EP0396732A1 - Immunoanalyses fluorescentes et composes fluorescents et elements a l'etat de traces - Google Patents

Immunoanalyses fluorescentes et composes fluorescents et elements a l'etat de traces

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Publication number
EP0396732A1
EP0396732A1 EP90900453A EP90900453A EP0396732A1 EP 0396732 A1 EP0396732 A1 EP 0396732A1 EP 90900453 A EP90900453 A EP 90900453A EP 90900453 A EP90900453 A EP 90900453A EP 0396732 A1 EP0396732 A1 EP 0396732A1
Authority
EP
European Patent Office
Prior art keywords
acid
tracer
hydrocarbon
ligand
chloride
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.)
Withdrawn
Application number
EP90900453A
Other languages
German (de)
English (en)
Other versions
EP0396732A4 (en
Inventor
Robert M. Dowben
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Individual
Original Assignee
Individual
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Filing date
Publication date
Application filed by Individual filed Critical Individual
Publication of EP0396732A1 publication Critical patent/EP0396732A1/fr
Publication of EP0396732A4 publication Critical patent/EP0396732A4/en
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C309/00—Sulfonic acids; Halides, esters, or anhydrides thereof
    • C07C309/78—Halides of sulfonic acids
    • C07C309/86—Halides of sulfonic acids having halosulfonyl groups bound to carbon atoms of six-membered aromatic rings of a carbon skeleton
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C309/00—Sulfonic acids; Halides, esters, or anhydrides thereof
    • C07C309/78—Halides of sulfonic acids
    • C07C309/86—Halides of sulfonic acids having halosulfonyl groups bound to carbon atoms of six-membered aromatic rings of a carbon skeleton
    • C07C309/87—Halides of sulfonic acids having halosulfonyl groups bound to carbon atoms of six-membered aromatic rings of a carbon skeleton containing singly-bound oxygen atoms bound to the carbon skeleton
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C309/00—Sulfonic acids; Halides, esters, or anhydrides thereof
    • C07C309/78—Halides of sulfonic acids
    • C07C309/86—Halides of sulfonic acids having halosulfonyl groups bound to carbon atoms of six-membered aromatic rings of a carbon skeleton
    • C07C309/88—Halides of sulfonic acids having halosulfonyl groups bound to carbon atoms of six-membered aromatic rings of a carbon skeleton containing nitrogen atoms, not being part of nitro or nitroso groups, bound to the carbon skeleton
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C2603/00—Systems containing at least three condensed rings
    • C07C2603/02—Ortho- or ortho- and peri-condensed systems
    • C07C2603/40—Ortho- or ortho- and peri-condensed systems containing four condensed rings
    • C07C2603/42—Ortho- or ortho- and peri-condensed systems containing four condensed rings containing only six-membered rings
    • C07C2603/50—Pyrenes; Hydrogenated pyrenes

Definitions

  • the present invention relates to fluorescence compounds and resultant tracers utilized as reagents in fluorescent immunoassay procedures to detect the presence of and/or quantify analytes in a mixture; even analytes that are present only in very low concentrations.
  • the invention has relevance for the determination of constituents present in biological fluids such as blood serum, plasma, spinal fluid, urine, amniotic fluid, and the like and other fluids such as milk and water, or even solid materials such as wastes that can be solubilized. Such measurements are important for the practice of public health medi ⁇ cine as well as human and veterinary medicine. In addition, the determination of therapeutic drug levels and the presence of drugs of abuse in fluids is very valuable.
  • One type of immunoassay involves the com- petitive binding of an unknown quantity of analyte in the sample to be analyzed, with a known quan ⁇ tity of analyte labeled by a tracer added to the reaction mixture, for a limited number of receptor-binding sites on specific antibodies that combine with labeled and unlabeled analyte.
  • radioactive tracers in immunoassays requires special licensing and special precautions in handling to minimize health hazards and con ⁇ tamination.
  • Federal and state regulations that must be followed in handling radioactive materials frequently impose an added burden in terms of time and effort required to perform the tests and to dispose of the used radioactive materials.
  • Standard curves constantly change owing to the decay of radioactivity and radiodecomposition of the constituent reagents.
  • counters for the measurement of radioactivity are large, expensive, cumbersome, and do not lend themselves to automation.
  • immunoassays utilizing fluorescent tracers are gradually displacing the older assays using radioactive tracers.
  • only certain molecules exhibit the property of fluorescence to a significant extent.
  • a fluorescent molecule will absorb a photon of light and utilize the energy to move an electron from the ground state to an oribtal with a higher energy level. This process is known as
  • excitation and occurs at specific wave lengths of light. After a short interval of time; of the order of nanoseconds, the excited electron falls back into the ground state and emits a photon of light. Some energy is lost in the process and the photon of emitted light is shifted toward the red part of the spectrum with respect to the wave length of exciting light.
  • the difference between wave lengths of exciting light and emitting light is known as the "Stokes shift".
  • the most commonly used fluorescent tracer in immunoassays is fluorescein.
  • fluorescein maximum excitation occurs when it is illuminated with light of wave length 496 n .
  • the maximum emitted light occurs at a wave length of 516 nm.
  • the Stokes shift is 20 nm.
  • a number of other fluorescent tracers are known in the art and have been used in immunoassays, but these fluorescent tracers have Stokes shifts of less than 35 nm.
  • the use of tracers with Stokes shifts shorter than 35 nm presents a number of problems that limit fluorescence measurements. First, fluorescence measurements are limited by light scattering which causes part of the exci ⁇ tation light to be detected in the emission chan ⁇ nel.
  • band pass optical inter ⁇ ference filters are placed in both the excitation and emission channels.
  • serum and other biological fluids are intrinsically fluorescent. Almost all of the intrinsic fluorescence of biological fluids show Stokes shifts of less than 35 nm. Thus, cons ⁇ tion of fluorescent tracers with Stokes shifts of 35 nm or shorter allows more interference by the intrinsic fluorescence of serum and other biologi- cal fluids as well as other fluids to be tested.
  • Another limiting factor in fluorescence measurements of solutions is elastic Raman light scattering. In the case of aqueous solutions, Raman scattering occurs at approximately 300 wave numbers on either side of the frequency of the exciting light. Raman scattering is a problem with fluorescent tracers with small Stokes shifts.
  • polycyclic compounds in particular pyrene and chrysene compounds, have been shown to have relatively large Stokes shifts in purely biochemical and cell biology research experiments.
  • 1-pyreneisothiocyanate is relatively nonfluorescent but reacts with the amine groups of antibodies to form conjugates with excitation maxima of approximately 386 nm and emission maxima of 470 nm giving a Stokes shift of 84 nm.
  • 6-chrysenemaleimide is a relatively nonfluorescent compound which forms adducts with the sulfhydryl groups of various compounds to form fluorescent products with an excitation maximum of 360 nm and an emission maximum of 400 nm.
  • 8-Hydroxypyrene-l,3,6-trisulfonic acid has been used as a pH indicator having an excitation maxi ⁇ mum of 450 nm and an emission maximum of 515 nm giving a Stokes shift of 65 nm.
  • the various reports describing the use of such reactive pyrene or chrysene compounds fail to teach or suggest their use in immunoassay testing; whether fluorescence intensity or fluorescence polariza- tion is measured.
  • the present invention overcomes the noted problems of prior fluorescent compounds and tra ⁇ cers -made therefrom in immunoassays and provides tracers having large Stokes shifts thereby enabling fluorescent assays of greater sensitivity with minimization of light scattering background, of interference by intrinsic fluorescence of the fluid being test, and of Raman light scattering.
  • the present invention comprises a polycyclic hydrocarbon
  • A is 0, N, or S
  • R is H, a substituted or unsubstitued Ci - C ⁇ alkyl group, a substituted or unsubstituted C2 - C ⁇ ester group, or a substi ⁇ tuted aryl group
  • Y is H or SO3Z wherein Z is H or a halide, with at least one Y being SO3Z and Z is a halide
  • R' is H, Y or A-R
  • n is 0 or 1 with at least one n equal to 1.
  • the invention also comprises the tracers and iramunoassay as hereinafter set forth.
  • ligand and analyte are used interchangeably herein and refer to consti ⁇ tuents of fluids or dissolved materials? par- ticularly biological fluids whose presence and/or concentration is to be measured.
  • Antibodies that specifically bind to ligands can be raised in ani ⁇ mals and such antisera isolated and purified by standard techniques well-known to those skilled in this art, or highly specific monclonal antibodies can be selected and produced by again standard techniques.
  • the ligands that can be identified and/or quantified by the assays of the present invention vary over a wide range of molecular weight and include, inter alia, such represen ⁇ tative compounds as theophyllin, dilantin, pheno- barbital, carbamazepine, gentamycin, streptomycin, amikacin, tobramycin, thyroxine, digoxin, digi- toxin, procainamide, lidocaine, quinidine, pri i- done, propranolol, morphine, codeine, heroin, hormones, and the like.
  • represen ⁇ tative compounds as theophyllin, dilantin, pheno- barbital, carbamazepine, gentamycin, streptomycin, amikacin, tobramycin, thyroxine, digoxin, digi- toxin, procainamide, lidocaine, quinidine, pri i- done, propranolo
  • Quantification of a ligand in a solution or Tnixture requires the preparation of a fluorescent- labeled ligand ⁇ tracer) that can be used to com- pete with unlabeled ligand for binding by a specific antibody.
  • the fluorescent compounds of the present invention can be bound to reactive a ino, reactive carboxyl, or other reactive substitutents present on the ligands or to ligand analogs possessing a reactive amino, reactive carboxyl reactive sulfhydryl, or other reactive groups to form suitable tracers.
  • Y in which A is 0, N, or S;
  • R is H, a substituted or unsubstitued C ⁇ - Cg alkyl group, a substituted or unsubstituted C2 - Cg ester group, or a substi ⁇ tuted aryl group;
  • Y is H or SO3Z wherein Z is H or a halide, with at least ony Y being SO3Z and Z is a halide;
  • R' is H, Y or A-R; and n is 0 or 1 with at least one n equal to 1.
  • pyrenesulfonic acid or pyrenesulfonyl chloride compounds are preferred with specific preferred compounds being 8-acetoxy-l,3,6- pyrenetrisulfonyl chloride, 6-8-diacetoxy -1,3-pyrenedisulfonyl chloride, 8-ethoxy-l,3 ,6-pyrenetrisulfonyl chloride, 6,8-diethoxy-l,3-pyrenedisulfonyl chloride, and 8-succinylamino-l,3,6-pyrenetrisulfonic acid.
  • 9-acetoxy-l,3,7-chrysenet- risulfonyl chloride is preferred, which form sulfonamides with reactive amino groups of a ligand or ligand analog.
  • such compounds can be synthesized from pyrene and chrysene by the conventional pro ⁇ cess of forming the tetrasulfonic acid derivati ⁇ ves using anhydrous sodium sulfate and concentrated sulfuric acid- The hydroxy or amino compounds are subsequently formed by reaction "with aqueous sodium hydroxide or ammonia at elevated temperatures under pressure.
  • Reactive fluorescent derivatives are the formed in one of two ways.
  • the sulfonic acid groups are to be converted to sulfonyl chlorides, for example, by the use of thionyl chloride.
  • Sulfonyl chloride derivates form adducts with reactive amino groups of ligands or ligand analog, for example.
  • the ligand or ligand analog can be coupled to the -0-R, -N-R, or -S-R group.
  • tracer adducts of ligands or ligand analogs with fluorescent moieties are formed by conventional procedures such as shown, for example, in U.S. Patents 3,996,345 or 4,420,568.
  • the pyrene and chrysene tracers of the present invention are o the forumla
  • R is H , a substituted or unsubstitued C ⁇ - Cg alkyl group, a substituted or unsubstituted C2 - Cg ester group, or a substituted aryl group
  • Y is H or SO3Z wherein Z is H or a halide, with at least one Y being SO3Z?
  • R is H, Y r or A-R; and n 0 or 1 with at least one n equal to 1
  • L is a ligand or ligand-analog
  • m is 0 or 1 with only one m being 1.
  • One group of-preferred fluorescent tracer com ⁇ pounds are based on pyrenesulfonic acids and pyrenesylfonyl chlorides. Pyrene and chrysene compounds generally have large Stokes shifts, greater than 35 nm and often as large as 50 to 70 nm. A particular advantages of hydroxy, ethoxy, acetoxy or similarly substituted pyrenesulfonic acids is that their excitation is in the blue- green part of the spectrum, rather than the near ultraviolet part of the spectrum characterstic of other pyrene or chysene compounds. Another advan ⁇ tage of these compounds is that they are readily soluble in water, rather than being hydrophobic like other pyrene and chrysene compounds.
  • Fluoroimmunoassays can be carried out in several different ways. It is preferable to use a homogeneous assay method in which the antibody bound ligand and tracer need not " be separated from the free ligand and tracer prior to measuring the fluorescence. Because only one incubation and no washings are required such assays can be performed simply and rapidly. Particularly useful are fluorescence polarization immunoassays first described by Dandliker, .B. e al. , Immunochemistry 10, 219 (1973) and Spencer, et al. Clin. Chem. 19, 838-844 (1973). In such assays, the reaction mixture is excited with polarized light, and the polarization of the fluorescence emission is measured.
  • the degree of polarization of the fluorescence emission depends upon the ratio of free tracer to tracer bound to antibody, and thus, the ratio in unknown samples can be determined by comparision to a standard curve. If owing to the high molecular weight of ligand, or to the low concentration of ligand, fluorescence polarization immunoassays are not sufficiently sensitive, another type of fluorescent immunoassay may be employed such as those reviewed by Hemmila in Clin. Chem. 31, 359 (1985).
  • the fluorescent compound was reacted with 2-aminophe- nobarbital to form a tracer. Excitation maximum of this tracer was at 455 nm and emission maximum at 520 nm (Stokes shift 65 nm) .
  • the assay was performed as described by Dandliker, et al. , supra.
  • a standard solution serum to be tested or other unknown was diluted 1:400 in 0.15 M phosphate buffer, pH 7.5, containing 0.01% sodium azide and 0.01% bovine gamma globulin.
  • 20 1 phenobarbital tracer stock solution which contained 5 g/ml 2-aminop- henobarbital in the above buffer.
  • the best antibody and optimum antibody dilution was determined by generating antibody titration curves upon adding increasing amounts of antibody to a constant amount of tracer and determining the polarization using a fluorescence polarimeter.
  • the appropriate amount of antibody in 20 1/buffer was added to the reaction and the mixture incu ⁇ bated at room temperature for 3 min. The polari ⁇ zation was then measured.
  • Figure 1 illustrates a typical standard curve. The polarization of unknown serum samples was assessed in this manner and the concentration of phenobarbital calculated by comparison to the standard curve.
  • EXAMPLE 2 2.5g 1-nitropyrene was added with stirring over a period of 5 minutes to a mixture of 13g concentrated sulfuric acid and 3g anhydrous sodium sulfate at 60°C and stirred for an additional 15 min. breaking up the dark purple clumps that formed. Then 8 g fuming sulfuric acid was added over 30 min. at 60° with constant stirring. The whole mixture was added to 80g ice. An additional 60 ml water was added. After 18 hours the pH was adjusted to 5 and solution filtered. The aqueous phase containing 8-nitro-l,3,6-pyrenetrisulfonic acid was reduced with iron filings and acetic acid.
  • This fluorescent marker was utilized in an assay for thyroxine by forming an adduct with 3,5-dichloro-3 • ,5'diiodothyroxine using dicyclo- hexylcarbodiimide. Excitation of the adduct tracer was 461 nm and the emission maximum was 524 nm (Stokes shift of 63 nm) . This fluorescent thyroxine tracer was utilized in a fluorescence polarization assay similar to that described above.
  • EXAMPLE 3 To a mixture of 6.5g concentrated sulfuric acid and 1.5g anhydrous sodium sulfate was added lOg pyrene with stirring at 60° for 15 min. After cooling to 50°, 4g fuming sulfuric acid was added in small portions over a 20 min. period. The dark reddish-black mass was cooled on ice. After standing for 2 hrs, it was broken up and dissolved in 70 ml water. After filtration, the product was precipitated by saturating the filtrate with NaCl.
  • EXAMPLE 4 To a mixture of 6.5g concentrated sulfuric acid and 1.5g anhydrous sodium sulfate was added lOg chrysene with stirring at 60° for 15 min. After cooling to 50°, 4g fuming sulfuric acid was added in small portions over a 20 min period. The dark mass was cooled on ice. -After standing for 2 hrs it was broken up, and dissolved in 70 ml water. After filtration, the product was precipi ⁇ tated by saturating the filtrate with NaCl.
  • the above product was mixed with 1.5g sodium hydroxide and 1.0 ml water, sealed in a glass pressure tube and heated at 155° for 30 min; then at 165° for 20 min; and then at 170° for 5 min.
  • the pressure tube was cooled and the resulting mass was dissolved in water and then brought to pH 9.0 After filtration, the supernatant was aci- dified with formic acid which precipitated the 7,9-dihydroxy-l,3-chrysenedisulfonic acid.
  • the diacetoxy derivative was first prepared by treat ⁇ ment with acetic anhydride, and then the disulfo- nyl chloride was formed by treatment with thionyl chloride as described in Example 1.
  • a tracer adduct was formed by reacting the 7,9-diacetoxy-l,3-chrysenedisulfonyl chloride with diphenyl glycine.
  • the tracer adduct had an absorption maximum of 438 nm and an emission maxi- mum of 482 nm.
  • the phenytoin tracer was utilized in a fluorescence polarization assay similar to that described in Example 1.
  • EXAMPLE 5 8-Acetoxy-1,3 f 6-pyrenetrisulfonyl chloride was reacted with genta ycin to form a tracer adduct that was utilized in a fluroescence polarization assay similar to that described in Example 1 using excitation light of 455 nm and measuring the fluorescence at 520 nm.
  • the average intrasample correlation coefficients for six replications of 26 samples was 0.987.

Abstract

L'invention concerne un hydrocarbure polycyclique de formules (I) ou (II) dans lesquelles A représente O, N, ou S; R représente H, un groupe alkyle de C1 en C8 substitué ou non substitué, un groupe ester de C2 en C8 substitué ou non substitué ou un groupe aryle substitué; Y représente H ou SO3Z où Z est H ou un halogénure, au moins un Y étant SO3Z et Z étant un halogénure; R' représente H, Y ou A-R; et n vaut 0 ou 1, au moins un n étant égal à 1. Les hydrocarbures peuvent être couplés à un ligand ou un analogue de ligand et être utilisés dans des immunoanalyses fluorescentes.
EP19900900453 1988-11-14 1989-10-27 Fluorescent immunoassays and fluorescent compounds and tracers therefore Withdrawn EP0396732A4 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US27116188A 1988-11-14 1988-11-14
US271161 1988-11-14

Publications (2)

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EP0396732A1 true EP0396732A1 (fr) 1990-11-14
EP0396732A4 EP0396732A4 (en) 1992-10-07

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EP19900900453 Withdrawn EP0396732A4 (en) 1988-11-14 1989-10-27 Fluorescent immunoassays and fluorescent compounds and tracers therefore

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EP (1) EP0396732A4 (fr)
JP (1) JPH03502333A (fr)
WO (1) WO1990005916A1 (fr)

Families Citing this family (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0624798A1 (fr) * 1993-04-28 1994-11-17 Nalco Chemical Company Procédé de contrôle de la consommation des additifs
US5514710A (en) * 1994-01-11 1996-05-07 Molecular Probes, Inc. Photocleavable derivatives of hydroxyprenesulfonic acids
JP3538277B2 (ja) * 1996-03-08 2004-06-14 和子 松本 免疫測定用標識試薬及びそれに用いる蛍光性化合物及び錯体、及びそれらを用いる生物物質の免疫測定法
WO2004027388A2 (fr) 2002-09-23 2004-04-01 Chromagen, Inc. Nouveaux marqueurs fluorescents de couleur verte et orange et leurs utilisations
WO2009021057A1 (fr) * 2007-08-06 2009-02-12 Glumetrics, Inc. Colorants fluorescents hpts monosubstitués et bisusbtitués cys-ma pour une utilisation dans des détecteurs analytiques
EP2609426B1 (fr) * 2010-08-27 2018-12-19 The Texas A&M University System Réactifs de marquage fluorescent et leurs utilisations
DE102011101207B3 (de) * 2011-05-11 2012-05-10 Sartorius Stedim Biotech Gmbh Fluoreszenzfarbstoff für pH-Sensor
CA3127235A1 (fr) * 2019-01-21 2020-07-30 Max-Planck-Gesellschaft Zur Forderung Der Wissenschaften E. V. Colorants 2(7)-aminoacridone et 1-aminopyrene sulfones et leur utilisation comme marqueurs fluorescents, en particulier pour l'analyse des hydrates de carbone
US20220026434A1 (en) * 2019-01-21 2022-01-27 Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V Advanced methods for automated high-performance identification of carbohydrates and carbohydrate mixture composition patterns and systems therefore as well as methods for calibration of multi wavelength fluorescence detection systems therefore, based on new fluorescent dyes

Citations (1)

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Publication number Priority date Publication date Assignee Title
US4568518A (en) * 1982-12-06 1986-02-04 Avl Ag Sensor element for fluorescence-optical measurement

Family Cites Families (5)

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JPS5724979U (fr) * 1980-07-09 1982-02-09
JPS5917282A (ja) * 1982-07-21 1984-01-28 Hitachi Ltd 半導体装置
JPS6018629A (ja) * 1983-07-08 1985-01-30 Honda Motor Co Ltd 防振マウント
AT385755B (de) * 1986-06-13 1988-05-10 Koller Ernst Verfahren zur herstellung von neuen pyrensulfonsaeurederivaten
US4954435A (en) * 1987-01-12 1990-09-04 Becton, Dickinson And Company Indirect colorimetric detection of an analyte in a sample using ratio of light signals

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4568518A (en) * 1982-12-06 1986-02-04 Avl Ag Sensor element for fluorescence-optical measurement

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
CLINICAL CHEMISTRY. vol. 31, no. 3, 1985, WINSTON US pages 359 - 370; I. HEMMIL[: 'FLUOROIMMUNOASSAYS AND IMMUNOFLUOROMETRIC ASSAYS' *
See also references of WO9005916A1 *

Also Published As

Publication number Publication date
JPH03502333A (ja) 1991-05-30
EP0396732A4 (en) 1992-10-07
WO1990005916A1 (fr) 1990-05-31

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