloroacetic acid in a test tube is added 100 yl of a standard or unknown sample. The tubes containing the sample are capped and vortexed.

2) The tubes containing standard or sample in trichloroacetic acid are centrifuged.

3) To a test tube 1.8 ml of BGG buffer and 25 yl of antisera at 350C is added 150 yl of the trichloroacetic supernatant solution.

4) The test tubes containing antisera and supernatant is incubated for 6 minutes at 350C, at which time the fluorescence polarization of the tubes are measured. This measurement is the background fluorescence polarization of the standard or unknown.

5) Ten minutes after the addition of supernatant to antisera, 25 Hl of the tracer is added to the test tube.

6) Six minutes after the addition of tracer, the fluorescence polarization of the standards and sample tubes are measured and the previously measured background fluorescence polarization is substracted to yield the fluorescence polarization of the antibody-tracer complex that had formed.

7) The results of a series of serum standards containing digoxin at concentrations between 0 and 5 ng/ml are presented below. Four samples at each concentration were assayed and averaged.

Digoxin concentration (ng/ml) Polarization 0 0.142 0.5 0.134 1.0 0.123 2.0 0.106 3.0 0.092 5.0 0.070 The polarization of fluorescence is seen to decrease in a regular manner as the digoxin concentration increases, allowing construction of a standard curve. Unknown specimens treated in an identical manner can be quantitated by reference to the standard curve, thereby illustrating the utility of digoxin carboxyfluorescein for the measurement of digoxin.

The following table summarizes the various fluorescence polarization assays that have been carried out in accordance with the above-described procedures employing tracers prepared in the preceding examples. The tracers employed are identical by Example number and the specific ligand(s) determined are indicated.

Example No. LigandRsJ I Phenobarbital II Phenytoin Ill Phenytoin IV Theophylline V Theophylline VI Valproic acid VII Nortriptyline; Amitriptyline VIII Imipramine; Desipramine IX Thyroxine X Digoxin Xl Propranolol XII Valproic acid XIII Valproicacid XIV Primidone XV Chloramphenicol XVI Acetaminophen XVII Ethosuximide XVII I N-acetylprocainamide XIX N-acetylprocainamide XX Disopyramide XXI Triiodothyronine XXII Thyroxine XXIII Imipramine; Desipramine XXIV Imipramine; Desipramine XXV Nortriptyline; Amitriptyline XXVI Nortriptyline; Amitriptyline As evident from the above results, the tracers of the present invention are effective reagents in fluorescence polarization immunoassays. In addition to the properties mentioned above, the tracers of the present invention possess a high degree of thermal stability, a high degree of bound polarization, high quantum yields and are relatively easy to produce and purify.

Claims (46)

Claims
1. A method for determining ligands in a sample comprising intermixing with said sample a tracer owt the formula: <img class="EMIRef" id="027228597-00150001" />
wherein R is a ligand-analog having a single reaction primary or secondary amino group which is attached to a carbonyl carbon of a carboxyfluorescein moiety, wherein said ligand-analog has at least one common epitope 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; and then determining the amount of tracer bound to antibody by fluorescence polarization techniques as a measure of the amount of ligand in the sample.
2. A method according to Claim 1 wherein said ligand is a drug or a metaboiite thereof.
3. A method according to Claim 2 wherein the carbonyl group bonded to the R group is also bonded to the 4- or 5- position of the carboxyfluorescein moiety.
4. A method according to Claim 3 wherein said drug is a steroid, hormone, antiasthamatic, antineoplastic, antiarrhythmic, anticonvulsant, antiarthritic, antidepressant, cardiac glycoside or a metabolite thereof.
5. A method according to Claim 4 wherein R has a molecular weight within a range of 50 to 4000.
6. A method according to Claim 5 wherein R has a molecular weight within a range of 100 to 2000.
7. A method according to Claim 6 wherein said drug is an anticonvulsant.
8. A method according to Claim 7 wherein said anticonvulsant drug is phenobarbital.
9. A method according to Claim 7 wherein said anticonvulsant drug is phenytoin.
10. A method according to Claim 7 wherein said anticonvulsant drug is primidone.
11. A method according to Claim 6 wherein said drug is a steroid.
12. A method according to Claim 10 wherein said steroid is digoxin.
13. A method according to Claim 6 wherein said drug is an antiarrhythmic.
14. A method according to Claim 13 wherein said antiarrhythmic drug is propranolol.
1 5. A method according to Claim 6 wherein said drug is an antiasthmatic.
1 6. A method according to Claim 1 5 wherein said antiasthmatic drug is theophylline.
17. A compound of the formula: <img class="EMIRef" id="027228597-00150002" />
wherein R is a ligand-analog having a single reactive primary or secondary amino group which is attached to a carbonyl carbon of a carboxyfluorescein moiety and having a molecular weight within a range of 100 to 2000 wherein said ligand-analog has at least one common epitope with a ligand so as to be specifically recognizable by a common antibody.
18. A compound according to Claim 17 wherein the carbonyl group bonded to the R group is also bonded to the 4- or 5- position of the carboxyfluorescein moiety.
1 9. A compound of Claim 1 8 wherein R is derived from a ligand selected from the group consisting of steroids, hormones, antiasthmatics, antineoplastics, antiarrhythmics, anticonvulsants, antiarthritics, antidepressants and cardiac glycosides.
20. A compound according to Claim 1 9 wherein R is derived from an anticonvulsant.
21. A compound according to Claim 20 wherein R is derived from phenobarbital.
22. A compound according to Claim 21 wherein R is <img class="EMIRef" id="027228597-00160001" />
23. A compound according to claim 20 wherein R is derived from phenytoin.
24. A compound according to Claim 23 wherein R is <img class="EMIRef" id="027228597-00160002" />
wherein N is an integer from 1 to 3 and m is O or 1.
25. A compound according to Claim 23 wherein n is 2 and m is 0.
26. A compound according to Claim 20 wherein R is derived from valproic acid.
27. A compound according to Claim 26 wherein R is <img class="EMIRef" id="027228597-00160003" />
wherein p is an integer of 2 to 4.
28. A compound according to Claim 20 wherein R is derived from primidone.
29. A compound according to Claim 28 wherein R is <img class="EMIRef" id="027228597-00160004" />
30. A compound according to Claim 1 9 wherein R is derived from a steroid.
31. A compound according to Claim 30 wherein R is derived from digoxin.
32. A compound according to Claim 31 wherein R is <img class="EMIRef" id="027228597-00170001" />
33. A compound according to Claim 1 9 wherein R is derived from an antiasthmatic drug.
34. A compound according to Claim 33 wherein R is derived from theophylline.
35. A compound according to Claim 34 wherein R is <img class="EMIRef" id="027228597-00170002" />
wherein n is an integer of 1 or 2.
36. A compound according to Claim 1 9 wherein R is derived from an antiarrhythmic.
37. A compound according to Claim 36 wherein R is derived from propranolol.
38. A compound according to Claim 37 wherein R is <img class="EMIRef" id="027228597-00170003" />
39. A compound according to Claim 19 wherein R is derived from a hormone.
40. A compound according to Claim 1 9 wherein R is derived from an antiarrthritic.
41. A compound according to Claim 19 wherein R is derived from a cardiac glycoside.
42. A compound according to Claim 1 9 wherein R is derived from an antidepressant.
43. A compound according to Claim 1 9 wherein R is derived from an antineoplastic.
44. A compound according to Claim 18 wherein R is <img class="EMIRef" id="027228597-00180001" />
wherein R' is hydrogen or iodo.
45. A compound according to any one of Examples I to XXVI herein.
46. A method as claimed in Claim 1 and substantially according to any one of Examples XXVII to XXX herein.
GB8203971A 1981-02-17 1982-02-11 Substituted carboxyfluoresceins and their use in fluorescence polarization immunoassay Expired GB2111476B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US23525981 true 1981-02-17 1981-02-17

Publications (2)

Publication Number Publication Date
GB2111476A true true GB2111476A (en) 1983-07-06
GB2111476B GB2111476B (en) 1985-04-03

Family

ID=22884765

Family Applications (1)

Application Number Title Priority Date Filing Date
GB8203971A Expired GB2111476B (en) 1981-02-17 1982-02-11 Substituted carboxyfluoresceins and their use in fluorescence polarization immunoassay

Country Status (4)

Country Link
JP (1) JPH0123061B2 (en)
BE (1) BE892158A (en)
FR (1) FR2500165B1 (en)
GB (1) GB2111476B (en)

Cited By (22)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4591569A (en) * 1984-04-11 1986-05-27 Becton Dickinson & Company Homogeneous fluorescent triiodothyronine uptake test
JPS61117454A (en) * 1984-10-25 1986-06-04 Abbott Lab Fluorescent polarization testing method for measuring digitoxin, reagent thereof and manufacture of reagent
US4784961A (en) * 1987-03-23 1988-11-15 Abbott Laboratories Fluorescence polarization method for monitoring fetal lung maturity
US4868132A (en) * 1987-02-03 1989-09-19 Abbott Laboratories Fluorescence polarization immunoassay for amphetamine/methamphetamine
EP0457213A2 (en) * 1990-05-16 1991-11-21 Abbott Laboratories Barbiturate assay, tracers, immunogens, antibodies and kit
US5073629A (en) * 1989-01-23 1991-12-17 Abbott Laboratories Methadone fluorescence polarization immunoassay
US5096838A (en) * 1989-11-27 1992-03-17 Abbott Laboratories Barbiturate assay compositions and methods
US5099020A (en) * 1989-11-27 1992-03-24 Abbott Laboratories Barbiturate assay compositions and methods
US5101015A (en) * 1989-04-10 1992-03-31 Abbott Laboratories Reagents for an amphetamine-class fluorescence polarization immunoassay
US5100807A (en) * 1987-10-19 1992-03-31 Abbott Laboratories Phenylacetylglutamine (pag) analytical test
US5124457A (en) * 1986-05-21 1992-06-23 Abbott Laboratories Phencyclidine and phencyclidine metabolites assay, tracers, immunogens and antibodies
US5155212A (en) * 1986-05-21 1992-10-13 Abbott Laboratories Phencyclidine and phencyclidine metabolites assay, tracers, immunogens, antibodies and reagent kit
WO1993003370A1 (en) * 1991-07-31 1993-02-18 Abbott Laboratories Reagents and methods for the quantification of amitriptyline or nortriptyline in biological fluids
US5221629A (en) * 1986-05-21 1993-06-22 Abbott Laboratories Phencyclidine and phencyclidine metabolites assay, tracers, immunogens and antibodies
US5223627A (en) * 1986-07-15 1993-06-29 Abbott Laboratories Fluorescence polarization method for monitoring fetal lung maturity
US5248791A (en) * 1989-04-10 1993-09-28 Abbott Laboratories Reagents, methods and kits for an amphetamine-class fluorescence polarization immunoassay
US5260441A (en) * 1986-07-14 1993-11-09 Abbott Laboratories Immunoassay for opiate alkaloids and their metabolites; tracers, immunogens and antibodies
US5262333A (en) * 1988-10-28 1993-11-16 Abbott Laboratories Method and reagents for detecting amphetamine and/or D-methamphetamine in biological samples
US5336622A (en) * 1986-04-25 1994-08-09 Abbott Laboratories Tracers for use in flecainide fluorescence polarization immunoassay
US5380825A (en) * 1988-10-19 1995-01-10 Sigma Chemical Company AZT immunoassays, derivatives, conjugates and antibodies
US5952187A (en) * 1995-12-01 1999-09-14 Oxis International, Inc. Topiramate immunoassay
US5986094A (en) * 1996-04-24 1999-11-16 Roche Diagnostics Corporation 4'-methyl substituted fluorescein derivatives

Families Citing this family (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA1195995A (en) * 1982-11-08 1985-10-29 Curtis L. Kirkemo Substituted carboxyfluoresceins
US4476228A (en) * 1982-11-08 1984-10-09 Abbott Laboratories Determination of unsaturated thyroxine binding protein sites using fluorescence polarization techniques
US4510251A (en) * 1982-11-08 1985-04-09 Abbott Laboratories Fluorescent polarization assay for ligands using aminomethylfluorescein derivatives as tracers
US4476229A (en) * 1982-11-08 1984-10-09 Abbott Laboratories Substituted carboxyfluoresceins
US4582791A (en) * 1983-10-07 1986-04-15 Syntex (U.S.A.) Inc. Reducing non-specific background in immunofluorescence techniques
EP0184120A3 (en) * 1984-11-29 1987-12-09 Abbott Laboratories Tracers for disopyramide assay and immunogens to raise antibody
EP0199042A1 (en) * 1985-03-26 1986-10-29 Abbott Laboratories Procainamide assay, tracers, immunogens and antibodies
EP0199963B1 (en) * 1985-04-01 1991-10-23 Abbott Laboratories Ethosuximide assay tracers, immunogens and antibodies
DE3685377D1 (en) * 1985-07-22 1992-06-25 Abbott Lab Fluorescence polarization immunoassay and reagent for measuring c-reactive protein.
EP0226730B1 (en) * 1985-10-15 1994-03-02 Abbott Laboratories Compounds and assay for tricyclic antidepressants
US4855225A (en) * 1986-02-07 1989-08-08 Applied Biosystems, Inc. Method of detecting electrophoretically separated oligonucleotides
JPH01272967A (en) * 1988-04-26 1989-10-31 Matsushita Electric Ind Co Ltd Immunological detection
US5364796A (en) * 1989-07-11 1994-11-15 Pb Diagnostics Systems, Inc. Diagnostic assay system
GB9024176D0 (en) * 1990-11-07 1990-12-19 Medical Res Council Photolabile compounds,their synthesis and use as fluorophores

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4193983A (en) * 1978-05-16 1980-03-18 Syva Company Labeled liposome particle compositions and immunoassays therewith
US4222744A (en) * 1978-09-27 1980-09-16 Becton Dickinson & Company Assay for ligands
CA1121345A (en) * 1979-03-05 1982-04-06 Robert A. Yoshida Method for competitive protein binding assays inhibiting non-specific interference

Cited By (28)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4591569A (en) * 1984-04-11 1986-05-27 Becton Dickinson & Company Homogeneous fluorescent triiodothyronine uptake test
EP0184630A2 (en) * 1984-10-25 1986-06-18 Abbott Laboratories Fluorescence polarization assay, reagents, and method of making reagents, for determination of digitoxin
EP0184630A3 (en) * 1984-10-25 1986-08-27 Abbott Laboratories Fluorescence polarization assay, reagents, and method of making reagents, for determination of digitoxin
JPS61117454A (en) * 1984-10-25 1986-06-04 Abbott Lab Fluorescent polarization testing method for measuring digitoxin, reagent thereof and manufacture of reagent
US5336622A (en) * 1986-04-25 1994-08-09 Abbott Laboratories Tracers for use in flecainide fluorescence polarization immunoassay
US5124457A (en) * 1986-05-21 1992-06-23 Abbott Laboratories Phencyclidine and phencyclidine metabolites assay, tracers, immunogens and antibodies
US5407834A (en) * 1986-05-21 1995-04-18 Abbott Laboratories Phencyclidine and phencyclidine metabolites assay, tracers, immunogens, antibodies and reagent kit
US5221629A (en) * 1986-05-21 1993-06-22 Abbott Laboratories Phencyclidine and phencyclidine metabolites assay, tracers, immunogens and antibodies
US5155212A (en) * 1986-05-21 1992-10-13 Abbott Laboratories Phencyclidine and phencyclidine metabolites assay, tracers, immunogens, antibodies and reagent kit
US5260441A (en) * 1986-07-14 1993-11-09 Abbott Laboratories Immunoassay for opiate alkaloids and their metabolites; tracers, immunogens and antibodies
US5223627A (en) * 1986-07-15 1993-06-29 Abbott Laboratories Fluorescence polarization method for monitoring fetal lung maturity
US4868132A (en) * 1987-02-03 1989-09-19 Abbott Laboratories Fluorescence polarization immunoassay for amphetamine/methamphetamine
US4784961A (en) * 1987-03-23 1988-11-15 Abbott Laboratories Fluorescence polarization method for monitoring fetal lung maturity
US5100807A (en) * 1987-10-19 1992-03-31 Abbott Laboratories Phenylacetylglutamine (pag) analytical test
US5380825A (en) * 1988-10-19 1995-01-10 Sigma Chemical Company AZT immunoassays, derivatives, conjugates and antibodies
US5262333A (en) * 1988-10-28 1993-11-16 Abbott Laboratories Method and reagents for detecting amphetamine and/or D-methamphetamine in biological samples
US5073629A (en) * 1989-01-23 1991-12-17 Abbott Laboratories Methadone fluorescence polarization immunoassay
US5101015A (en) * 1989-04-10 1992-03-31 Abbott Laboratories Reagents for an amphetamine-class fluorescence polarization immunoassay
US5248791A (en) * 1989-04-10 1993-09-28 Abbott Laboratories Reagents, methods and kits for an amphetamine-class fluorescence polarization immunoassay
US5354693A (en) * 1989-04-10 1994-10-11 Abbott Laboratories Reagents, methods and kits for an amphetamine-class fluorescence polarization immunoassy
US5096838A (en) * 1989-11-27 1992-03-17 Abbott Laboratories Barbiturate assay compositions and methods
US5099020A (en) * 1989-11-27 1992-03-24 Abbott Laboratories Barbiturate assay compositions and methods
EP0457213A2 (en) * 1990-05-16 1991-11-21 Abbott Laboratories Barbiturate assay, tracers, immunogens, antibodies and kit
EP0457213A3 (en) * 1990-05-16 1992-09-02 Abbott Laboratories Barbiturate assay, tracers, immunogens, antibodies and kit
WO1993003370A1 (en) * 1991-07-31 1993-02-18 Abbott Laboratories Reagents and methods for the quantification of amitriptyline or nortriptyline in biological fluids
US5614419A (en) * 1991-07-31 1997-03-25 Abbott Laboratories Reagents and methods for the quantification of amitriptyline or nortriptyline in biological fluids
US5952187A (en) * 1995-12-01 1999-09-14 Oxis International, Inc. Topiramate immunoassay
US5986094A (en) * 1996-04-24 1999-11-16 Roche Diagnostics Corporation 4'-methyl substituted fluorescein derivatives

Also Published As

Publication number Publication date Type
GB2111476B (en) 1985-04-03 grant
JPH0123061B2 (en) 1989-04-28 grant
BE892158A1 (en) grant
FR2500165A1 (en) 1982-08-20 application
BE892158A (en) 1982-08-16 grant
JPS57150680A (en) 1982-09-17 application
JP1541465C (en) grant
FR2500165B1 (en) 1985-06-14 grant

Similar Documents

Publication Publication Date Title
US4174384A (en) Fluorescence quenching with immunological pairs in immunoassays
US4272506A (en) Purification of reagents by disulfide immobilization
US4318846A (en) Novel ether substituted fluorescein polyamino acid compounds as fluorescers and quenchers
US3905871A (en) Lactam conjugates to enzymes
US5359093A (en) Reagents and methods for the detection and quantification of thyroxine in fluid samples
US4629691A (en) Tricyclic antidepressant conjugates with antigens and enzymes
US5501987A (en) Dual analyte immunoassay for methamphetamine and amphetamine
US4213893A (en) Flavin adenine dinucleotide-labeled conjugates for use in specific binding assays
US4235792A (en) Immunological materials
US5661040A (en) Fluorescent polymer labeled conjugates and intermediates
US4404366A (en) Beta-galactosyl-umbelliferone-labeled hapten conjugates
US5464741A (en) Palladium (II) octaethylporphine alpha-isothiocyanate as a phosphorescent label for immunoassays
US4199559A (en) Fluorescence quenching with immunological pairs in immunoassays
US3996345A (en) Fluorescence quenching with immunological pairs in immunoassays
US5593896A (en) Reagents and methods for the detection and quantification of thyroxine in fluid samples
US4279992A (en) Specific binding assay employing an enzyme-cleavable substrate as label
US4130462A (en) Receptor steric hindrance immunoassay for receptor determination
US4331590A (en) β-Galactosyl-umbelliferone-labeled protein and polypeptide conjugates
US4495281A (en) Tricyclic antidepressant drug immunogens, antibodies, labeled conjugates, and related derivatives
US5340716A (en) Assay method utilizing photoactivated chemiluminescent label
US5304645A (en) Resorufin derivatives
US4193983A (en) Labeled liposome particle compositions and immunoassays therewith
US4476228A (en) Determination of unsaturated thyroxine binding protein sites using fluorescence polarization techniques
US5321136A (en) Peri substituted fused ring chemiluminescent labels and their conjugates, and assays therefrom
US5284951A (en) Hydrolytically stable chemiluminescent labels and their conjugates, and assays therefrom

Legal Events

Date Code Title Description
PCNP Patent ceased through non-payment of renewal fee

Effective date: 19980211