JPH0123061B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to methods and reagents for determining ligands in biological fluids such as serum, plasma, spinal fluid, amniotic fluid and urine. In particular, the present invention relates to fluorescence polarization immunoassay procedures and tracers used as reagents in this procedure. The fluorescence polarization immunoassay procedure of the present invention combines the specificity of immunoassays with the speed and convenience of fluorescence polarization techniques to provide a means for determining the amount of specific ligands present in a sample. . Competitive binding immunoassay for measuring ligands is a method of binding between a ligand in a test sample and a labeled reagent called a tracer for a limited number of receptor binding sites on an antibody specific for the ligand and tracer. It is based on competition. In general, fluorescence polarization technology is based on the principle that when a fluorescent labeling compound is excited with linearly polarized light, it emits fluorescence with a degree of polarization that is inversely related to its rotation rate. It is. Thus, when a molecule, such as a tracer-antibody conjugate with a fluorescent label, is excited with linearly polarized light, the emitted light remains highly polarized. This is because the fluorophore is constrained from rotating between the time the light is absorbed and the time it is emitted. When a "free" tracer compound (i.e., not bound to an antibody) is excited by linearly polarized light, its rotation is much faster than that of the corresponding tracer-antibody conjugate. , hence the molecules are more randomly arranged and the emitted light is therefore depolarized. Thus, fluorescence polarization provides a quantitative means of measuring the amount of tracer-antibody conjugate produced in competitive binding immunoassays. A variety of fluorescent labeling compounds are known in the art. U.S. Pat. No. 3,998,943 uses fluorescein isothiocyanate (FITC) as a fluorescent label.
The preparation of fluorescently labeled insulin derivatives using 4-aminofluorescein hydrochloride and 4-aminofluorescein hydrochloride as the fluorescent label is described. Carboxyfluorescein has also been used for spectrometry measurements. Anal.Lett, Volume 10, Page 787 (1977)
RF Chen reports the use of carboxyfluorescein to direct the activity of phospholipases. However, carboxyfluorescein is not conjugated according to the present invention.
This substance is encapsulated in lecithin liposomes and produces fluorescence only when released by hydrolysis of lecithin. The present invention allows the sample to be (wherein R is a ligand-analog of molecular weight 50-4000 having a single reactive primary or secondary amino group attached to the carbonyl carbon of carboxyfluorescein; a biologically acceptable salt of a tracer having at least one common epitope with a ligand such that it is specifically recognized by a common antibody; Provided is a method for determining a ligand in a sample, characterized in that it is mixed with a recognizable antibody and then the amount of tracer antibody conjugate is determined by fluorescence polarization techniques as a standard for the concentration of said ligand in the sample. It is something to do. Additionally, the present invention relates to certain novel tracers and biologically acceptable salts thereof useful as reagents in the methods described above. The methods and tracers of the invention are particularly useful for quantitatively monitoring concentrations of therapeutic drugs in serum and plasma. The term "ligand" as used herein refers to a molecule, especially a low molecular weight hapten, having a single reactive amino group from which a receptor, usually an antibody, can be obtained or formed.
Such haptens generally have a low molecular weight protein-free matrix (they do not induce antibody formation when injected into an animal, but are reactive with antibodies).
Antibodies to haptens are generally produced by first conjugating the hapten to a protein and injecting the conjugate product into an animal. The resulting antibodies are isolated using conventional antibody isolation techniques. Ligands that can be measured by the method of the invention vary over a wide molecular weight range. Although high molecular weight ligands may also be measured, for best results it is generally preferred to use the method of the present invention to measure ligands of low molecular weight, generally in the range of 50-4000. More preferably, ligands with molecular weights in the range 100-2000 are measured. The novel tracers of the present invention include compounds of formula () in which the ligand-analog represented by R has a molecular weight within the range of 50-4000. Preferred new tracers include compounds of formula () in which the ligand-analog represented by R includes a group having a molecular weight within the range of 100-2000. Representative examples of ligands having a single reactive amino group that can be measured by the method of the present invention include steroids such as esterone, estradiol, cortisol, testoestrone, progesterone, henodeoxycholic acid, digoxin, cholic acid, digitoxin, deoxycholic acid. acids, lithocholic acid and their ester and amide derivatives; vitamins such as B-12, folic acid; thyroxine, triiodothyroxine, histamine, serotonin, prostaglandins such as PGE, PGF, PGA; antiasthmatic agents such as theophylline, anticancer agents such as doxorubicin and Methotrexate, antiarrhythmic agents such as diisopyramide, lidocaine, procainamide, propanolol, quinidine, N-acetyl-
procainamide; anticonvulsants such as phenobarbital, phenytoin, pyrimidone, valproic acid, carbamazepine, and ethosuccinimide; antibiotics such as penicillin, cephalosporin, and vancomycin; antiarticular agents such as salicylates; antidepressants including tricyclics such as nortriptyline, Included are aminotriptyline, imipramine, desipramine, and the like, as well as their metabolites. Other ligands that can be measured by the method of the invention include drugs of abuse such as morphine, heroin, hydromorphone, oxymorphone, metapone, codeine, hydrocodone, dihydrocodeine, dihydrohydroxycodeine, phorcodeine, dextromethorphan, phenazosine and deonine; Metabolites are included. The tracers of the invention generally exist in equilibrium between their acid and ionized states, and are effective in the methods of the invention in their ionized states. Accordingly, the present invention encompasses tracers in their acid or ionized states, and for convenience, the tracers of the present invention are structurally represented herein in their acid form. When the tracer of the invention is present in its ionized state, it is present in the form of a biologically acceptable salt. As used herein, the term "biologically acceptable salts" refers to salts such as sodium, potassium, ammonium and similar salts which, when used in the method of the invention, may cause the tracer of the invention to be present in its ionized state. name something. Generally, the tracer of the invention will be present as a salt in solution, with the particular salt arising from the buffer used. That is, in the presence of a sodium phosphate buffer, the tracer of the invention generally exists in its ionized state as a sodium salt. The tracer of the present invention is a ligand represented by R bound to carboxyfluorescein of the following formula:
Contains analogs. As used herein, the term "ligand-analogue" refers to a substance that is identical to the ligand in defining one or more determinant or epitope sites, a substantial proportion of which can compete with the ligand for binding sites on the receptor. Refers to monovalent or polyvalent groups with spatial and polar organization. A feature of this ligand-analog is that it has sufficient structural similarity to the ligand in question such that it is recognized by antibodies for the ligand. In most cases, the ligand-analog has the same or substantially the same structure and charge distribution (spatial and polar organization) as the ligand in question over a significant portion of the molecular surface. Often the binding site for the hapten is the same as that used to bind the ligand and in the preparation of the antigen for antibody production, so that the same portion of the ligand-analog that provides the template for the antibody is Exposed by the ligand-analog in the tracer. Generally, the ligand-analog pair denoted R is obtained by removal of a reactive hydrogen atom from the corresponding ligand, i.e., a hydrogen atom attached to a reactive amine (primary or secondary) or attached to a carboxyfluorescein moiety. imino group at the site where

The formula is derived by forming an amino derivative of the ligand which replaces one or more atoms originally present in the ligand. Examples of ligands that can form a ligand-analogue represented by R upon removal of a reactive hydrogen include, for example, procainamide, thyroxine, and quinidine. Examples of ligands for which amino derivatives are useful as ligand-analogs include theophylline,
Valproic acid, phenobarbital, phenytoin, primidone, diisopyramide, digoxin,
Included are chloramphenicol, salicylates, acetaminophenone, carbamazepine, desipramine and nortriptyline. Additionally, the ligand can be structurally modified to form a ligand-analog by the addition or removal of one or more functional groups while retaining the epitope site necessary for binding the antibody. However, such modified ligand-analogs are attached to carboxyfluorescein via the imino group. The tracers of the present invention are generally manufactured according to known techniques. For example, the formula R—X () where R is as defined above and
is a reactive hydrogen) with the formula (In the formula, Z is a hydroxyl group or an active ester,
and the carboxy group is preferably 4 of the benzoic acid ring.
- or 5-bonded) in the presence of an inert solvent to produce a compound of formula (). The term "active ester" as used herein refers to a moiety that is readily "removed" from the carboxy carbon in the presence of a coupling agent. "Active esters" of carboxyfluorescein such as
is readily ascertained by those skilled in the art and can be achieved by reacting carboxyfluorescein with a compound such as N-hydroxy-succinimide, 1-hydroxybenzotriazole hydrate or p-nitrophenol in the presence of a coupling agent such as dicyclohexylcarbodiimide and a solvent. Manufactured by. The active ester of carboxyfluorescein thus obtained is then reacted with a compound of formula () to obtain a tracer of formula (). When the compound of formula () is water-soluble, the reaction mechanism is to use a water-soluble carbodiimide such as 1-ethyl-3- as a coupling agent between carboxyfluorescein and the compound of formula () in aqueous solution.
It proceeds by direct reaction in the presence of (3'-dimethylaminopropyl)carbodiimide. There is no limit to the temperature at which the method for producing a tracer of the present invention proceeds. The temperature must be sufficient to initiate and maintain the reaction. Generally, room temperature is sufficient for convenience and economy. In preparing the tracers of this invention, the ratio of reactants is not strictly critical. Reasonable yields should be obtained using 1 mole of compound of formula () for each mole of compound of formula (). For ease of reaction and collection of reaction products, it is preferred to use an excess of the compound of formula (). To facilitate handling and collection of the product, the process for producing tracers of the invention is carried out in the presence of an inert solvent. Suitable inert solvents include those sufficient to dissolve the starting materials without reacting with them, such as water (if the compound of formula () is water-soluble), dimethylformamide, dimethyl Includes sulfoxide and the like. When the compound of formula () 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 product of formula () is generally purified using thin layer or column chromatography before application to the process of the invention. According to the method of the invention, a sample containing the ligand to be measured is mixed with a biologically acceptable salt of a tracer of formula () and an antibody specific for the ligand and the tracer. Ligand and tracer present in the sample compete to confine antibody sites, resulting in ligand-antibody and tracer-antibody complexes. By keeping the concentrations of tracer and antibody constant, the ratio of ligand-antibody complexes to tracer-antibody complexes formed is directly proportional to the amount of ligand present in the sample. Therefore, by exciting the mixture with polarized light and measuring the polarization of the fluorescent light emitted by the tracer and tracer-antibody complex, it is possible to quantitatively measure the amount of ligand in the sample. In theory, the fluorescence polarization of a tracer that is not conjugated to an antibody is low, close to zero. When complexed with a specific antibody, the tracer-antibody complex thus formed will rotate the antibody molecule more slowly than the relatively small tracer molecule, thereby increasing the observed polarization. Thus, when a ligand competes with a tracer for an antibody site, the observed polarization of the fluorescence of the tracer-antibody complex will be somewhere between the polarization of the tracer and that of the tracer-antibody complex. When a sample contains a high concentration of ligand, the observed polarization value will be closer to, ie, lower, that of free ligand. When the test sample contains a low concentration of ligand, the polarization value will be closer to, ie, higher, that of bound ligand. Accurately measuring the polarization of fluorescent light in the reaction mixture by sequentially exciting the immunoassay reaction mixture with vertically and then horizontally polarized light and refracting only the vertical component of the emitted light. Can be done. The exact relationship between polarization and the concentration of the ligand to be measured is established by measuring the polarization value of a calibrator with known concentration. The concentration of ligand can be extrapolated from the standard curve thus generated. The pH at which the method of the invention is carried out must be sufficient to cause the tracer of formula () to exist in its ionized state. The PH may range from about 3 to 12, more usually from about 5 to 10, and most preferably from about 6 to 9. Various buffers may be used to achieve and maintain pH during the fractionation procedure. Typical buffers used include borates, phosphates,
Carbonates, tris, barbital, etc. are included.
The particular buffer used is not limiting to the present invention, but a particular buffer may be preferred in view of the antibody used and the ligand to be measured in a particular analysis. The cationic portion of the buffer generally defines the cationic portion of the tracer salt in solution. The method of the invention is carried out at moderate temperatures, preferably constant temperatures. The temperature usually ranges from about 0 to 50°C, more usually from about 15 to 40°C. The concentration of resolvable ligand is generally about 10 ^-2 ~
10 ^-13 M, and more commonly can vary from about 10 ^-4 to ^{10 -10} M. Higher concentrations of ligand can be separated by diluting the original sample. In addition to the concentration range of the ligand in question, the determination of which tracers and antibodies should be used takes into account, for example, whether the analysis will be qualitative, semi-quantitative or quantitative, the equipment used, and the characteristics of the tracers and antibodies. Define the concentration normally. The concentration of the ligand in the sample usually defines a range of concentrations of the other reagents, ie, tracer and antibody, to optimize the sensitivity of the analysis, but the concentrations of the individual reagents are determined empirically. Tracer and antibody concentrations are readily ascertained by those skilled in the art. As mentioned above, the preferred tracer of the present invention is 5
-carboxyfluorescein or 4-carboxyfluorescein or a mixture thereof, and is represented by the following formula. The following illustrative and non-limiting examples serve to further illustrate to those skilled in the art how particular tracers within the scope of the present invention may be made.
The symbol [CF] appearing in the structural formulas specifically showing the compounds produced in the following examples indicates a part of the following formula. (In view of the fact that a mixture of 4- and 5-carboxyfluorescein is used as starting material, the carbonyl carbon in the above formula is 4- or 5-
). Example m- or p-aminophenobarbital (5
mg) and carboxyfluorescein (5 mg) were dissolved in 0.5 ml of pyridine. N,N'-dicyclohexylcarbodiimide (15 mg) was added to the mixture. The reaction was allowed to proceed for 2 hours at room temperature and the reaction product was then purified twice by silica gel thin layer chromatography using a chloroform:methanol (2:1) mixture as the developing solvent to give aminophenobarbital with the formula: A carboxyfluorescein conjugate was obtained. Example 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 2 hours and then reduced under reduced pressure. Evaporate to dryness. The residue was suspended in 50 ml of water, and 6N sodium hydroxide was added to adjust the pH to PH11 to dissolve unreacted phenytoin. The residual precipitate, 2-β-aminoethylphenytoin, was filtered off, washed thoroughly with water, and dried. Active ester of carboxyfluorescein,
N-hydroxysuccinimide (5 mg), carboxyfluorescein (7.5 mg) in 0.5 ml of pyridine
and N,N'-dicyclohexylcarbodiimide (20 mg). The reaction was allowed to proceed for 2 hours at room temperature, after which 2-β-aminoethylphenytoin (10 mg) was dissolved in the reaction mixture. The resulting mixture was reacted overnight at room temperature in the dark, and the reaction product was then purified twice by silica gel thin layer chromatography using a chloroform:methanol (3:1) mixture as the developing solvent to obtain 2 with the following formula: -β-aminoethylphenytoin-carboxyfluorescein conjugate was obtained. EXAMPLE A solution containing 2-carboxymethylphenytoin (620 mg), N-hydroxysuccinimide (248 mg) and N,N'-dicyclohexylcarbodiimide (453 mg) in 6 ml of dry dimethyl sulfoxide was left overnight at room temperature. The mixture was filtered and 0.7 ml of hydrozine (95%) was added to liquid 4.5. 40 ml of water and 10% sodium hydroxide after 4 hours at room temperature.
0.5ml was added to the reaction mixture. The precipitated 2-carboxymethylphenytoin hydrazide was filtered, washed with water, dried and used without further purification. N,N'-dicyclohexylcarbodiimide (15 mg) was added to a solution of 2-carboxymethylphenytoinhydrazine (5 mg) and carboxyfluorescein (5 mg) in 0.5 ml of pyridine. The reaction was allowed to proceed for 2 hours at room temperature, and the reaction product was then purified twice by silica gel thin layer chromatography using a chloroform:acetone (1:1) mixture as the developing solvent to give a 2-carboxylic acid with the formula Methylphenytoin hydrazide
A carboxyfluorescein conjugate was obtained. EXAMPLE N,N'-dicyclohexylcarbodiimide (15 mg) was added to a solution of 8-β-aminoethylthiophylline (5 mg) and carboxyfluorescein (5 mg) in 0.5 ml of pyridine. The reaction was allowed to proceed for 2 hours at room temperature and the reaction product was purified twice by silica gel thin layer chromatography using a chloroform:methanol (2:1) mixture as the developing solvent to give an 8-β-amino compound having the formula An ethyl theophylline-carboxyfluorescein conjugate was obtained. Example 8-8 instead of β-aminoethyltheophylline
Using the procedure of the Example with -aminomethyltheophylline, an 8-aminomethyltheophylline-carboxyfluorescein conjugate having the following formula was obtained. Example δ-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) was added dropwise to the reaction flask and cooled in a dry ice-acetone bath. After the addition of n-butyllithium was complete, the reaction mixture was stirred for 1 hour at room temperature, refluxed for 30 minutes, and cooled to room temperature under an atmosphere of dry nitrogen. 1-Bromoethane (8.0 g) was slowly added to the reaction flask while cooling the flask in an ice bath. The resulting mixture was then stirred at room temperature for 16 hours, after which 100 ml of water was slowly added. The resulting mixture was stirred at room temperature for 30 minutes and the organic layer was separated. Extract the aqueous layer with 50 ml of diethyl ether,
The organic layers were then combined and dried over sodium sulfate. Evaporation of the solvent gave a dark oil which crystallized on standing. The crystalline residue was recrystallized from petroleum ether to give 3.8 g of residue.
The residue (2.8 g) was refluxed in 25 ml of 6N hydrochloric acid for 6 hours. Evaporation of the water from the mixture gave 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), carboxyfluorescein (7.5 mg) and N,N'-dicyclohexylcarbodiimide (20 mg) in 0.5 ml of pyridine. The reaction was allowed to proceed for 2 hours at room temperature, then 2-ethyl-5-
Aminopentanoic acid (20 mg) was dissolved in the reaction mixture. The resulting mixture was reacted overnight at room temperature in the dark, and the reaction product was purified twice by silica gel thin layer chromatography using a chloroform:methanol (3:1) mixture as the developing solvent to give 2 with the following formula: -Ethyl-5-aminopentanoic acid-carboxyfluorescein conjugate was obtained. EXAMPLE 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 2 hours at room temperature, after which 5-(γ-aminopropylidene)-5H-dibenzo[a,d)-10,
11-dihydrocycloheptene (20 mg) was dissolved in the reaction mixture. The resulting mixture was reacted overnight at room temperature in the dark, and the reaction product was purified by silica gel thin layer chromatography using a chloroform:methanol (3:1) mixture as the developing solvent.
When purified twice, it has the following formula: 5-(γ-aminopropylidene)-5H-dibenzo[a,d]-10,
A 11-dihydrocyclopentene-carboxyfluorescein conjugate was obtained. Example Desipramine hydrochloride (1.33 g) and chloroacetyl chloride (0.8
The solution containing g) was refluxed for 2 hours. The chloroform was evaporated and the residue was dissolved in 25 ml of acetone.
Sodium iodide (0.75g) was added to the acetone solution and the solution was refluxed for 30 minutes. The solution was filtered and the precipitated salts were washed with acetone. The acetone solution was evaporated and the residue was taken up in 20 ml of methanol. Concentrated ammonium hydroxide (20ml) was added to the methanol solution and the resulting solution was refluxed for 1 hour. The reaction mixture was extracted three times with 25 ml of chloroform and the combined extracts were dried over sodium sulfate, filtered and evaporated to give N-aminoacetyldesipramine, which was used without further purification. N-acetyldesipramine (5 mg) and carboxyfluorescein (5 mg) in 0.5 ml of pyridine.
dissolved in N,N'-dicyclohexylcarbodiimide (15 mg) was added to the mixture. The reaction was allowed to proceed for 2 hours at room temperature and the reaction product was then purified twice by silica gel thin layer chromatography using a chloroform:acetone (1:1) mixture as the developing solvent to obtain N-aminoacetyldesipramine having the formula - Obtained carboxyfluorescein conjugate. Example N-hydroxysuccinimide (5 mg), carboxyfluorescein (7.5 mg) in 1 ml of pyridine
A solution containing N,N'-dicyclohexylcarbodiimide (20 mg) was reacted at room temperature for 4 hours. The active ester of carboxyfluorescein was precipitated by adding 10 ml of diethyl ether to the reaction mixture. The precipitate was filtered, washed well with diethyl ether, and redissolved in 0.5 ml of dimethyl sulfoxide. Next, L-thyroxine (10 mg) was added to the solution, the reaction was allowed to proceed for 2 hours at room temperature, and then the reaction product was analyzed by silica gel thin layer chromatography using a chloroform:methanol (3:1) mixture as the developing solvent. After purification twice, an L-thyroxine-carboxyfluorescein conjugate having the following formula was obtained. Example Ammonium acetate (0.89 g) in 5 ml methanol,
A solution containing 3-oxodigoxigenin (389 mg) and sodium cyanoborohydride (63 mg) was
Stir at room temperature for an hour. Add concentrated hydrochloric acid to the solution
The pH was adjusted to 1, and the mixture was evaporated to dryness under reduced pressure. Water the residue 10
ml and extracted three times with 10 ml of chloroform. The aqueous layer was adjusted to pH 11 using solid potassium hydroxide. The resulting solution was diluted with 10 ml of methylene chloride.
Extracted twice. The organic layers were combined, dried and evaporated to dryness under reduced pressure to yield 3-amino-3-deoxydigoxigenin, which was used without further purification. Add active ester of carboxyfluorescein to 05 ml of pyridine and add N-hydroxysuccinimide (5 ml) to 05 ml of pyridine.
mg), carboxyfluorescein (7.5 mg) and N,N'-dicyclohexylcarbodiimide (20
mg). The reaction was allowed to react for 2 hours at room temperature, after which a 3-amino-3-deoxydigoxigenin-carboxyfluorescein conjugate having the following formula was isolated. The following tracers were also prepared according to the procedure described above. Example XI O-aminoacetyl-propranolol-carboxyfluorescein conjugate Example XII 2-propyl-5-aminopentanoic acid-carboxyfluorescein conjugate Example 2-butyl-5-amino-pentanoic acid-carboxyfluorescein conjugate Example Aminoprimidone-carboxyfluorescein conjugate Example 1-(4′-nitrophenyl)-1-hydroxy-2-amino-3-hydroxypropane-carboxyfluorescein conjugate Example p-aminophenol-carboxyfluorescein conjugate Example N-(2-aminoethyl)-ethosuximide-
carboxyfluorescein conjugate Example N'-desethyl-N-acetyl-procainamide-carboxyfluorescein conjugate Example N'-desethyl-N'-aminoacetyl-N-
Acetyl-procainamide-carboxyfluorescein conjugate Example 1-amino-2-phenyl-2-(2'-pyridyl)-4-(diisopropylamino)-butane-carboxyfluorescein conjugate Example XI 3,3',5'-triiodo-L-thyronine-carboxyfluorescein conjugate Example XII 3,3',5,5'-tetraiodo-D-thyronine-carboxyfluorescein conjugate Example N-aminoacetyl-iminodibenzylcarboxyfluorescein conjugate Example Carbhydrazinoimino-dibenzylcarboxyfluorescein conjugate Example Dibenzosuberone hydrazone fluorescein conjugate Example 5-amino-10,11-dihydro-5H-dibenzo[a,d]-cycloheptene As mentioned above, the tracers of the present invention are effective reagents for use in fluorescence polarization immunoassays. The following examples illustrate the suitability of the tracers of the invention in immunoassays using fluorescence polarization techniques. This assay is performed according to the following general procedure. 1. Dispense a measured amount of standard or test serum into test tubes and dilute with buffer; 2. Add a tracer of the invention (of known concentration) optionally containing a surfactant to each test tube; 3. Add a known concentration of the tracer of the invention to each test tube; Add the antiserum to the test tube; 4. Incubate the reaction mixture at room temperature; and 5. measure the amount of tracer bound to the antibody by fluorescence polarization techniques as a measure of the amount of ligand in the sample. EXAMPLE Phenytoin Assay A Materials Required: 1 BGG buffer consisting of sodium phosphate (0.1M, PH7.5) containing 0.01% bovine gamma-globulin and 0.01% sodium azide. 2 BGG with 5% sodium cholate added
Tracer consisting of 2-β-aminoethylphenytoin-carboxyfluorescein at a concentration of approximately 105 nM in buffer. 3 BGG containing 0.005% benzalkonium chloride
Antiserum consisting of raised antiserum against phenytoin appropriately diluted in buffer. 4. Samples of human serum or other biological fluids containing phenytoin. 5 Kvet, used as kvet
10 x 75mm glass culture tube. 6. A fluorometer capable of measuring fluorescence polarization with an accuracy of ±0.001 units. B Assay method: 1. Pipette a small amount of sample (0.366 μl), 15 μl of the sample, and add 600 μl BGG to a dilution container.
Add to each cube by diluting with buffer. Next, pipet 15 μl of the diluted sample into the tube, followed by 600 μl BGG buffer. 2 Add 40μl tracer and
Add tracer by pipetting 1000 μl BGG buffer. 3 Add 40 μl antiserum to Kvet and then
Start the reaction by adding antiserum by pipetting 1000 μl BGG buffer. 4. Mix the contents of the entire khvet well and incubate for 15 minutes at ambient temperature. 5 Read the fluorescence polarization with a fluorometer and plot the standard curve to measure the unknown. Results for a series of serum standards containing phenytoin at concentrations between C 0 and 40 μy/ml are shown below. Each concentration is tested in duplicate and averaged. Concentration of phenytoin (μy/ml) 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 constant manner as the phenytoin concentration increases, which is possible due to the construction of the standard curve. do. Unknown samples treated in the same manner can be quantified with reference to the standard curve. This allows for the measurement of phenytoin.
- The usefulness of β-aminoethylphenytoin-carboxyfluorescein is demonstrated. EXAMPLE Phenobarbital Assay A Materials Required: 1 BGG buffer (see phenytoin) 2 Tris HCl buffer containing 0.01% sodium azide, 0.01% bovine gamma-globulin, and 0.125% sodium dodecyl sulfate in PH 7.5 approx.
Tracer consisting of aminophenobarbital carboxyfluorescein at a concentration of 110 nM. 3 Antiserum consisting of antiserum against phenobarbital appropriately diluted in BGG buffer containing 0.005% benzalkonium chloride. 4 Human serum or other biological fluids containing phenobarbital. 5 Quvet (see phenytoin) 6 Fluorometer (see phenytoin) B Assay procedure: 1. Prepare a small amount of sample (0.196 μl) by pipetting 10 μl of sample and diluting with 500 μl BGG buffer in a dilution vessel. Put it in Kvet. Next, 10 μl of the diluted sample and then
Pipette 500 μl of BGG buffer into the tube. 2 Add 40 μl of tracer to each test tube and
Add tracer by pipetting 1000 μl of BGG buffer. 3 Add antiserum and start the reaction by pipetting 40 μl of antiserum and then 1000 μl of BGG buffer. 4 Mix the contents of the entire khvet well and incubate for 15 minutes at ambient temperature. 5 Read the fluorescence polarization with a fluorometer and plot the standard curve to measure the unknown. The results for a series of serum standards containing phenobarbital at concentrations between C 0 and 80 μy/ml are shown below. Each concentration is tested in duplicate and averaged. Concentration of phenobarbital (μl) 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 was found to decrease in a constant manner as the phenobarbital concentration increased, and the construction of the standard curve possible. Unknown samples treated in the same manner can be quantified with reference to the standard curve. This allows aminophenobarbital to be used for the measurement of phenolbarbital.
The usefulness of carboxyfluorescein is demonstrated. Example Theophylline Assay A Required materials: 1 Contains 0.01% sodium dodecyl sulfate
in BGG buffer (see phenytoin assay)
Tracer consisting of 2nM 8-aminoethyltheophylline-carboxyfluorescein. 2 Antiserum consisting of an antiserum raised against theophylline appropriately diluted in BGG buffer. 3. Samples of human serum or other biological fluids containing theophylline. 4 Quvette (see phenytoin assay) 5 Fluorometer (refer to phenytoin assay) B Test method: 1. Add 1.0 ml of tracer to every quavette. 2 Add 2.0 μl of sample to the entire volume. 3. Add 1.0 ml of antiserum to all samples. 4. Mix well and incubate for 15 minutes at ambient temperature. 5 Read the fluorescence polarization using a fluorometer and draw a standard curve. Results are shown for a series of serum standards containing theophylline at concentrations between C 0 and 40 μy/ml. Each concentration was tested in duplicate and the average value is shown. Concentration of theophylline (μy/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 was seen to decrease in a constant manner as the theophylline concentration increased, allowing the construction of a standard curve. do. Unknown samples treated in the same manner can be quantified with reference to the standard curve, thereby providing an 8-
The usefulness of aminoethyltheophylline-carboxyfluorescein is demonstrated. Example Digoxin Assay A Required materials: 1 0.1M sodium phosphate containing 0.01% bovine gamma-globulin and 0.01% sodium azide
BGG buffer consisting of PH7.5. 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 appropriately diluted in BGG buffer. 4. Samples of human serum or other biological fluids containing digoxin. 5 Precipitation reagent...5% trichloroacetic acid (in water) 6 Kvet, used as Kvet10
×75mm glass culture tube. 7. A fluorometer capable of measuring fluorescence polarization with an accuracy of ±0.001 units. B Assay procedure: 1. Add 100 μl of standard or unknown sample to 100 μl of 5% trichloroacetic acid in a test tube. Cap the test tube containing the sample and shake to mix. 2. Centrifuge the test tube containing the standard or sample in trichloroacetic acid. 3 Add 150 μl of trichloroacetic acid supernatant solution to a test tube containing 1.8 ml of BGG buffer and 25 μl of antiserum at 35°C. 4. Incubate the test tube containing the antiserum and supernatant at 35°C for 6 minutes and measure the fluorescence polarization of the tube during this time. This measurement is the background fluorescence polarization of the standard or unknown. 5 The tracer was added 10 minutes after adding the supernatant to the antiseptic.
Add 25 μl to the test tube. 6 Measure the fluorescent light polarization of the standard product and sample tube 6 minutes after adding the tracer, and subtract the previously measured background fluorescent light polarization to obtain the fluorescent light polarization of the antibody-tracer complex that had been formed. It will be done. The results for a series of serum standards containing digoxin at concentrations between 70 and 5 ny/ml are shown below. Four samples were assayed at each concentration and averaged. Digoxin Concentration (ny/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 was found to decrease in a constant manner as the digoxin concentration increased, allowing the construction of a standard curve. . Unknown samples treated in the same manner can be quantified with reference to the standard curve, demonstrating the utility of digoxin carboxyfluorescein for digoxin. The following table summarizes various fluorescence polarization assays performed using the tracers prepared in the examples above and according to the procedures described above. The tracer used is identified by example number and the specific ligand measured is indicated. Example No. Ligand phenobarbital phenytoin phenytoin theophylline theophylline valproic acid nortriptyline; amitriptyline imipramine; desiplacin thyroxine digoxin XI propranolol Tilprocainamide diisopyramide XI triiodothyronine
In addition to the above properties, the tracers of the present invention have a high degree of thermal stability, a high degree of binding polarization, high yields, and are relatively easy to manufacture and purify. Although the invention has been described with respect to certain variations,
The details should not be construed as limiting. It is obvious that various equivalents, changes and modifications may be made without departing from the spirit and scope of the invention, and it is intended that such equivalent embodiments be covered by the invention. It should be understood that

Claims (1)

[Claims] 1 formula (wherein R is a ligand-analog of molecular weight 50-4000 having a single reactive primary or secondary amino group attached to the carbonyl carbon of the carboxyfluorescein moiety; has at least one epitope in common with the ligand, thereby making it specifically recognizable by a common antibody), and an antibody capable of specifically recognizing the tracer and the ligand, mixed with the sample; and a method for measuring a ligand in a sample, the method comprising: measuring the amount of the tracer bound to the antibody by fluorescence polarization technology as a reference for the amount of the ligand in the sample. 2. The method according to claim 1, wherein the ligand is a drug or a metabolite thereof. 3. The 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. The method according to claim 3, wherein the drug is a steroid, a hormone, an anti-asthmatic agent, an anti-cancer agent, an anti-arrhythmic agent, an anti-convulsant agent, an anti-arthritic agent, an anti-depressant, a glycoside cardiac agent, or a metabolite thereof. 5. The method of claim 1, wherein R has a molecular weight within the range of 100 to 2000. 6. The method of claim 5, wherein the drug is an anticonvulsant. 7. The method of claim 6, wherein the anticonvulsant is phenobarbital. 8. The method of claim 6, wherein the anticonvulsant is phenytoin. 9. The method of claim 6, wherein the anticonvulsant is primidone. 10. The method of claim 5, wherein the drug is a steroid. 11. The method according to claim 9, wherein the steroid is digoxin. 12. The method according to claim 5, wherein the drug is an antiarrhythmic agent. 13. The method according to claim 12, wherein the antiarrhythmic agent is propranolol. 14. The method according to claim 5, wherein the drug is an anti-asthmatic agent. 15. The method of claim 14, wherein the asthma agent is theophylline.