CA1178269A - Fluorescent polarization immunoassay utilizing substituted carboxyfluoresceins - Google Patents
Fluorescent polarization immunoassay utilizing substituted carboxyfluoresceinsInfo
- Publication number
- CA1178269A CA1178269A CA000396311A CA396311A CA1178269A CA 1178269 A CA1178269 A CA 1178269A CA 000396311 A CA000396311 A CA 000396311A CA 396311 A CA396311 A CA 396311A CA 1178269 A CA1178269 A CA 1178269A
- Authority
- CA
- Canada
- Prior art keywords
- compound according
- derived
- ligand
- drug
- tracer
- 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.)
- Expired
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D405/00—Heterocyclic 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
- C07D405/02—Heterocyclic 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
- C07D405/12—Heterocyclic 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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
- C07D311/02—Heterocyclic 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
- C07D311/78—Ring systems having three or more relevant rings
- C07D311/80—Dibenzopyrans; Hydrogenated dibenzopyrans
- C07D311/82—Xanthenes
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D473/00—Heterocyclic compounds containing purine ring systems
- C07D473/02—Heterocyclic compounds containing purine ring systems with oxygen, sulphur, or nitrogen atoms directly attached in positions 2 and 6
- C07D473/04—Heterocyclic compounds containing purine ring systems with oxygen, sulphur, or nitrogen atoms directly attached in positions 2 and 6 two oxygen atoms
- C07D473/06—Heterocyclic compounds containing purine ring systems with oxygen, sulphur, 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
- C07D473/08—Heterocyclic compounds containing purine ring systems with oxygen, sulphur, 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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07J—STEROIDS
- C07J41/00—Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring
- C07J41/0005—Normal 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
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Hematology (AREA)
- Molecular Biology (AREA)
- Urology & Nephrology (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Cell Biology (AREA)
- Steroid Compounds (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
A B S T R A C T
This disclosure relates to a method and roagents for determining ligands in biological fluids such as serum, plasma, spinal fluid, amniotic fluid and urine. In particular, this disclosure relates to a fluorescence polarization immunoassy procedure and to a novel class of tracer compounds employed as reagents in such procedures.
The procedure disclosed combines the specificity of an immunoassay with the speed and convience of fluorescence polarization techniques to provide a means for determining the amount of a specific ligand present in a sample.
This disclosure relates to a method and roagents for determining ligands in biological fluids such as serum, plasma, spinal fluid, amniotic fluid and urine. In particular, this disclosure relates to a fluorescence polarization immunoassy procedure and to a novel class of tracer compounds employed as reagents in such procedures.
The procedure disclosed combines the specificity of an immunoassay with the speed and convience of fluorescence polarization techniques to provide a means for determining the amount of a specific ligand present in a sample.
Description
t~3 Baclcground O The Invention The present invention relates to a method and reagents for determining ligands in biological fluids such as serum, plasmal spinal fluid, amnionic fluid and urine.
In par~icular, the present invention relates to a fluorescence polarization i~nunoassay 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 bindin~ immunoassays for measuring ligands are based on the competition bekween a ligand in a test sample and a labeled reagent, re~erred to as a tracer, ~or a limited number of receptor binding sites on antibodie!s specific to the ligand ancl tracer~ The concentration o~
ligand in ~he sample determines the amount o~ tracer that will speci~ically b~nd to an anti~ody. The amount o~ tracer-antibody conjugate produced may be quantitively measured and is inversely proportional to the quantity of ligand in the test sample.
In general, fluorescence polarization techniques are based on the principle that a fluorescent labeled compound when excited by linearly polarized light will emit fluorescence having a degree o~ polarization inversely related to its rate of rotation. Therefore, when a molecule such a~ a txacer-antibody conjugate ha~ing 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-anti~ody conjugate and the molecules ~t~t~i~3 are more randomly oriented, therefore, the emitted light is depolarized~ Thus, fluorescence polarization pxovides a quantitive means for measuring the amount of tracer-antibody conjugate produced in a competitive binding immunoassay.
Various fluorescent la~eled compounds are known in the art. U. S. Patent ~o. 3, 9~8,943 describes the preparation of a fluorescently labeled insulin derivative using fluorescein isothiocyanate (FITC) as the fluorescent label and a fluorescently labeled morphine derivative uslng 4-aminofluorescein hydrochloride as the fluorescent label.
Carboxyfluorescein has also been used for analytical determi-nations. Ro F. Chen, AnaZ. Lett~ ~ 10, 787 (lq77~ 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 Summar Of The Invention Y
The present invention encompasses ~ method for determining ligands in a sample comprising intermixing with said sample a biologically acceptable salt of a tracer of the formula:
OH
V
~\
1~'7~
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 reconizable ~y 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 inyention further relates to certain novel tracers and biologically acceptable salts thereof, which are useful in reagents in the above desoribed method. The methods and tracers o the present invention are particularly useul in quantitatively monitoring therapeutic drug concentrations in serum and plasmaO
_ctailed Description O~ The Invention The term l'ligand" as used herein refers to a molecule,in particular a low molecular weight hapten having a single react;ve amino group, to which a receptor, normally an antibody, can be o~tained or formed. Such haptens are protein-free bodies, generally of low molecular weight that 25 do not induce antihody 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 30 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 ~e determined, for best results, it is generally preferable to employ the methods 35 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.
t~
The novel tracers of the Present invention include compounds of fQrmula LI~ whereIn the ligand-analog repre.sented ~y R include r~dicals having a molecular weight within a range o~ 50 to 4~Q0. The preferred novel tracers include compounds of formula (I~ wherein the ligand-analogs represented ~y R include radicals havinq 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 steriods such as esterone, estradoil, cortisol, testoestrone, progesterone, chenodeoxycholic acid, digo~in!
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 antiarrhy~hmic drugs such as disopyramide, lidocaine, procainamide, propranolol, quinidine, N-acetyl-procainamide; anticonvulsant drugs such as phenobarbital, phenytoin, primidone, valproic acid, carbamazepine and ethosuximide; antibiotics such as penic.illins, cephalosporins and vancomycin; antiarthritic drugs such as salicylate; anti-depressant drugs including tricyclics such as nortriptylinet amitriptyline, imipramine and desipramlne; 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 a~use 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 equili~rium ~etween their acid and ionized states, and in the ionized state are effective in the method of the present invention. Th.erefore, 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 - 6 - ~7~
acceptable salts~ ~s 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 thelr 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 ~mployed, 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:
OH
(II) / \OH
_ 7 ~ 8~fi~
The term ligand-analog as used herein refers to a mono or polyvalent radical a su~stantial proportion of which has the same spatial and polar organization as the ligand to define one or more determinant or epitopic sites capable fo competing with the ligand for ~he binding sites of a receptor. A characteristic of such ligand-analog is that it possesses su~icient 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 10 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 same in preparing the antigen for production of anti-15 bodies as used for linking to the Ligand, the same portionof 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 ~rom the corresponding ligancl hy removal oE
20 a reactive hydrogen atom, i~e., a hydrogen atom bonded to a reacti~re amine (primary or secondary) or by the formation or an amino derivative of the ligand wherein an imino group -N-replaces one or more atoms originally present in the ligand, 25 at the site of binding to the carbo~yfluorescein 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 30 as ligand-analog include theophylline, valproic acid, phenobarbital, phenytoin, primidone, disopyramide ! digoxin, chloramphenicol, sal~cylate, acetaminophen, carbamazepine, desipramine and nortriptyline. In addition, a ligand may be structurally modified by the addition or deletion of one or 35 more functional groups to form a ligand-analog, while retaining - 8 ~LiL7E~2~i~
the necessary epitope sites for hinding to an anti~ody.
However, such modified ligand-analogs are bonded to the carboæyfluorescein 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:
(III) R - X
wherein R is above-defined and X is a reactive hydrogen; is treated with a compound of the formula:
' ~ ~
~ OH
~: 1l ~
(IV
O
. ~
:
, ,:~
: ~ ; ` :
: :;
: , - 9 - ~17~
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'l rom the car~oxy 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, l-hydroxy~enzotriazole-hydrate or p-nitrophenol in the presence of a coupling agent, such as dicyclohexyl-car~odiimide and a solvent. The active esters of carboxy-fluorescein thus produced are subse~uently reacted with a compound of formula tIII) to yield a tracer of ~rmula (I~.
If the compound o formula (III) i5 water soluble, the reaction mechanism proceeds by directly reacting carboxyfluorescein with a compound of formula (IIIl in aqueous solution in the presence of a water solu~le carbodiimide, such as 1-ethyl-3~(3Ldimethylaminopropyl)-carbodiimide hydrochloride r as a couplin~ agent.
The temperature at which the process for preparing the tracers of this invention proceeds is not critical. The temperature should be one wh~ch is sufficient so as to initiate and maintain the reaction. Generally, or con-venience and economy, ro~m 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 tIII) to obtain a reasona~le yield. It is preferred to employ an excess of comp~und 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 sol~ent, 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 10- ~:L'7i~
water (if the compound o formula (III) is water solublel, 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. Suitahle bases include for example, triethylamine. The reaction products of formula ~I) are generally purified using either thin-layer or column chromatography prior to application in the methods of the present invention.
In accordance with the method of the present invention, a sample containing the ligand to be determined is intermixed with a biologically acceptable salt of a tracer of formula (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-antlbody complexes.
By maintaining constant the concentration of tracer and antibody, tha 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 ~he 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 txacer not complexed to an antibody is low, approaching zero. Upon complexing with a specific anti~ody, the tracer-antibody complex thus formed assumes the rotation of the antibody molecule which is slower than that of the relatively small tracer molecule, thereby increasing the polarization observed. Therefore, when a ligand competes with the tracer for antibody sites, the observed polarization o~ fluorescence of the tracer-antibody 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., low. If the test sample contains a low concentration of the ligand, the polarization value is L7~ 9 closer to that of the bound ligand, i.e., high. By sequentially exciting the reaction mixture of an Lmmunoassay with vertically and then horizontally polarized light and analyzing only the vertical component of the emitted light, the polarization of fluorescence in the reaction mix may be accurately determined. The precise relationship between polarization and concentration ofthe ligand to be determined is established by measuring the polarizat:ion values of calibrators with known concentrations. rrhe 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 10, most preferably from a~out 6 to 9. Various buffers may be used to achieve and maintain the pH during the assay procedure. R~presentative buffers include ~orate, phosphate, carbonate, tris, barbital and the like. The particular bu~er employed is not critical to the present invention, ~ut in an individ~al assay, a specific ~uffer may be preferred in view of the antibody employed and ligand to be determined. The cation portion of the huffer 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. ~he temperature will normally range from about 0 to 50 C, more usually from a~out 15 to 40 C~
The concentration of liaand which may be assayed will generally vary from about 10 2 to 10 13M, more usually from a~out 10 4 to 10 QM. Hi~her concentrations of ligand may be assayed upon dilution of the original sample.
In addition to the concentration range of ligand of interest, considerations such as whether the assay is qualitative, semiquantitative or quantitative, the equipment employed, and the characteristics of the tracer and antibody will normally determine the concentration o~ the tracer and antibody to be employed. While the concentration of - 12 - ~ ~ 7~
ligand in the sample will determine the range of con-centration of the other reagents, i.e~, tracer and anti~ody, normally to optimize the sensitivity of the assay, individual reagent concentrations will be determinecl empirically. Con-centrations o the tracer and antibody are readilyascertained by one of ordinary skill in the art.
As previously mentioned the preferred tracers of the present inention are prepared from 5--carboxyfluorescein or 4-car~oxyfluorescein or mixtures thereof and are represented ~y the formulas:
OH
~_1C ~ ~ (V) or OH
z ~l ~
~ (VI) ~, 0/~ \0}~ ~ , 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 ~he is invention may be prepared. The symbol ~C~]
appearing in the structural formulas illustrating the com-pounds prepared in the following examples, represents a moiety o the formula:
OH
(VII) ~ \o~
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-carboxy1uorescein is employed as starting material.
- 15 - ~ ~'7 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'-dichohexylcarbodi-imide (15 mg). The reaction proceeded for two hours atroom 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:
H O
~ _ ~ ~ ~3 ¦ ~cF~
- 16 ~
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 re~luxed 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 preclpitate, 2-~-amino-ethylphenytoin, was filtered, rinsed thoroughly with water and dried.
An active ester o~ 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 2 ~-aminoethyl-phenytoin (10 mg) was dissolved ln the reaction mixture.
The resultin~ 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-~-aminoethylphenytoin-carboxyfluorescein conjugate of the formula;
O H
C _ N-CH2CH2-N [C~}
/=\ / 2~ C
H / O
- 17 - ~ ~78'Z~3 EXAMPLE III
A solution containing 2-carboxymethylphenytoin ~620 mg), N-hydroxysuccinimide (248 mg) and N,N'-dicyclo-hexylcar~odiimide (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,
In par~icular, the present invention relates to a fluorescence polarization i~nunoassay 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 bindin~ immunoassays for measuring ligands are based on the competition bekween a ligand in a test sample and a labeled reagent, re~erred to as a tracer, ~or a limited number of receptor binding sites on antibodie!s specific to the ligand ancl tracer~ The concentration o~
ligand in ~he sample determines the amount o~ tracer that will speci~ically b~nd to an anti~ody. The amount o~ tracer-antibody conjugate produced may be quantitively measured and is inversely proportional to the quantity of ligand in the test sample.
In general, fluorescence polarization techniques are based on the principle that a fluorescent labeled compound when excited by linearly polarized light will emit fluorescence having a degree o~ polarization inversely related to its rate of rotation. Therefore, when a molecule such a~ a txacer-antibody conjugate ha~ing 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-anti~ody conjugate and the molecules ~t~t~i~3 are more randomly oriented, therefore, the emitted light is depolarized~ Thus, fluorescence polarization pxovides a quantitive means for measuring the amount of tracer-antibody conjugate produced in a competitive binding immunoassay.
Various fluorescent la~eled compounds are known in the art. U. S. Patent ~o. 3, 9~8,943 describes the preparation of a fluorescently labeled insulin derivative using fluorescein isothiocyanate (FITC) as the fluorescent label and a fluorescently labeled morphine derivative uslng 4-aminofluorescein hydrochloride as the fluorescent label.
Carboxyfluorescein has also been used for analytical determi-nations. Ro F. Chen, AnaZ. Lett~ ~ 10, 787 (lq77~ 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 Summar Of The Invention Y
The present invention encompasses ~ method for determining ligands in a sample comprising intermixing with said sample a biologically acceptable salt of a tracer of the formula:
OH
V
~\
1~'7~
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 reconizable ~y 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 inyention further relates to certain novel tracers and biologically acceptable salts thereof, which are useful in reagents in the above desoribed method. The methods and tracers o the present invention are particularly useul in quantitatively monitoring therapeutic drug concentrations in serum and plasmaO
_ctailed Description O~ The Invention The term l'ligand" as used herein refers to a molecule,in particular a low molecular weight hapten having a single react;ve amino group, to which a receptor, normally an antibody, can be o~tained or formed. Such haptens are protein-free bodies, generally of low molecular weight that 25 do not induce antihody 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 30 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 ~e determined, for best results, it is generally preferable to employ the methods 35 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.
t~
The novel tracers of the Present invention include compounds of fQrmula LI~ whereIn the ligand-analog repre.sented ~y R include r~dicals having a molecular weight within a range o~ 50 to 4~Q0. The preferred novel tracers include compounds of formula (I~ wherein the ligand-analogs represented ~y R include radicals havinq 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 steriods such as esterone, estradoil, cortisol, testoestrone, progesterone, chenodeoxycholic acid, digo~in!
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 antiarrhy~hmic drugs such as disopyramide, lidocaine, procainamide, propranolol, quinidine, N-acetyl-procainamide; anticonvulsant drugs such as phenobarbital, phenytoin, primidone, valproic acid, carbamazepine and ethosuximide; antibiotics such as penic.illins, cephalosporins and vancomycin; antiarthritic drugs such as salicylate; anti-depressant drugs including tricyclics such as nortriptylinet amitriptyline, imipramine and desipramlne; 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 a~use 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 equili~rium ~etween their acid and ionized states, and in the ionized state are effective in the method of the present invention. Th.erefore, 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 - 6 - ~7~
acceptable salts~ ~s 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 thelr 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 ~mployed, 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:
OH
(II) / \OH
_ 7 ~ 8~fi~
The term ligand-analog as used herein refers to a mono or polyvalent radical a su~stantial proportion of which has the same spatial and polar organization as the ligand to define one or more determinant or epitopic sites capable fo competing with the ligand for ~he binding sites of a receptor. A characteristic of such ligand-analog is that it possesses su~icient 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 10 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 same in preparing the antigen for production of anti-15 bodies as used for linking to the Ligand, the same portionof 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 ~rom the corresponding ligancl hy removal oE
20 a reactive hydrogen atom, i~e., a hydrogen atom bonded to a reacti~re amine (primary or secondary) or by the formation or an amino derivative of the ligand wherein an imino group -N-replaces one or more atoms originally present in the ligand, 25 at the site of binding to the carbo~yfluorescein 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 30 as ligand-analog include theophylline, valproic acid, phenobarbital, phenytoin, primidone, disopyramide ! digoxin, chloramphenicol, sal~cylate, acetaminophen, carbamazepine, desipramine and nortriptyline. In addition, a ligand may be structurally modified by the addition or deletion of one or 35 more functional groups to form a ligand-analog, while retaining - 8 ~LiL7E~2~i~
the necessary epitope sites for hinding to an anti~ody.
However, such modified ligand-analogs are bonded to the carboæyfluorescein 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:
(III) R - X
wherein R is above-defined and X is a reactive hydrogen; is treated with a compound of the formula:
' ~ ~
~ OH
~: 1l ~
(IV
O
. ~
:
, ,:~
: ~ ; ` :
: :;
: , - 9 - ~17~
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'l rom the car~oxy 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, l-hydroxy~enzotriazole-hydrate or p-nitrophenol in the presence of a coupling agent, such as dicyclohexyl-car~odiimide and a solvent. The active esters of carboxy-fluorescein thus produced are subse~uently reacted with a compound of formula tIII) to yield a tracer of ~rmula (I~.
If the compound o formula (III) i5 water soluble, the reaction mechanism proceeds by directly reacting carboxyfluorescein with a compound of formula (IIIl in aqueous solution in the presence of a water solu~le carbodiimide, such as 1-ethyl-3~(3Ldimethylaminopropyl)-carbodiimide hydrochloride r as a couplin~ agent.
The temperature at which the process for preparing the tracers of this invention proceeds is not critical. The temperature should be one wh~ch is sufficient so as to initiate and maintain the reaction. Generally, or con-venience and economy, ro~m 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 tIII) to obtain a reasona~le yield. It is preferred to employ an excess of comp~und 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 sol~ent, 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 10- ~:L'7i~
water (if the compound o formula (III) is water solublel, 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. Suitahle bases include for example, triethylamine. The reaction products of formula ~I) are generally purified using either thin-layer or column chromatography prior to application in the methods of the present invention.
In accordance with the method of the present invention, a sample containing the ligand to be determined is intermixed with a biologically acceptable salt of a tracer of formula (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-antlbody complexes.
By maintaining constant the concentration of tracer and antibody, tha 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 ~he 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 txacer not complexed to an antibody is low, approaching zero. Upon complexing with a specific anti~ody, the tracer-antibody complex thus formed assumes the rotation of the antibody molecule which is slower than that of the relatively small tracer molecule, thereby increasing the polarization observed. Therefore, when a ligand competes with the tracer for antibody sites, the observed polarization o~ fluorescence of the tracer-antibody 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., low. If the test sample contains a low concentration of the ligand, the polarization value is L7~ 9 closer to that of the bound ligand, i.e., high. By sequentially exciting the reaction mixture of an Lmmunoassay with vertically and then horizontally polarized light and analyzing only the vertical component of the emitted light, the polarization of fluorescence in the reaction mix may be accurately determined. The precise relationship between polarization and concentration ofthe ligand to be determined is established by measuring the polarizat:ion values of calibrators with known concentrations. rrhe 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 10, most preferably from a~out 6 to 9. Various buffers may be used to achieve and maintain the pH during the assay procedure. R~presentative buffers include ~orate, phosphate, carbonate, tris, barbital and the like. The particular bu~er employed is not critical to the present invention, ~ut in an individ~al assay, a specific ~uffer may be preferred in view of the antibody employed and ligand to be determined. The cation portion of the huffer 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. ~he temperature will normally range from about 0 to 50 C, more usually from a~out 15 to 40 C~
The concentration of liaand which may be assayed will generally vary from about 10 2 to 10 13M, more usually from a~out 10 4 to 10 QM. Hi~her concentrations of ligand may be assayed upon dilution of the original sample.
In addition to the concentration range of ligand of interest, considerations such as whether the assay is qualitative, semiquantitative or quantitative, the equipment employed, and the characteristics of the tracer and antibody will normally determine the concentration o~ the tracer and antibody to be employed. While the concentration of - 12 - ~ ~ 7~
ligand in the sample will determine the range of con-centration of the other reagents, i.e~, tracer and anti~ody, normally to optimize the sensitivity of the assay, individual reagent concentrations will be determinecl empirically. Con-centrations o the tracer and antibody are readilyascertained by one of ordinary skill in the art.
As previously mentioned the preferred tracers of the present inention are prepared from 5--carboxyfluorescein or 4-car~oxyfluorescein or mixtures thereof and are represented ~y the formulas:
OH
~_1C ~ ~ (V) or OH
z ~l ~
~ (VI) ~, 0/~ \0}~ ~ , 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 ~he is invention may be prepared. The symbol ~C~]
appearing in the structural formulas illustrating the com-pounds prepared in the following examples, represents a moiety o the formula:
OH
(VII) ~ \o~
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-carboxy1uorescein is employed as starting material.
- 15 - ~ ~'7 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'-dichohexylcarbodi-imide (15 mg). The reaction proceeded for two hours atroom 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:
H O
~ _ ~ ~ ~3 ¦ ~cF~
- 16 ~
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 re~luxed 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 preclpitate, 2-~-amino-ethylphenytoin, was filtered, rinsed thoroughly with water and dried.
An active ester o~ 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 2 ~-aminoethyl-phenytoin (10 mg) was dissolved ln the reaction mixture.
The resultin~ 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-~-aminoethylphenytoin-carboxyfluorescein conjugate of the formula;
O H
C _ N-CH2CH2-N [C~}
/=\ / 2~ C
H / O
- 17 - ~ ~78'Z~3 EXAMPLE III
A solution containing 2-carboxymethylphenytoin ~620 mg), N-hydroxysuccinimide (248 mg) and N,N'-dicyclo-hexylcar~odiimide (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 puri~ied twice employing silica gel thin-layer chromatography using a chloroform:acetone (1:1) mixture as developing solvent to yield a 2-carboxymethylphenytoin hydrazide-car~oxy-1uorescein conjugate of the formula:
O H H
~ ~ 11 1 1 ~ ~C N-C~2-C-N-N~CF]
~ H O
- 18 - ~'7~Z6~
EXAMPLE IV
N,N'-dicyclohexylcarbodiimide (15 mg) was added to a solution of 8-~-aminoethyltheophylline (5 mg) and carboxy-fluorescein ~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 chromato~raphy using a thin chloroform:methanol ~2:1) mixture as developing solvent to yield an 8-~-amino ethyltheophylline-car~oxyfluoresceirl conjugate of the formula:
- 19 - ~L7~
EXAM ~E V
The procedure of Example IV was employed utilizing 8~aminomethyltheophylline in lieu of 8-3-aminoethylthoephylline to yield an 8-aminomethylthoephylline-carboxyfluorescein conjugate of the formula;
J ~CH2_N~CF
C \ N
~XAMPLE ~I
~ -Valerolactam ~7 5 g~ was dissolved in 6a ml of dry tetrahydrofuran, under a dry nitrogen atmosphere and n-butyllithium (1.6 M, 90 mll tn hexane were added dropwise to the reaction flask and chilled in a dry ~ce-acetone ~ath.
Upon completion of the additton of n-~utyllithium, the reaction mixture was stirred at room temperature ~or one hour, refluxed for thirty minutes, and cooled to room temperature under dry nitorgen atmosphere. l-Bromoethane (8.0 g) was slowly added to the reaction flask while the flask was chilled in an ice ~ath. The resulting mi-xture 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 com~ined and dried over sodium sulfate. The solvent - 20 - ~ ~7~Z~
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), 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-ethyl-5-ami~opentanoic acid (20 mg) was dissolved in the reaction mixture. The resulting mixture was allowed to react over-15 night 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 oe the formula:
CH3CH2 f CH2CH2 2 ~~ ]
Cl=O
OH
- 21 ~
EXAMPLE ~II
An active ester of carboxyfluorescein was prepared by dissolving N-hydroxysuccinimide tS mg), carboxyfluorescein (7.5 mg) and ~,N'-dicyclohexylcarhodiimide (20 mg) in 0.5 ml 5 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]-10,11-dihydrocycloheptene ~20 mg) was dissolved in the reaction mixture. The resulting mixture was allowed to react overnight in the dark at room 10 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-tY-aminopropylidene~-5H-dibenzo[a~d]-10~11-di-hydrocycloheptene-carboxy1uorescein conjugate of the formula:
~/~
~'CHCE12CH2-NH ~ CF]
- 22 - ~ &~
XAMPLE VIII
A solution containing desipramine hydrochloride (1~33 g) and chloroacetyl chloride (0.8 g) in 25 ml of chloro-form was refluxed for two hours, The chloroform was evaporated 5 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 prec;pitated salt was rinsed with acetone.
The acetone filtrate was evaporated and the residue was 10 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, 15 filtered and evaporated to yield N-aminoacetyldesipramine which was used without further purification.
N-aminoacetyldesipramine (5 mg) and carboxy-fluorescein ~5 mg) were dissolved in 0.5 ml of pyridine.
To the mixture was added N,N'-diclohexylcarbodiimide (15 mg).
20 ~he ,reaction proceeded for two hours at room temperature, a~ter which time the reaction product was purified twice employing silica gel thin-layer chromatography using a chloro~orm:acetone tl:l) mixture as developing solving to yield a N-aminoacetyldesipramine-carboxyfluorescein 25 conjugate of the formula:
~ I,`i N-C~ CH CH -N-C-CH2NH-~CF]
E~YAMPLE IX
A solution containing N-hydroxysuccinimide (S mg~, carboxyfluorescein (7~5 mg1 and N,N~-dicyclohexylcarbodi-s imide (20 mg) in 1 ml of pyridine was allowed to reack at room temperature for ~our hours. An acti~e ester of carboxy1uorescein was precipitated by adding lO ml o~ di-ethylether to the reaction mixture. Tha precipitate was I filtered, rinsed well with diethylether and redissolved 10 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 chloro~orm:methanol lS (3:1) mixture as developing solvent to yield a L-thyroxine-carboxyfluorescein conjugate of the formula:
I\ I \ ~
T-OH
H(:) I CH2 -C N~ CF ]
2 ~
EXAMPLE X
A solution containing ammonium ace~ate (0.89 g),
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 puri~ied twice employing silica gel thin-layer chromatography using a chloroform:acetone (1:1) mixture as developing solvent to yield a 2-carboxymethylphenytoin hydrazide-car~oxy-1uorescein conjugate of the formula:
O H H
~ ~ 11 1 1 ~ ~C N-C~2-C-N-N~CF]
~ H O
- 18 - ~'7~Z6~
EXAMPLE IV
N,N'-dicyclohexylcarbodiimide (15 mg) was added to a solution of 8-~-aminoethyltheophylline (5 mg) and carboxy-fluorescein ~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 chromato~raphy using a thin chloroform:methanol ~2:1) mixture as developing solvent to yield an 8-~-amino ethyltheophylline-car~oxyfluoresceirl conjugate of the formula:
- 19 - ~L7~
EXAM ~E V
The procedure of Example IV was employed utilizing 8~aminomethyltheophylline in lieu of 8-3-aminoethylthoephylline to yield an 8-aminomethylthoephylline-carboxyfluorescein conjugate of the formula;
J ~CH2_N~CF
C \ N
~XAMPLE ~I
~ -Valerolactam ~7 5 g~ was dissolved in 6a ml of dry tetrahydrofuran, under a dry nitrogen atmosphere and n-butyllithium (1.6 M, 90 mll tn hexane were added dropwise to the reaction flask and chilled in a dry ~ce-acetone ~ath.
Upon completion of the additton of n-~utyllithium, the reaction mixture was stirred at room temperature ~or one hour, refluxed for thirty minutes, and cooled to room temperature under dry nitorgen atmosphere. l-Bromoethane (8.0 g) was slowly added to the reaction flask while the flask was chilled in an ice ~ath. The resulting mi-xture 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 com~ined and dried over sodium sulfate. The solvent - 20 - ~ ~7~Z~
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), 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-ethyl-5-ami~opentanoic acid (20 mg) was dissolved in the reaction mixture. The resulting mixture was allowed to react over-15 night 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 oe the formula:
CH3CH2 f CH2CH2 2 ~~ ]
Cl=O
OH
- 21 ~
EXAMPLE ~II
An active ester of carboxyfluorescein was prepared by dissolving N-hydroxysuccinimide tS mg), carboxyfluorescein (7.5 mg) and ~,N'-dicyclohexylcarhodiimide (20 mg) in 0.5 ml 5 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]-10,11-dihydrocycloheptene ~20 mg) was dissolved in the reaction mixture. The resulting mixture was allowed to react overnight in the dark at room 10 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-tY-aminopropylidene~-5H-dibenzo[a~d]-10~11-di-hydrocycloheptene-carboxy1uorescein conjugate of the formula:
~/~
~'CHCE12CH2-NH ~ CF]
- 22 - ~ &~
XAMPLE VIII
A solution containing desipramine hydrochloride (1~33 g) and chloroacetyl chloride (0.8 g) in 25 ml of chloro-form was refluxed for two hours, The chloroform was evaporated 5 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 prec;pitated salt was rinsed with acetone.
The acetone filtrate was evaporated and the residue was 10 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, 15 filtered and evaporated to yield N-aminoacetyldesipramine which was used without further purification.
N-aminoacetyldesipramine (5 mg) and carboxy-fluorescein ~5 mg) were dissolved in 0.5 ml of pyridine.
To the mixture was added N,N'-diclohexylcarbodiimide (15 mg).
20 ~he ,reaction proceeded for two hours at room temperature, a~ter which time the reaction product was purified twice employing silica gel thin-layer chromatography using a chloro~orm:acetone tl:l) mixture as developing solving to yield a N-aminoacetyldesipramine-carboxyfluorescein 25 conjugate of the formula:
~ I,`i N-C~ CH CH -N-C-CH2NH-~CF]
E~YAMPLE IX
A solution containing N-hydroxysuccinimide (S mg~, carboxyfluorescein (7~5 mg1 and N,N~-dicyclohexylcarbodi-s imide (20 mg) in 1 ml of pyridine was allowed to reack at room temperature for ~our hours. An acti~e ester of carboxy1uorescein was precipitated by adding lO ml o~ di-ethylether to the reaction mixture. Tha precipitate was I filtered, rinsed well with diethylether and redissolved 10 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 chloro~orm:methanol lS (3:1) mixture as developing solvent to yield a L-thyroxine-carboxyfluorescein conjugate of the formula:
I\ I \ ~
T-OH
H(:) I CH2 -C N~ CF ]
2 ~
EXAMPLE X
A solution containing ammonium ace~ate (0.89 g),
3-oxodigoxigenin (389 mg) and sodium cyanoborohydride (63 mg) in 5 ml of methanol was stirred at room temperature for 48 5 hoursA 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 10 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 3-amino-3-deoxydigoxigenin which was used without further puriication.
An active ester of carboxyfluorescein was prepared by dissolvi.ng N-hydroxysuccinimide (5 mg~, carboxyfluorescein (7.5 mg) and N,N'-dicyclohexylcarbodiimide (20 mg~ in 0.5 ml of pyridine. The reac~ion was allowed to proceed for two hours at room temperature, after which time a 3-amino-20 3-deoxydigoxigenin-carboxyfluorescein conjugate of the formula was isolated:
2 5 ~ Z~3 o~
~N~CF ]
~ JLt;~ 3 The following tracers were also prepared in accordance with the procedures previously dascribed:
XAMPLE XI - O-~minoacetyl-propranolol-carboxyfluor~scein coniuqate O H
Il I
OcH2cH-o-c-cH2-N~cF]
XAMPLE XII - 2-Propyl-5-aminopentanoic acid-carbox~fluorescein coniuqate H H
CH3cH2cH2~f-cH2cH2cH2-N~cF ]
C=O
I
OH
EXAMPLE XIII - 2-Butyl-5-aminopentanoic acid-carboxyfluorescein conjugate CH3cH2cH2cH2-c-cH2cH2cH2-N~cF]
C=O
OH
EXAMPLE XIV - Aminoprimidone-carboxy~luorescein conjugate ,~ C' '' CH 2 CH 3 CH ~ C
~ N - C~
H O
- 27 - ~'3L7~
EXAMPLE XV ~ 4'-nitrophenyl~-l-hydroxy-2-amino-3-hydroxypropane-_ carboxyfluorescein conjugate 02N-~-CH-fH-N~CF~
CH2 ~
5 XAMPLE XVI - p-amlnophenol-carbaxyfluorescein conjugate Ho_e~-N~CF]
EXAMPLE XVII - N- (2-aminoethyl)-ethosuximide-_ _ carboxy~luorescein conjugate CH 2 C tH
~ N-CEI2CH2-N~CP3 10 EXAMPLE XVIII - N'-desethyl-N-acetyl-procainamide-carboxyfluorescein conjugate -C - N -~ C -N- CH2 CH 2 - N{`C li ]
- 28 ~ 8~
EXAMPLE XIX - N'-desethyl-N'-aminoacetyl-N-acetyl-procainamide-carboxy-fluorescein coniugate O H ~ O H CH2CH3 Hl ~-N-~-c-N-cH2cH2-N-c-cH2-N~cFJ
5 EXAMPLE XX - l-amino-2-phenyl-2-(2'-pyridyl)-4-(diisopropylamino)-butane-_ carbo ~ rescein conjugate H-C CH2-N--~CF]
C ;
C ~ N
CH3 ~
EXAMPLE XXI - 3,3',5-Triiodo-L-thyronine-lO _ carboxy~luorescein conjugate ~C /H
HO- ~ O- ~ _C~2_~ CF]
- 29 - ~7~9 EXAMPLE XXII - 3,3',5,5'-tetraiodo-D-thyronine-carboxyfluorescein~c~o~n ~
.
O OH
~C H
HO-~- o~ CH2 - C-N~ C F ]
EXA~PLE XXIII ~ N-aminoacetyl-iminodibenzyl 5car~oxyfluorescein conjugate ~ O H
N-C-CH2-N~CF]
/
EXAMPLE XXIV - Carbhydrazinoimino-dibenzyl carboxyfluorescein conjugate O H H
N-C-N-N-~ CF ]
- 30 - ~ 9 EX~MPLE X~- Dibenzosuberonehydrazone fluorescein conjugate H
~=N-N-~CP]
EXAMPLE XXVI - 5-amino-10,11-dihydro-SH-dibenzo-S~a,d3-cycloheptene ~H
N~CF]
~.~'7~
As previously mentioned~ the tracers of the present in~ention are effective reagents for use in fluorescence polari2ation immunoassays. The followlng Examples illustrate the suitablility of tracers of the 5 present invention in immunoassays employing fluorescence polarization techniques. Such assays are conclucted in accordance with the Eollowing 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 CQntaining a surfactant is then added to each tube;
3) A known concentration of antisera is added to the tubes;
The aqueous layer was adjusted to pH 11 by using solid 10 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 3-amino-3-deoxydigoxigenin which was used without further puriication.
An active ester of carboxyfluorescein was prepared by dissolvi.ng N-hydroxysuccinimide (5 mg~, carboxyfluorescein (7.5 mg) and N,N'-dicyclohexylcarbodiimide (20 mg~ in 0.5 ml of pyridine. The reac~ion was allowed to proceed for two hours at room temperature, after which time a 3-amino-20 3-deoxydigoxigenin-carboxyfluorescein conjugate of the formula was isolated:
2 5 ~ Z~3 o~
~N~CF ]
~ JLt;~ 3 The following tracers were also prepared in accordance with the procedures previously dascribed:
XAMPLE XI - O-~minoacetyl-propranolol-carboxyfluor~scein coniuqate O H
Il I
OcH2cH-o-c-cH2-N~cF]
XAMPLE XII - 2-Propyl-5-aminopentanoic acid-carbox~fluorescein coniuqate H H
CH3cH2cH2~f-cH2cH2cH2-N~cF ]
C=O
I
OH
EXAMPLE XIII - 2-Butyl-5-aminopentanoic acid-carboxyfluorescein conjugate CH3cH2cH2cH2-c-cH2cH2cH2-N~cF]
C=O
OH
EXAMPLE XIV - Aminoprimidone-carboxy~luorescein conjugate ,~ C' '' CH 2 CH 3 CH ~ C
~ N - C~
H O
- 27 - ~'3L7~
EXAMPLE XV ~ 4'-nitrophenyl~-l-hydroxy-2-amino-3-hydroxypropane-_ carboxyfluorescein conjugate 02N-~-CH-fH-N~CF~
CH2 ~
5 XAMPLE XVI - p-amlnophenol-carbaxyfluorescein conjugate Ho_e~-N~CF]
EXAMPLE XVII - N- (2-aminoethyl)-ethosuximide-_ _ carboxy~luorescein conjugate CH 2 C tH
~ N-CEI2CH2-N~CP3 10 EXAMPLE XVIII - N'-desethyl-N-acetyl-procainamide-carboxyfluorescein conjugate -C - N -~ C -N- CH2 CH 2 - N{`C li ]
- 28 ~ 8~
EXAMPLE XIX - N'-desethyl-N'-aminoacetyl-N-acetyl-procainamide-carboxy-fluorescein coniugate O H ~ O H CH2CH3 Hl ~-N-~-c-N-cH2cH2-N-c-cH2-N~cFJ
5 EXAMPLE XX - l-amino-2-phenyl-2-(2'-pyridyl)-4-(diisopropylamino)-butane-_ carbo ~ rescein conjugate H-C CH2-N--~CF]
C ;
C ~ N
CH3 ~
EXAMPLE XXI - 3,3',5-Triiodo-L-thyronine-lO _ carboxy~luorescein conjugate ~C /H
HO- ~ O- ~ _C~2_~ CF]
- 29 - ~7~9 EXAMPLE XXII - 3,3',5,5'-tetraiodo-D-thyronine-carboxyfluorescein~c~o~n ~
.
O OH
~C H
HO-~- o~ CH2 - C-N~ C F ]
EXA~PLE XXIII ~ N-aminoacetyl-iminodibenzyl 5car~oxyfluorescein conjugate ~ O H
N-C-CH2-N~CF]
/
EXAMPLE XXIV - Carbhydrazinoimino-dibenzyl carboxyfluorescein conjugate O H H
N-C-N-N-~ CF ]
- 30 - ~ 9 EX~MPLE X~- Dibenzosuberonehydrazone fluorescein conjugate H
~=N-N-~CP]
EXAMPLE XXVI - 5-amino-10,11-dihydro-SH-dibenzo-S~a,d3-cycloheptene ~H
N~CF]
~.~'7~
As previously mentioned~ the tracers of the present in~ention are effective reagents for use in fluorescence polari2ation immunoassays. The followlng Examples illustrate the suitablility of tracers of the 5 present invention in immunoassays employing fluorescence polarization techniques. Such assays are conclucted in accordance with the Eollowing 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 CQntaining 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) ~he amount of tracer bound to antibody is measured by fluorescence polariæation techniques as a measure of the amount o ligang in the sample~
- 32 - ~7~
EXAMPLEXXVqI- Phen tion assa Y y A~ Materials required:
1) BGG buffer consisting o~ 0.1 M sodium phosphate, pH 7.5, containing bovine gammaglobulin, 0.01% and sodium azide, 0.01~.
2) Tracer, consisting of 2-~-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 contain-ing 0.005% benzalkonium chloride.
4) Samples of human serum or other biologiGal 1uid containing phenytoin.
5) Cuvettes, lO x 75 mm glass culture tubes used as cuvettes.
- 32 - ~7~
EXAMPLEXXVqI- Phen tion assa Y y A~ Materials required:
1) BGG buffer consisting o~ 0.1 M sodium phosphate, pH 7.5, containing bovine gammaglobulin, 0.01% and sodium azide, 0.01~.
2) Tracer, consisting of 2-~-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 contain-ing 0.005% benzalkonium chloride.
4) Samples of human serum or other biologiGal 1uid containing phenytoin.
5) Cuvettes, lO x 75 mm glass culture tubes used as cuvettes.
6) Fluorometer capable of measuring ~luorescence polarization with a precl~ion of ~ 0.001 unit5.
Bl Assay Method:
1) A small volume of sample (0.366 microliters) is placed in each cuvette by pipetting 15 ~Q of sample and diluting with 600 ~Q BGG buffer in a dilution vessel. Next, 15 ~Q of diluted sample is pipetted into the cuvette followed by 600 ~Q BGG buffer.
2) Tracer is added by pipetting 40 ~ tracer and lO00 ~Q BGG buffer into the cuvette.
3~ Antiserum is added to start the reaction by pipetting 40 ~Q antiserum into the cuvette followed by lO00 ~Q
BGG buffer.
4) The contents of all cuvettes are well mixed and allowed to incubate for 15 minutes at ambient temperature.
5) The fluorescence polarization is read on a fluorometer and a standard curve constructed to determine unknowns.
- 33 - ~ ~'7~
C~ The results of a series of serum standards containing phenytoin at concentrations between 0 and 40 ~g,/ml are presented below. Each concentration was assayed in duplicate and averaged.
S Concentration of Phenytoin (~g/ml)Polarlzation 0.222 2.5 0.1~6 5.0 0.178 10,0 Q.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 quan-titated by reerence to the standard curve, thereby illustrating the utility of 2-~ aminoethyl phenytoin-carboxy~luorescein for the measurement of phenytoin.
EXAMPLE XNIII -Phenobarbital assay A) Materials required:
1) BGG buffer lsee Phenytoin) 22 Tracer, consisting of amlnophenobarbital 23 carboxyfluorescein at a concentration of approximately 110 nM
in tris HCl buffer, pH 7.5, containing 0.01~ sodium azide, 0.01% bovine gamma globulin, and 0.125% sodium dodecyl sulfate.
3) Antiserum, consiting of antiserum against phenobarbital diluted appropriately in BGG buffer containing 0.0~5~ benzalkonium chloride.
4) Samples of human serum or other ~iological fluid containing phenobarbital.
5) Cuvettes (see Phenytoin) 6~ Fluorometer ~see Phenytoin~
iLi7~
B~ Assay Protocol:
1~ A small volume of sample (0.196 micr~liter~
is placed in the cuvette by ipetting 10 ~Q o sample and diluting with 500 ~Q BGG buffer in a dilution vessel. Next, 10 ~Q of diluted sample is pipetted into the cuvette followed by 500 ~Q BGG buffer.
2) Tracer is added by pipettin~ 40 ~ of tracer and 1000 ~Q BGG buffer into each cuvette.
3) Antiserum is added to start the reaction by 10 pipetting 40 ~Q antiserum followed by 1000 ~ BGG buffer.
4) The contents of all cuvettes are mixed well and allowed to incubate for 15 minutes at ambient temperature.
5) The fluoxescence polarization is read on a fluorometer and a standard curve ccnstructed to determine unknowns.
C) The results of a series of senlm standards containing phenobarbital at concentrations between 0 and 80 ~g/ml are presented below. Each concentration was assayed in duplicate and the values averaged.
Concentration of P enobarbital (~Q)Polarization 0 0.250 5.0 0.~31 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, 30 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.
- 35 - ~ ~7 _AMPLE XXIX _ - Theophylline assay A) Materials required:
1) ~racer, consisting of 2 nM of 8-aminoethyl theophylline-carboxyfluorescein in BGG buffer (see Phentoin assay~ containing 0.01~ sodium dodecyl ~ulfate.
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 tracer in all cuvettas.
2) Add 2.0 ~Q sample to all cuvettes.
3) Add 1.O ml antiserum to all cuvettes, 4) Mix well and incubate 15 minutes at ambient temperature.
5) Read the fluorescence polarization on a ~luoro-meter and construct a standard curve.
C~ The results of a series of serum standards containinq theophylline at concentrations between 0 and 40 ~g/ml are presented. Each concentration was assayed in duplicate and the average is presented.
Concentration of Theo hvlline C~q/mllPolarization P ,~
o 0.158 2,5 0.118 0.105 la 0.091 0.076 0.063 ~7~
The polarization of fluore~cence 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 ~uantita~ed by reference to the standard curbe thereby illustrating the utility of 8-aminoethyltheophylline-carbox~tfluorescein for the measure-ment of the theophylline.
E~L~ XXX - Digoxin assa Y
A) Materials required:
1) BGG buffer consisting of O.lM sodium phosphate, pH 7.5, containing bovine gammaglobulin, 0.01% and sodium azide, 0.01~.
2) Tracer, consisting of digoxin carbo~yfluorescein at a concentration of approximately 2nM in BGG bufer.
3) Antiserum, consisting oE rabbit antiserum raised against digoxin diluted appropriately in BGG buffer.
4) Samples or human serum or other biological fluid containing phenytoin.
5) Precipitation reagent --5% trichloroacetic 20 acid in water.
6) Cuvettes, 10 x 75 mm glass culture tubes used as cuvettes.
Bl Assay Method:
1) A small volume of sample (0.366 microliters) is placed in each cuvette by pipetting 15 ~Q of sample and diluting with 600 ~Q BGG buffer in a dilution vessel. Next, 15 ~Q of diluted sample is pipetted into the cuvette followed by 600 ~Q BGG buffer.
2) Tracer is added by pipetting 40 ~ tracer and lO00 ~Q BGG buffer into the cuvette.
3~ Antiserum is added to start the reaction by pipetting 40 ~Q antiserum into the cuvette followed by lO00 ~Q
BGG buffer.
4) The contents of all cuvettes are well mixed and allowed to incubate for 15 minutes at ambient temperature.
5) The fluorescence polarization is read on a fluorometer and a standard curve constructed to determine unknowns.
- 33 - ~ ~'7~
C~ The results of a series of serum standards containing phenytoin at concentrations between 0 and 40 ~g,/ml are presented below. Each concentration was assayed in duplicate and averaged.
S Concentration of Phenytoin (~g/ml)Polarlzation 0.222 2.5 0.1~6 5.0 0.178 10,0 Q.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 quan-titated by reerence to the standard curve, thereby illustrating the utility of 2-~ aminoethyl phenytoin-carboxy~luorescein for the measurement of phenytoin.
EXAMPLE XNIII -Phenobarbital assay A) Materials required:
1) BGG buffer lsee Phenytoin) 22 Tracer, consisting of amlnophenobarbital 23 carboxyfluorescein at a concentration of approximately 110 nM
in tris HCl buffer, pH 7.5, containing 0.01~ sodium azide, 0.01% bovine gamma globulin, and 0.125% sodium dodecyl sulfate.
3) Antiserum, consiting of antiserum against phenobarbital diluted appropriately in BGG buffer containing 0.0~5~ benzalkonium chloride.
4) Samples of human serum or other ~iological fluid containing phenobarbital.
5) Cuvettes (see Phenytoin) 6~ Fluorometer ~see Phenytoin~
iLi7~
B~ Assay Protocol:
1~ A small volume of sample (0.196 micr~liter~
is placed in the cuvette by ipetting 10 ~Q o sample and diluting with 500 ~Q BGG buffer in a dilution vessel. Next, 10 ~Q of diluted sample is pipetted into the cuvette followed by 500 ~Q BGG buffer.
2) Tracer is added by pipettin~ 40 ~ of tracer and 1000 ~Q BGG buffer into each cuvette.
3) Antiserum is added to start the reaction by 10 pipetting 40 ~Q antiserum followed by 1000 ~ BGG buffer.
4) The contents of all cuvettes are mixed well and allowed to incubate for 15 minutes at ambient temperature.
5) The fluoxescence polarization is read on a fluorometer and a standard curve ccnstructed to determine unknowns.
C) The results of a series of senlm standards containing phenobarbital at concentrations between 0 and 80 ~g/ml are presented below. Each concentration was assayed in duplicate and the values averaged.
Concentration of P enobarbital (~Q)Polarization 0 0.250 5.0 0.~31 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, 30 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.
- 35 - ~ ~7 _AMPLE XXIX _ - Theophylline assay A) Materials required:
1) ~racer, consisting of 2 nM of 8-aminoethyl theophylline-carboxyfluorescein in BGG buffer (see Phentoin assay~ containing 0.01~ sodium dodecyl ~ulfate.
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 tracer in all cuvettas.
2) Add 2.0 ~Q sample to all cuvettes.
3) Add 1.O ml antiserum to all cuvettes, 4) Mix well and incubate 15 minutes at ambient temperature.
5) Read the fluorescence polarization on a ~luoro-meter and construct a standard curve.
C~ The results of a series of serum standards containinq theophylline at concentrations between 0 and 40 ~g/ml are presented. Each concentration was assayed in duplicate and the average is presented.
Concentration of Theo hvlline C~q/mllPolarization P ,~
o 0.158 2,5 0.118 0.105 la 0.091 0.076 0.063 ~7~
The polarization of fluore~cence 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 ~uantita~ed by reference to the standard curbe thereby illustrating the utility of 8-aminoethyltheophylline-carbox~tfluorescein for the measure-ment of the theophylline.
E~L~ XXX - Digoxin assa Y
A) Materials required:
1) BGG buffer consisting of O.lM sodium phosphate, pH 7.5, containing bovine gammaglobulin, 0.01% and sodium azide, 0.01~.
2) Tracer, consisting of digoxin carbo~yfluorescein at a concentration of approximately 2nM in BGG bufer.
3) Antiserum, consisting oE rabbit antiserum raised against digoxin diluted appropriately in BGG buffer.
4) Samples or human serum or other biological fluid containing phenytoin.
5) Precipitation reagent --5% trichloroacetic 20 acid in water.
6) Cuvettes, 10 x 75 mm glass culture tubes used as cuvettes.
7) Fluorometer capable of measurin~ fluorescence polarization with a precision of ~ 0.001 units.
B) Assay protocol:
1) To 100 ~Q of 5% trichloroacetic acid in a test tube is added 100 ~Q of a standard or unknown sample. The tubes containing the sample are capped and vortexed.
2~ The tubes containing standard or sample in 30 trichloroacetic acid are centrifuged.
3) To a test tube 1.8 ml of BGG buffer and 25 ~Q
of antisera at 35 C is added 150 ~Q of the trichloroacetic supernatant solution.
4) The test tubes containing antisera and supernatant 35 i5 incubated for 6 minutes at 35 C, at which time the - 37 ~
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 5 to antisera, 25 ~Q of the tracer is added to the test tube.
62 Six minutes after the addition of tracer, the fluorescence polarization of the standards and sample ~ubes are measured and the previously measured background fluorèscence polarization is substracted to yield the fluorescence 10 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.
15Digoxin Concentration (n~/mll Polarization 0 0.1~2 0.5 0.134 1.0 0.123 2.0 Q.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.
25 Unknown ~pecimens 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 30 fluorescence polarization assays that have been carried out in accordance with the above-described procedures employing tracers prepared in the preceeding examples. The tracers employed are identical by Example number and the specific ligand(s) determined are indicated.
- 38 - ~ ~7 Example No. _ Ligand(s) I Phenobarbital II Phenytoin III Phenytoin 5IV Theophylline V Theophylline VI Valproi.c acid VII Nortriptyline; Amitriptyline VIII Imipramine; Desipramine 10IX Thyroxine X Digoxin XI Propranolol XII Valproic acid XIII Valproic acid 15 XIV Primidone XV Chloramphenicol XVI Acetaminophen XVII Ethosuximide XVIII N-acetylprocainamide 20 XIX N-acetylprocainamide XX Disopyramide XXI Triiodothyronine XXII Thyroxine XXIII Imipramine; Desipramine 25 XXIV Imipramine; Desipramine XX~ ~ortriptyline; Amitriptyline XXVI Nortrip~yline; ~mitriptyline - 39 ~
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 5 invention possess a high degree of thermal stability, a high degree of bound polarization, high quantum yields and are realitively easy to produce and purify.
Although this invention has been described with respect to specific modifications, the details thereof are 10 not to be construed as limitations, for it will be apparent that various equivalents, changes and modifications may be resorted to without departing from the spirit and scope thereof and it is understood that such equivalent embodiments are intended to be included therein.
B) Assay protocol:
1) To 100 ~Q of 5% trichloroacetic acid in a test tube is added 100 ~Q of a standard or unknown sample. The tubes containing the sample are capped and vortexed.
2~ The tubes containing standard or sample in 30 trichloroacetic acid are centrifuged.
3) To a test tube 1.8 ml of BGG buffer and 25 ~Q
of antisera at 35 C is added 150 ~Q of the trichloroacetic supernatant solution.
4) The test tubes containing antisera and supernatant 35 i5 incubated for 6 minutes at 35 C, at which time the - 37 ~
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 5 to antisera, 25 ~Q of the tracer is added to the test tube.
62 Six minutes after the addition of tracer, the fluorescence polarization of the standards and sample ~ubes are measured and the previously measured background fluorèscence polarization is substracted to yield the fluorescence 10 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.
15Digoxin Concentration (n~/mll Polarization 0 0.1~2 0.5 0.134 1.0 0.123 2.0 Q.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.
25 Unknown ~pecimens 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 30 fluorescence polarization assays that have been carried out in accordance with the above-described procedures employing tracers prepared in the preceeding examples. The tracers employed are identical by Example number and the specific ligand(s) determined are indicated.
- 38 - ~ ~7 Example No. _ Ligand(s) I Phenobarbital II Phenytoin III Phenytoin 5IV Theophylline V Theophylline VI Valproi.c acid VII Nortriptyline; Amitriptyline VIII Imipramine; Desipramine 10IX Thyroxine X Digoxin XI Propranolol XII Valproic acid XIII Valproic acid 15 XIV Primidone XV Chloramphenicol XVI Acetaminophen XVII Ethosuximide XVIII N-acetylprocainamide 20 XIX N-acetylprocainamide XX Disopyramide XXI Triiodothyronine XXII Thyroxine XXIII Imipramine; Desipramine 25 XXIV Imipramine; Desipramine XX~ ~ortriptyline; Amitriptyline XXVI Nortrip~yline; ~mitriptyline - 39 ~
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 5 invention possess a high degree of thermal stability, a high degree of bound polarization, high quantum yields and are realitively easy to produce and purify.
Although this invention has been described with respect to specific modifications, the details thereof are 10 not to be construed as limitations, for it will be apparent that various equivalents, changes and modifications may be resorted to without departing from the spirit and scope thereof and it is understood that such equivalent embodiments are intended to be included therein.
Claims (44)
1. A method for determining ligands in a sample comprising intermixing with said sample a tracer of the formula:
(I) wherein R is a ligand-analog having a single reaction primary or secondary amino group which is attached to a carbonyl carbon of a carboxy-fluorescein 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.
(I) wherein R is a ligand-analog having a single reaction primary or secondary amino group which is attached to a carbonyl carbon of a carboxy-fluorescein 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 l wherein said ligand is a drug or a metabolite 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, anti-arrhythmic, 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 anti-convulsant 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.
15. A method according to Claim 6 wherein said drug is an antiasthmatic.
16. A method according to Claim 15 wherein said antiasthratic drug is theophylline.
17. A compound of the formula:
( I ) wherein R is a ligand-analog having a single reactive primary or secondary amino group which is attached to a carbonyl carbon of a carboxy-fluroescein 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.
( I ) wherein R is a ligand-analog having a single reactive primary or secondary amino group which is attached to a carbonyl carbon of a carboxy-fluroescein 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.
19. A compound of Claim 18 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 19 wherein R
is derived from an anticonvulsant.
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
23. A compound according to Claim 20 wherein R
is derived from phenytoin.
is derived from phenytoin.
24. A compound according to Claim 23 wherein R is wherein N is an integer from 1 to 3 and m is 0 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 wherein p is an integer of 2 to 4.
23. A compound according to Claim 20 wherein R is derived from primidone.
29. A compound according to Claim 28 wherein R is
30. A compound according to Claim 19 wherein R
is derived from a steroid.
is derived from a steroid.
31. A compound according to Claim 30 wherein R
is derived from digoxin.
is derived from digoxin.
32. A compound according to Claim 31 wherein R is
33. A compound according to Claim 19 wherein R is derived from an antiasthmatic drug.
34. A compound according to Claim 33 wherein R
is derived from theophylline.
is derived from theophylline.
35. A compound according to Claim 34 wherein R is wherein n is an integer of 1 or 2.
36. A compound according to Claim 19 wherein R
is derived from an antiarrhythmic.
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
39. A compound according to Claim 19 wherein R
is derived from a hormone.
is derived from a hormone.
40. A compound according to Claim 19 wherein R is derived an antiarrthritic.
41. A compound according to Claim 19 wherein R
is derived from a cardiac glycoside.
is derived from a cardiac glycoside.
42. A compound according to Claim 19 wherein R is derived from an antidepressant.
43. A compound according to Claim 19 wherein R
is derived from an antineoplastic.
is derived from an antineoplastic.
44. A compound according to Claim 18 wherein R is wherein R' is hydrogen or iodo.
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US23525981A | 1981-02-17 | 1981-02-17 | |
| US235,259 | 1981-02-17 | ||
| US32997481A | 1981-12-11 | 1981-12-11 | |
| US329,974 | 1981-12-11 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1178269A true CA1178269A (en) | 1984-11-20 |
Family
ID=26928739
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000396311A Expired CA1178269A (en) | 1981-02-17 | 1982-02-16 | Fluorescent polarization immunoassay utilizing substituted carboxyfluoresceins |
Country Status (3)
| Country | Link |
|---|---|
| CA (1) | CA1178269A (en) |
| DE (1) | DE3205506C2 (en) |
| NZ (1) | NZ199629A (en) |
Families Citing this family (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4476229A (en) * | 1982-11-08 | 1984-10-09 | Abbott Laboratories | Substituted carboxyfluoresceins |
| 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 |
| US4698315A (en) * | 1985-04-22 | 1987-10-06 | Hoffmann-La Roche Inc. | Method and kit for determining total digoxin levels involving agent to dissociate digoxin-protein complex |
| EP0226730B1 (en) * | 1985-10-15 | 1994-03-02 | Abbott Laboratories | Compounds and assay for tricyclic antidepressants |
| US5017474A (en) * | 1988-02-12 | 1991-05-21 | Eastman Kodak Company | Wash solution, test kit and method for the determination of an immunological ligand |
| EP0517327B1 (en) * | 1991-06-07 | 2001-08-16 | Johnson & Johnson Clinical Diagnostics, Inc. | Immunoassay with labeled hapten analogues |
| EP0517326B1 (en) * | 1991-06-07 | 2001-08-16 | Johnson & Johnson Clinical Diagnostics, Inc. | Labeled drug hapten analogues for immunoassays |
| US5284948A (en) * | 1991-06-07 | 1994-02-08 | Eastman Kodak Company | Drug hapten analogues for immunoassays |
| EP0576095B1 (en) * | 1992-06-26 | 1999-01-27 | Johnson & Johnson Clinical Diagnostics, Inc. | Immunoassays with labeled thyronine hapten analogues |
| US5315015A (en) * | 1992-11-10 | 1994-05-24 | Hoffmann-La Roche Inc. | Compounds having improved fluorescence in fluorescence polarization immunoassays and immunoassays utilizing same |
| DE10032633A1 (en) * | 2000-07-05 | 2002-01-17 | Bayer Ag | Method for finding protoporphyrinogen oxidase inhibitors |
-
1982
- 1982-02-02 NZ NZ19962982A patent/NZ199629A/en unknown
- 1982-02-16 CA CA000396311A patent/CA1178269A/en not_active Expired
- 1982-02-16 DE DE19823205506 patent/DE3205506C2/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| DE3205506A1 (en) | 1982-09-16 |
| DE3205506C2 (en) | 1996-10-10 |
| NZ199629A (en) | 1984-11-09 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US4668640A (en) | Fluorescence polarization immunoassay utilizing substituted carboxyfluoresceins | |
| US5391740A (en) | Fluoresence polarization immunoassay | |
| US4476229A (en) | Substituted carboxyfluoresceins | |
| US4510251A (en) | Fluorescent polarization assay for ligands using aminomethylfluorescein derivatives as tracers | |
| GB2111476A (en) | Substituted carboxy- fluoresceins and their use in fluorescence polarization immunoassay | |
| US4593089A (en) | Fluorescent polarization immunoassay utilizing substituted triazinylaminofluorescein aminoglycosides | |
| US4420568A (en) | Fluorescent polarization immunoassay utilizing substituted triazinylaminofluoresceins | |
| US4160016A (en) | Receptor fluorescent immunoassay | |
| US5260441A (en) | Immunoassay for opiate alkaloids and their metabolites; tracers, immunogens and antibodies | |
| US4161515A (en) | Double receptor fluorescent immunoassay | |
| EP0218010A2 (en) | Ligand detection method and substituted carboxyfluorescein tracers therefor | |
| US4255329A (en) | Double receptor fluorescent immunoassay | |
| US4614823A (en) | Aminomethylfluorescein derivatives | |
| EP0597333B1 (en) | Compounds for fluoroscense polarization immunoassays and immunoassays utilising them | |
| CA1178269A (en) | Fluorescent polarization immunoassay utilizing substituted carboxyfluoresceins | |
| US4687747A (en) | Phenanthridinium ester as a labelling compound in luminometric immunoassay | |
| US5066426A (en) | Fluorescence polarization immunoassay utilizing substituted carboxyfluoresceins | |
| US4952691A (en) | Fluorescence polarization immunoassay | |
| CA1248086A (en) | Fluorescence polarization immunoassay | |
| EP0380019B1 (en) | Methadone fluorescence polarization immunoassay | |
| EP0200960A1 (en) | Total estriol fluorescence polarization immunoassay | |
| CA1195995A (en) | Substituted carboxyfluoresceins | |
| US5097097A (en) | Triazinylaminofluoresceins | |
| US5258512A (en) | Immunoassay for opiate alkaloids and their metabolites; tracers, immunogens and antibodies | |
| CA1338763C (en) | Compounds and assay for tricyclic antidepressants |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| MKEC | Expiry (correction) | ||
| MKEX | Expiry |