WO1993019366A1 - Immuno-essais par fluorescence au moyen de matrices fluorescentes exemptes d'agregat et de liant de serum - Google Patents

Immuno-essais par fluorescence au moyen de matrices fluorescentes exemptes d'agregat et de liant de serum Download PDF

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Publication number
WO1993019366A1
WO1993019366A1 PCT/US1993/002470 US9302470W WO9319366A1 WO 1993019366 A1 WO1993019366 A1 WO 1993019366A1 US 9302470 W US9302470 W US 9302470W WO 9319366 A1 WO9319366 A1 WO 9319366A1
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probe
receptor
target analyte
labeled
amount
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PCT/US1993/002470
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English (en)
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Robert Francis Devlin
Walter Beach Dandliker
Peter Olof Gustaf Arrhenius
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Diatron Corporation
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Priority to EP93908390A priority Critical patent/EP0632893A4/fr
Priority to CA002132708A priority patent/CA2132708C/fr
Priority to US08/035,633 priority patent/US5846703A/en
Publication of WO1993019366A1 publication Critical patent/WO1993019366A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
    • A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
    • A61K49/0036—Porphyrins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
    • A61K49/0052—Small organic molecules
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F7/00—Compounds containing elements of Groups 4 or 14 of the Periodic System
    • C07F7/02—Silicon compounds
    • C07F7/08—Compounds having one or more C—Si linkages
    • C07F7/0834—Compounds having one or more O-Si linkage
    • C07F7/0838—Compounds with one or more Si-O-Si sequences
    • 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
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • 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
    • 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
    • G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/18—Togaviridae; Flaviviridae
    • G01N2333/19—Rubella virus

Definitions

  • the present invention relates to methods for deter ⁇ mining the presence or amount of antigenic substances in samples.
  • the invention is directed to fluorescence 15 immunoassays using particular fluorescence dyes which are essentially free of aggregation and serum binding and, thus, are particularly suited for the measurement of antigenic substances in biological materials such as serum, plasma and whole blood.
  • the determination of the presence or amount of anti ⁇ genic substances is commonly performed by immunoassay.
  • Immunoassay techniques are based on the binding of the antigenic substance being assayed (the "target analyte") and a receptor for the target analyte. Either the target analyte or the receptor may be labeled to permit detec ⁇ tion. Various labels have been employed for use in
  • immunoassays including radioisotopes, enzymes and fluorescent compounds.
  • immuno ⁇ assays include competitive inhibition assays, sequential addition assays, direct "sandwich” assays, radioallergosorbent assays, radio- immunosorbent assays and enzyme-linked immunosorbent assays.
  • the basic reaction underlying most immunoassays is the binding of certain substance, termed the "ligand” or “analyte", by a characteristic protein (receptor) to form a macromolecular complex.
  • ligand or "analyte”
  • receptor characteristic protein
  • the unknown quantity of target analyte in the sample competes with a known amount of labeled target analyte for a limited number of receptor binding sites.
  • the reagents usually consist of a labeled target analyte, such as an antigen, and a solid phase coupled receptor, such as an antibody.
  • the antigen to be assayed competes with the labeled anti ⁇ gen for binding sites on the coupled antibodies.
  • the concentration of target analyte present in the sample can be determined by measuring the amount of labeled target analyte — either "free” or “bound.” This is an indirect assay method where the amount of labeled antigen bound to the antibodies is inversely correlated with the amount of antigen in the test solution. Thus, low concentrations of target analyte in the sample will result in low concentra ⁇ tions of "free” labeled target analyte and high concentra ⁇ tions of "bound” labeled target analyte, and vice versa. The amount of "free” or "bound” labeled target analyte is measured using a suitable detector.
  • Quantitative determi- nations are made by comparing the measure of labeled tar ⁇ get analyte with that obtained for calibrated samples containing known quantities of the target analyte.
  • This method has been applied to the assay of a great number of different polypeptide hormones, enzymes and immunoglobu- lins. This method may also be used as a total liquid system. It is apparent to those skilled in the art that it is not absolutely necessary that the labeled analyte be iden ⁇ tical to the unlabeled target analyte.
  • the reaction between labeled and unlabeled analytes may be considered to be competitive for the receptor binding sites; and the reaction will still provide quantitative answers, provid ⁇ ing the difference in affinity of the analytes is not too great.
  • competition occurs in a system consisting of labeled analyte, unlabeled analyte, and receptor depends on the nature of the labeled analyte and the specificity of the receptor.
  • the reagents used are the same as in the competitive inhibition assay described above. However, instead of incubating them at the same time, the unlabeled antigen is first incubated with the antibody, then the labeled antigen is added.
  • Direct immunoassay systems are also known in the art.
  • Such assays also termed “immunometric” assays, employ a labeled receptor (antibody) rather than a labeled analyte (antigen) .
  • antibody labeled receptor
  • analyte analyte
  • Immunometric assays are well- suited to the detection of antigenic substances which are able to complex with two or more antibodies at the same time. In such "two-site” or “sandwich” assays, the anti ⁇ genic substance has two antibodies bound to its surface at different locations.
  • an antibody bound to a solid phase is first con- tacted with the sample being tested to form a solid phase antibody:antigen complex. After incubation, the solid support is washed to remove the residual sample, including unreacted antigen, if any. The complex is then reacted with a solution containing a known amount of labeled anti ⁇ body. After a second incubation to permit the labeled antibody to complex with the antigen bound to the solid support through the unlabeled antibody, the solid support is washed to remove unreacted labeled antibody.
  • the assay can be used as a simple "yes/no" assay to determine whe ⁇ ther the antigen is present.
  • Quantitative determinations can be made by comparing the measure of labeled antibody with that of calibrated samples containing known quanti ⁇ ties of antigen.
  • "Simultaneous” and “reverse” sandwich assays are also known in the art.
  • a simultaneous assay involves a single incubation step, both the labeled and unlabeled antibodies being added at the same time.
  • a reverse assay involves the addition of labeled antibody followed by addition of unlabeled antibody bound to a suitable solid support.
  • the sandwich technique can also be used to assay antibodies rather than antigens.
  • Such an assay uses as a first receptor an antigen coupled to a solid phase. The antibodies being tested are first bound to the solid phase-coupled antigen. The solid phase is then washed, and then labeled anti-antibody (second receptor) is added.
  • the radioallergosorbent technique is a method for the determination of antigen-specific IgE.
  • the method uses a solid phase coupled antigen and an immunoabsorbent purified antibody labeled with a radioactive isotope.
  • the method is used to detect reaginic antibodies against vari ⁇ ous antigens which elicit allergic reactions (allergens) .
  • the reaginic antibodies react with allergen bound to a solid matrix. After washing of the solid phase, the allergen-bound reaginic antibodies are detected by their ability to bind labeled antibodies against IgE.
  • a variant of RAST can be used for the determination of allergens.
  • the allergen to be tested is incubated with the reaginic antibody.
  • the mixture is then tested with RAST using the same allergen coupled to the solid matrix.
  • the allergen in solution reacts with the reaginic antibodies and thus inhibits the binding of these antibodies to the solid phase-coupled allergen.
  • RIST radioimmunosorbent technique
  • the solid support is sensitized with anti-IgE and increasing amounts of labeled IgE are added to determine the maximum amount of IgE that can bind.
  • a quantity of labeled IgE equivalent to approximately 80% of the plateau binding is chosen.
  • this amount of labeled IgE is mixed with the serum containing the IgE to be tested.
  • the test IgE competes with the labeled IgE. The more IgE present in the test serum the less the amount of labeled IgE that binds.
  • RIST radioimmunosorbent technique
  • the above immunoassay methods can be applied to the assay of many different biologically active substances.
  • substances include haptens, hormones, gamma globu ⁇ lin, allergens, viruses, virus subunits, bacteria, toxins such as those associated with tetanus and animal venom, and many drugs.
  • Similar techniques can be used in non- immunological systems with, for example, specific binding proteins.
  • a separa- tion step is required to separate "free" from "bound" labeled target analyte.
  • Such assays which require a separation step, are called “heterogeneous” assays. If the properties of the label are altered in some way when it is bound, no separation step is required, and the immunoassay is termed "homogeneous.”
  • Both the specificity and sensitivity of an immunoassay depend on the characteristics of the binding interaction between the target analyte and the receptor involved. For example, the reaction must be specific for the analyte to be measured and the receptor used should not bind to any other structurally related compounds. In addition, by choosing a receptor with a high affinity for the target analyte, the sensitivity can be increased.
  • the label used to monitor the assay affects the sensitivity of an immunoassay. Labels currently used for immunoassay of target analytes in biological fluids include radioisotopes (radioimmunoassay, RIA) , enzymes
  • RIAs are sufficiently sensitive for use in detection in low concentrations of analytes because of their low background. They are disadvantageous in that they are heterogeneous, thus requiring a separation step before measurement of the bound and/or free portions of labelled target analyte. RIAs involve the inconvenience and haz ⁇ ards associated with the handling and disposing of radio ⁇ isotopes. In addition, they are labor intensive and have a short shelf life due to the half-lives of radiolabels and to chemical damage produced by the emitted radiation. EIAs have the advantage of increased signal over background, longer shelf life, lack of radiation hazards, and homogeneity.
  • CIAs Chemiluminescent immunoassays
  • CIAs are disadvanta- geous because they are heterogeneous, require expensive reagents, and are expensive to automate. See generally Boeckx, R.L., "Luminescence: A New Analytical Tool for Therapeutic Drug Monitoring,” in P. Moyer et al. , Applied Therapeutic Drug Monitoring, American Association of Clin- ical Chemistry (1984) .
  • FIAs use fluorescent molecules as labels. Fluores ⁇ cent molecules (fluorophores) are molecules which absorb light at one wavelength and emit light at another wave ⁇ length. See Burd, J.F., "Fluoroimmunoassay — Application to Therapeutic Drug Measurement,” in P. Moyer et. al. , App ⁇ lied Therapeutic Drug Monitoring, American Association of Clinical Chemistry (1984) . Typically, an excitation pulse of radiation is directed onto or into a sample, followed by fluorescence of the sample, and the detection of the fluorescence radiation.
  • FIAs may be either heterogeneous or homogeneous.
  • homogeneous assays are usually simpler to perform and are thus, more amenable to automation. How ⁇ ever, previously known homogeneous FIAs are less sensitive than heterogeneous FIAs because high background can limit sensitivity.
  • the heterogeneous FIA procedures can detect smaller amounts of analyte than present homogenous FIAs, but only because the separation and washing steps in the assays serve to eliminate background interference from biological substances.
  • solid phase fluorescent assays the solid support can limit sensitivity at the wavelengths of presently used fluors. In many cases the support itself will fluoresce at wavelengths of commonly used fluors such as fluorescein (493 nm) .
  • FIAs also offer the advantage of using stable reagents.
  • Another assay method uses enzyme-enhanced fluores- cence technology which combines microparticle capture and antigen-antibody reaction with an enzyme rate reaction using a fluorescent enzyme substrate. The rate reaction is monitored by steady state fluorometric measurement.
  • an enzyme-enhanced fluorescence assay the analyte in question is "captured" by an antibody bound to a solid phase and the solid phase is washed. An enzyme is then bound to the captured analyte using an enzyme-anti analyte conjugate. Excess reactants are washed away and the amount of enzyme is measured by the addition of a non- fluorescent substrate. As the enzymatic reaction pro ⁇ ceeds, the non-fluorescent substrate is converted to the fluorescent product.
  • an alkaline phospha- tase-labeled antibody can be used to catalyze the hydro ⁇ lysis of 4-methylumbelliferyl phosphate substrate to the fluorescent product methylumbelliferone.
  • the rate at which the fluorescent product is generated is directly proportional to the concentration of analyte in the test solution.
  • Enzyme-enhanced fluorescence assays like EIAs, have the disadvantages associated with enzymes. As discussed above, fluorescence is a phenomenon exhibited by certain substances, which causes them to emit light, usually in the visible range, when radiated by another light source. This is not reflection, but crea ⁇ tion of new light. Current commercially available assay methods use fluorescein, which emits green light when radiated by a light source containing blue light.
  • fluorescein In addition to fluorescing, fluorescein (and other fluorophores) emit polarized light. That is, the light emitted has the same direction of polarization as the incident polarized light, if the fluorescein molecule is held fixed wit its transition moment parallel to the electric field of the excitation.
  • the amount of polariza- tion in the emission can be defined in terms of the inten ⁇ sity of the horizontally and vertically polarized light, as follows:
  • Ih intensity of horizontally polarized emission
  • the maximum, or limiting value of polarization, for fixed, randomly oriented molecules is 0.5 (Po) .
  • a second equation (the Perrin equation) defines polarization in terms of physical parameters and Po:
  • Rotation relaxation is further defined for spherical molecules as
  • FPIA fluor ⁇ escence polarization immunoassay
  • the relationship between polarization and drug con- centration can be determined by creating a standard, or calibration, curve. This is done by running an assay using a range of known drug concentrations, from the lowest to highest expected concentrations, and plotting the resulting values of polarization. Thereafter, for a given value of polarization, the drug concentration can be determined from the standard curve.
  • One advantage of the polarization technique is the elimination of a step to separate unbound probe. Although the unbound tracer is not physically eliminated from the samples in FPIA, its contribution is readily assessed by the. polarization.
  • fluorometry is capable of being the most sensitive of all analytic tools as it is possible to detect single photon events.
  • a problem which has plagued fluorescence immunoassays has been discriminating the fluorescent signal of interest from background radiation.
  • the intensity of signal from background radiation may be up to 10,000 times larger than the intensity of the fluorescent signal of interest.
  • the problem of background detection is particularly pronounced in assay of biological samples.
  • Many of the current fluorescence assays use the fluorescent molecule, fluorescein. Fluorescein has an excitation maximum of 493 nm, and there are numerous substances in biological fluids with overlapping excitation and emission similar to fluor ⁇ escein.
  • a second technique attempting to * discriminate the desired fluorescent signal from the background is the so called time gating approach.
  • the fluorescent signal is observed in a short time window after the excitation.
  • the time window may be varied both in its length and in its starting time.
  • the detected radiation may be observed at the maximal time for detection sensitivity.
  • this technique has used a fluorophore of very long decay time (such as 1,000 nanoseconds) to allow the background fluorescence to substantially decay before detection of the fluorescent signal of interest.
  • long decay time fluorophores require longer times for overall analysis. Due to the long decay time, the light source cannot be pulsed rapidly to collect: data, thus requiring additional time for final analysis.
  • transient state fluorescent analysis utilizes a single, relatively high power pulse which excites the fluorophore.
  • the transient state is typically monitored by a high speed photomultiplier tube by monitoring the analog signal representative of current as a function of time.
  • Single pulse systems require sufficiently high power to excite a large number of fluorescent molecules to make detection reliable.
  • the other principal format for transient state fluorescent analysis is a digital format which utilizes repetitive excitation pulses.
  • pulses of relatively short, typically nanosecond duration, light with power in the microwatt range are repetitively supplied to the sam- pie at rates varying from 1 to 10,000 Hz.
  • the excitation source is a lamp, such as a Xenon-lamp.
  • the decay curve is measured digitally by deter ⁇ mining the time to receipt of a photon.
  • One commercially available system uses repetitive pulses (such as at 5,000 Hz) and pulses the photomultiplier tube at increasingly longer times after the flash in order to obtain a time dependent intensity signal. Detection in such systems has proved to be very time consuming and insensitive. A single analysis can take on the order of one hour, even at relatively high fluorescable dye concentrations (e.g. , 10 "6 M) .
  • an object of the present invention is to provide improved processes for assay of antigenic substances. More speci ⁇ fically, the present invention provides fluorescence assays which allow the detection of low levels of anti ⁇ genic substances in biological samples such as serum, plasma and whole blood. The present invention also pro ⁇ vides homogeneous fluorescence assays which allow rapid and accurate determination of low levels of antigenic substances in biological samples.
  • the present invention is directed to methods for determining the presence or amount of a target analyte in a sample by using, as a label for the target analyte or a receptor which is capable of specifically recognizing the target analyte, a fluorophore moiety comprising a lumi ⁇ nescent substantially planar molecular structure coupled to two stabilizing polyoxyhydrocarbyl moieties, one located on either side of the planar molecular structure.
  • target analyte is meant the antigenic substance being assayed, for example an antigen.
  • receptor is meant a molecule or molecular component capable of specifically recognizing the target analyte.
  • an antibody may be a receptor for an antigen.
  • detectable labels or marker components are advantageous in that these labels have substantially the same intensities of parallel and perpen ⁇ dicular components of transient state fluorescence emis ⁇ sion in the presence and absence of biological fluids such as serum.
  • assay methods using these labels are capable of detecting low concentrations of target analyte in biological fluids.
  • the methods of the present invention are particularly suitable for use with the improved fluorescence detection system described in commonly assigned U.S. Patent Applica- tion entitled “Fluorometer Detection System, " Lyon & Lyon Docket No. 195/129, filed concurrently herewith.
  • the present invention is directed toward competitive inhibition assay procedures utilizing particular labels.
  • the present invention is directed to a method of determining the presence or amount of a target analyte by contacting a sample sus ⁇ pected of containing the target analyte with a known quantity of added target analyte or analog thereof linked to a fluorescent probe which includes a detectably labeled marker component made up of a fluorophore moiety which includes a luminescent substantially planar molecular structure coupled to two solubilizing polyoxyhydrocarbyl moieties, one located on either side of the planar mole- cular- structure; contacting the sample with a receptor capable of specifically recognizing the target ligand; and determining either the amount of fluorescent probe bound to receptor or free fluorescent probe.
  • the amount of bound or free fluorescent probe in the unknown samples may be compared with blank samples and samples containing known amounts of target analyte.
  • the resultant mixture of sample, fluorescent probe and receptor is diluted before measurement of the amount of bound and/or free fluorescent probe.
  • the dilution step allows for greater sensitivity of the assay. Particularly preferred are dilutions of 2-fold to 100-fold, preferably about 7-fold to about 50-fold, and more preferably about 35-fold.
  • the present invention provides an improvement in immunoassay procedures which utilize a label for either the target analyte (or analog thereof) or the receptor.
  • the improvement is the use of a fluorophore moiety comprising a luminescent substantially planar mole ⁇ cular structure coupled to two solubilizing polyoxyhydro- carbyl moieties, one located on either side of the planar molecular structure.
  • Assays using this type of label are advantageous in that they are free of serum binding and aggregation and are therefore, especially suitable for testing biological samples such as serum, plasma, whole blood and urine.
  • the present invention provides a method for performing a "sandwich” or "two-site” immuno ⁇ assay comprising the steps of:
  • a sandwich-type assay may be either a heterogeneous assay or a homogeneous assay. If it is heterogeneous, it may incorporate the additional step of separating the solid carrier from the unreacted labeled first receptor. Homogeneous assays are generally preferred because they are more rapid. In another embodiment, the assay may incorporate the additional step of relating the amount of labeled first receptor measured in the unknown sample to the amount of labeled first receptor measured in a control sample free of said target analyte, or to the amount of labeled first receptor measured in samples containing known quantities of target analyte. In another aspect, the present invention provides a method for a simultaneous sandwich-type assay comprising a method for determining the presence or amount of a target analyte in a sample comprising the steps of :
  • the present invention provides a method for a simultaneous sandwich-type assay comprising a method further comprising the step of relating the amount of labeled first receptor measured to the amount of labeled first receptor measured for a control sample free of said target analyte, or relating to the amount of labeled first receptor measured with the amount of labeled first receptor measured in samples containing known amounts of target analyte.
  • the present invention provides a sandwich-type fluorescence immunoassay method for measure ⁇ ment of a target analyte which is capable of recognizing two different receptors independently without mutual interference. The method utilizes two receptors, each of which is labeled with a different dye.
  • one receptor is labeled with a first dye having absorption and emission maxima of 680 nm and 690 nm, respectively, and the other receptor is labeled with a second dye having absorption and emission maxima of 695 and 705 nm, respec ⁇ tively.
  • Detection and quantitation of the analyte can be made using either steady state or transient state measure- ments. In either case, for the example given, excitation would be at 680 nm and detection would be at 705 nm.
  • This type of assay is based on energy transfer and is advan ⁇ tageous in that it is homogeneous.
  • the present invention is directed to immunoassay of biological fluids, including serum, plasma, whole blood and urine.
  • red blood cells in whole blood are lysed prior to assay of whole blood samples.
  • Preferred methods of lysing red blood cells include addition of stearyl-lysolecithin, palmitoyl-lysolecithin and myristoyl lysolecithin.
  • the target analyte may be an antigen, a hapten or an antibody; and the receptor may be an antigen or antibody.
  • the antibody may be polyclonal or monoclonal. Preferably, the antibody is a monoclonal antibody.
  • Monoclonal antibodies useful in the present invention may be obtained by the Kohler & Milstein method reported in Nature 256:495-497 (1975) . Alternatively, they may be produced by recombinant methods. Science 246:1275-1281 (1989) .
  • the target analyte is a drug or a metabolite of a drug.
  • the drug may be a steroid, hormone, antiasthmatic, antineoplastic, antiarrhythmic, anticonvul- sant, antiarthritic, antidepressant, or cardiac glycoside.
  • examples of such drugs include digoxin, digitoxin, theo- phylline, phenobarbital, thyroxine, N-acetylprocainamide , primidone, amikacin, gentamicin, netilmicin, tobramycin, carbamazepine, ethosuximide, valproic acid, disopyramide, lidocaine, procainamide, quinidine, ethotrexate, ami- triptyline, mortriptyline, imipramine, desipramine, van- comycin, and cyclosporine.
  • the drug is digoxin.
  • the target analyte is a pep ⁇ tide, for example, a peptide hormone such as luteinizing hormone, follicle stimulating hormone, human choriogonado- tropin, thyroid stimulating hormone, angiotensin I, angio- tensin II, prolactin or insulin.
  • the peptide may also be a tumor marker such as carcinoembryonic antigen.
  • the peptide may be a virus or portion thereof, for example, rubella virus or a portion thereof.
  • the methods of the present invention provide ways of measuring target analytes in concentrations of from about 1 x 10 "5 M/L to about 1 x 10 "13 M/L, and particularly in the concentration range of from about 1 x 10" 9 M/L to about 1 x 10 "12 M/L.
  • the present methods allow measurement in the range from about 5 x 10" 9 M/L to about 5 x 10" 12 M/L, and particularly, concentrations of from about 1 x 10" 10 M/L to about 5 x 10 "10 M/L.
  • the present methods allow measurement in the range of from about 1 x 10 "11 M/L to about 1 x 10 "12 M/L.
  • the measurement of amount of fluorescent probe — bound or free or both — can be determined by measuring steady-state fluorescence or by measuring transient state fluorescence.
  • the wavelength of light measured is greater than about 500 nm, preferably greater than about 650 nm, and more preferably greater than about 680 nm or 690 nm. Because the transient state detection system utilizes a laser diode, it is necessary for the dyes to have excitation maxima matched to the diode output wavelengths. Dyes have been made available to match other commercially available laser diodes have output wavelengths of 680, 690, 720, 750, or 780 nm.
  • the wavelength of the light measured may be greater than about 680 nm, 690 nm, 720 nm, 750 nm or 780 nm. The further into the red region of the spectrum one moves, i.e. , the greater the wavelength, the greater signal enrichment there is over background.
  • detection and quantitation is performed using transient state measurement.
  • Transient state energy transfer offers improved measurements due to optimization of the wavelengths of absorption and emis ⁇ sion, as well as due to optimization of the decay times of the first and second dyes. Such optimization allows removal of Rayleigh and Raman scattering, and achieving the best compromise between efficiency of transfer and the undesired direct excitation of the second dye by the first dye.
  • the present invention is directed to immunoassays using detectably labelled marker components which comprise a fluorophore moiety which comprises a substantially planar macrocyclic multidentate ligand coordinated to a central atom and two solubilizing poly ⁇ oxyhydrocarbyl moieties, one linked on either side of the plane of the multidentate ligand to the central atom.
  • detectably labelled marker components which comprise a fluorophore moiety which comprises a substantially planar macrocyclic multidentate ligand coordinated to a central atom and two solubilizing poly ⁇ oxyhydrocarbyl moieties, one linked on either side of the plane of the multidentate ligand to the central atom.
  • the present invention is directed to immunoassays using a marker component compris ⁇ ing a fluorophore moiety which comprises a substantially planar multidentate macrocyclic ligand coordinated to a central atom capable of coordinating with two axial lig ⁇ ands which are coordinated to the central atom on either side of the macrocyclic ligand.
  • Marker components used in the immunoassays of the present invention comprise a macrocyclic multidentate ligand having two solubilizing polyoxyhydrocarbyl moieties one located on either side of the plane of the multiden ⁇ tate ligand exhibit no detectable non-specific binding to serum components, and exhibit no detectable solvent sensi ⁇ tivity. These marker components also exhibit enhanced decay times which approach their natural (fluorescent) lifetimes.
  • fluorophores which produce fluorescent light efficiently, i.e. , which are characterized by high absorbitivity at the appropriate wavelength and high fluorescence quantum yields.
  • preferred fluorophores have measured fluorescence decay times on the order of at least 2 nanoseconds and exhibit a high degree of fluorescence polarization.
  • Preferred solubilizing polyoxyhydrocarbyl moieties include water soluble carbohydrates such as glucose, sucrose, maltotriose, and the like; water soluble car ⁇ bohydrate derivatives such as gluconic acid and mannitol and oligo saccharides; and water soluble polymers such as polyvinylpyrrolidone, poly(vinylalcohol) , poly(ethyleni- mine) , polyacrylic acid, polyacrylamide, ethylene oxide copolymers such as Pluronic (a propylene oxide copolymer, available from BASF) and Tetronic (BASF) polyol surfac ⁇ tants; and polyethers, including water soluble polyoxy- alkylene polymers, particularly poly(ethylene glycol) (“PEG”) and poly(ethylene glycol) derivatives such as poly(ethylene glycol) methyl ether, poly(ethylene glycol) silicon derived ethers and the like.
  • PEG poly(ethylene glycol)
  • the present invention is directed to immunoassays using marker components comprising a fluoro ⁇ phore moiety which comprises a substantially planar, mul- tidentate macrocyclic ligand coordinated to a central atom capable of coordinating with two axial ligands and two polyoxyhydrocarbyl moieties which are attached as axial ligands to the central atom.
  • Suitable central atoms are those to which may coordinate two axial ligands and which are not of high enough atomic number to cause extensive fluorescence quenching by transition to the triplet state.
  • Preferred elements for the central atom include silicon, germanium, phosphorus, and tin, especially preferred are silicon and germanium.
  • these marker components may be used as labels for labelling an analyte, antigen, antibody or other molecule.
  • These marker compo- nents may be optionally functionalized so as to include a linker arm which allows the marker component to be linked to the analyte, antigen, antibody or other molecule.
  • linker arms which may be suited to this pur- pose.
  • the marker component is linked to the analyte, antigen, antibody or other molecule using conventional techniques.
  • the present invention is also directed to the use of divalent peptide derivatives as analogs for large mole- cules in immunoassays.
  • a divalent hapten consisting of two epitopes of the same specificity con ⁇ nected by a linker about 10 nm long is used to bind to a single antibody molecule, requiring approximately 26 residues.
  • the present invention also includes assay methods of involving cellular receptors located on the plasma mem ⁇ brane or isolated from cytosols and synthetic ligand binders obtained by molecular imprinting.
  • the present invention also provides particular fluor ⁇ escent probes for use in immunoassays, for instance, see Examples 3 and 11-18 below.
  • target analyte refers to the compound or compound to be measured in an assay which may be any compound for which a receptor naturally exists or can be prepared which is mono- or polyepitopic, antigenic or haptenic, a single or plurality of compounds which share at least one common epitopic site or a receptor.
  • analog of a target analyte is meant a compound or com ⁇ pounds capable of competing with the target analyte for binding to a receptor.
  • axial ligand refers to a substituent which, together with a macrocyclic ligand, forms a coordination complex with a central atom.
  • the axial ligand lies normal to the plane described by the macro ⁇ cyclic ligand.
  • fluorescent probe refers to a marker component comprising a fluorophore moiety which is bonded to or coordinates either directly or via a linker arm to an analyte, antigen, hapten, antibody or other molecule which is used in an assay, such as a fluoroimmunoassay to determine the presence of and/or quantitate a substance of interest.
  • solvent sensitivity refers to changes in the fluorescence behavior of a molecule depending on the solvent system in use, most notably referring to differ ⁇ ences in fluorescence behavior in aqueous solution in comparison with organic solvents (such as DMF) .
  • organic solvents such as DMF
  • Fluorescence intensity is related to sample concen ⁇ tration and the intensity of the exciting radiation.
  • the fluorescence intensity of a particular dye can be corre- lated to its characteristic light absorptivity (extinction coefficient) and fluorescence quantum efficiency, as well as environmental factors.
  • binding pair refers to two dif ⁇ ferent molecules (or compositions) wherein one of the molecules has an area on the surface or in a cavity which specifically recognizes and binds to a particular spatial and polar organization of the other molecule or molecular complex involving other molecules.
  • binding partner refers to a molecule or molecular complex which is capable of specifically recog ⁇ nizing or being recognized by a particular molecule or molecular complex.
  • bound refers to the condition in which a binding interaction has been formed between a molecule and its specific binding partner.
  • decay time is the time which must elapse in order for the concentration of excited molecules to decrease from its initial concentration to 1/e of that value.
  • receptor refers to a molecule or molecular complex which is capable of specifically recognizing or being recognized by a target analyte or analog thereof.
  • Fig. 1 depicts an HPLC analysis of crude caged dicar- boxy silicon phthalocyanine dye preparation.
  • Fig. 2 shows the absorbance of caged dicarboxy sili ⁇ con phthalocyanine dye in various solvents.
  • Fig. 3 describes the Diatron Analog System.
  • Fig. 4 depicts the decay time for caged dicarboxy silicon phthalocyanine dye.
  • Fig. 5 shows serum interactions of purified caged dicarboxy silicon phthalocyanine dye.
  • Fig. 6 depicts the absorbance spectrum of caged dicarboxy silicon phthalocyanine dye-C12 linker.
  • Fig. 7 depicts the polarization of caged dicarboxy silicon phthalocyanine dye-C12 linker at 680 nm.
  • Fig. 8 depicts the polarization of caged dicarboxy silicon phthalocyanine dye-C12 linker at 690 nm.
  • Fig. 9 depicts an HPLC Chromatograph of caged dicar- boxy silicon phthalocyanine digoxin probe.
  • Fig. 10 depicts the structure of caged dicarboxy silicon phthalocyanine digoxin probe.
  • Fig. 11 shows the absorbance spectrum of caged dicarboxy silicon phthalocyanine digoxin probe in methanol.
  • Fig. 12 shows the absorbance spectrum of caged dicarboxy silicon phthalocyanine digoxin probe in FPIA buffer.
  • Fig. 13 shows the decay time for caged dicarboxy silicon phthalocyanine digoxin probe.
  • Fig. 14 shows the linearity of intensity for caged dicarboxy silicon phthalocyanine digoxin probe.
  • Fig. 15 shows serum/urine interactions for caged dicarboxy silicon phthalocyanine digoxin probe.
  • Fig. 16 depicts a comparison of TDx ® and FAST-60 calibration curves.
  • Fig. 17 depicts the correlation of digoxin samples assayed by TDx ® and FAST-60.
  • Fig. 18 depicts the effect of dilution jump on non-specific binding.
  • Fig. 19 depicts a digoxin probe-serum calibration curve.
  • Fig. 20 depicts a calibration curve for a high sensi ⁇ tivity digoxin assay.
  • Fig. 21 describes the FAST-60 digoxin assay procedure.
  • Fig. 22 depicts digoxin correlation — TDx ® Serum vs. FAST-60 Whole Blood.
  • Fig. 23 depicts digoxin correlation — Stratus ® Serum vs. FAST-60 Whole Blood.
  • Fig. 24 depicts digoxin correlation — ' FAST-60 Serum vs. FAST-60 Whole Blood.
  • Fig. 25 depicts digoxin correlation — TDx ® Serum vs. FAST-60 Serum.
  • Fig. 26 depicts digoxin correlation — Stratus ® Serum vs. FAST-60 Serum.
  • Fig. 27 depicts the structure of caged dicarboxy silicon phthalocyanine digitoxin probe.
  • Fig. 28 depicts the structure of caged dicarboxy silicon phthalocyanine theophylline probe.
  • Fig. 29 depicts the structure of caged dicarboxy silicon phthalocyanine phenobarbital probe.
  • Fig. 30 depicts the structure of caged dicarboxy silicon phthalocyanine thyroxine probe.
  • Fig. 31 depicts the structure of caged dicarboxy silicon phthalocyanine n-acetylprocainamide probe.
  • Fig. 32 depicts the structure of caged dicarboxy silicon phthalocyanine primidone probe.
  • Fig. 33 depicts the structure of caged dicarboxy silicon phthalocyanine phenytoin probe.
  • Fig. 34 depicts a rubella antibody calibration curve for a sandwich assay.
  • Fig. 35 depicts a rubella peptide calibration curve for an inhibition assay.
  • Fig. 36 depicts a rubella antibody calibration curve for direct polarization.
  • the present invention provides fluorescence immuno ⁇ assay methods which have dramatic increases in sensitivity over previous methods, which can be easily performed 5 because they require no separation step, and which can be used to detect and quantitate low levels of target analyte in biological samples such as serum, plasma, whole blood and urine.
  • the FIAs of the present invention may be per ⁇ formed in small samples. For example, a digoxin assay may
  • FIA 10 be performed on a 20 ⁇ l sample of serum, plasma or whole blood, and the assay may be performed in about five min ⁇ utes.
  • the ability to perform FIAs on whole blood samples is particularly significant because it allows assays to be performed at locations closer to the patient, such as
  • the present invention provides methods for FIAs which
  • the probes used in the method of the invention have excitation (and emission) wavelengths greater than about 650 nm, preferably greater than 680 nm. This wavelength shift into the infra-red
  • the probes used in the present assay methods have low dielectric constants, which applicants believe tend to increase the Van der Waal interactions and hydrogen bond ⁇ ing, thus accelerating the antigen/antibody reaction. In addition, applicants believe that these probes compete for the water of hydration, thus potentiating the antigen/ antibody reaction. In other words, the probes not only substantially decrease non-specific bindings to serum components, but applicants believe that they potentiate the immunochemical reaction.
  • the present invention provides a further increase in sensitivity by measuring transient state fluorescence rather than the steady state signal.
  • the signal In the steady state mode the signal is constant over time enabling the determination of one experimental parameter, e.g. , the polarization or the anisotropy, both of which are related to molecular rotational motion.
  • the transient state mode the signals vary in a systematic way with time. This varia ⁇ tion represents a complex summation of the rates of decay and of molecular rotation as it changes from moment to moment in time.
  • the increase in sensitivity from transient state measurements stems from two sources. First, that portion of the background due to Rayleigh and Raman scattering disappears in about 10 "15 sec and so is cleanly removed before the transient state measurements start. This portion of background is normally an important part of the total in steady state measurements.
  • the tran ⁇ sient state measurements provide an additional powerful means to discriminate between the desired signal and the remaining fluorescence portion of the background. This discrimination rests upon the time dependence of the polarized components in the fluorescence decay and makes it possible to extract the desired signal only, simultane ⁇ ously on the basis of the rate of decay of the excited state and the rate of decay of the rotational distribution imprinted by the excitation.
  • transient state methods allow signal to be distinguished from background in ways not possible with steady state information alone.
  • the probes exhibit a high degree of polarization, necessary for mix and read (homogeneous) fluorescence polarization assays. This increase in polarization translates into increased sensitivity.
  • the methods of the present invention are particularly useful when used with a time-correlated transient state detection system, as described in commonly assigned Studholme, et. al..
  • the system uses a laser diode which can be modulated at very high frequencies, e.g.. 10 MHz rate, and exhibits high output power. Typically the laser "on" time is approximately 2-3 nanoseconds. Photons from the solution are detected using a photomultiplier tube (PMT) operating in a single photon counting mode.
  • PMT photomultiplier tube
  • the photon event along with the relative time of the photon event as compared with the laser pulse time is determined.
  • a histogram of frequency of photons as a function of time is generated.
  • Data obtained in this manner can be analyzed as described in Dandliker et, al. , U.S. Patent No. 4,877,965, entitled “Fluorometer” or as described by Studholme, et al. , U.S. Patent Application entitled, "Fluorometer Detection System,” Lyon & Lyon Docket No. 195/129, filed concurrently herewith.
  • the methods of the present invention also include the use of divalent peptide derivatives as analogs for large molecules in immunoassays. Both polyclonal and monoclonal antibody molecules are divalent. Due to the "chelate effect, " the binding of a low molecular weight mimic or analog of a larger molecule will be stronger and dissoci- ation from the antibody will be slower if both antibody binding sites are utilized in the bonding. It is within the scope of the present invention to arrange the struc ⁇ ture of the peptide analog to have two identical sequences joined together by a linker of suitable length so as to place the two peptide sequences, in their normal config ⁇ uration in solution, in the most favorable position for reaction with the two sites on the same antibody molecule.
  • divalent peptide derivatives as analogs for large molecules in immunoassays is preferred in solution, especially in conjunction with dilution jump, due to the tighter binding afforded by the "chelate effect, " resulting in an increase in the sensitivity of the immunoassay.
  • a divalent hapten consisting of two epitopes of the same specificity connected by a linker about 10 nm long is used to bind to a single antibody molecule. Taking into account the bond distances and angles for simple peptides (L. Pauling, The Nature of the Chemical Bond, Cornell University Press (1960) , p. 498) and assuming a length of 0.380 nm per amino acid residue, this would require approximately 26 residues for a 10 nm length.
  • One approach for designing a divalent hapten with such a linker is to synthesize the epitope with a 13 resi- due linker terminating in a primary amino group. This peptide is then reacted with the bis (3-isocyanatopropyl- dimethylsilyl) derivative of dihydroxysilicon phthalocya ⁇ nine. The resulting structure has the phthalocyaninine moiety with two axial substituents, one on either side of the molecular plane, each consisting of a thirteen residue peptide linker leading to the peptide epitope. The mole ⁇ cular plane of the dye moiety is perpendicular to the direction of the linker.
  • the dye moiety may be located midway between the two arms of the Y-shaped antibody molecule.
  • the polarization changes obtainable with this type of structure may not be as great as if the dye were linked through a peripheral rather than an .axial bond, and the absence of PEG may result in non-specific binding.
  • the dye moiety is held close to the antibody surface between the two Fab fragments after binding, it may prove to be quite protected and rotate with the long ⁇ est rotational decay time of the antibody (since the molecular plane of the dye may lie parallel to the long axis of the antibody) .
  • a divalent peptide hapten may be designed to utilize the PEG protected dye linked through a peripheral carboxyl to an amino group on the linker or on one of the peptide epitopes, e.g. , the linkage could be the e-amino group of a lysine residue located approxi ⁇ mately midway on an interconnecting chain between the two peptide epitopes.
  • Immunoassays depend upon the strong and selective binding of some analyte of interest to antibody specific for that analyte.
  • Other molecular structures that have similar strong and selective binding for such an analyte can serve equally well in designing an assay and such structures may have some inherent advantages over antibody.
  • molecules which may have desirable properties in this context include cellular receptors located on the plasma membrane or isolated from cytosols and synthetic ligand binders obtained by a process known as "molecular imprinting.
  • the sensitivity of a ligand binding assay depends upon the binding affinity of association constant (K) of the reaction between the analyte and the binding molecule.
  • K association constant
  • these Ks are of the order of 10 9 M "1 .
  • the binding constant for diazepam was found to be about 10 8 M "1 (Vlatakis et, al. , Nature 361: 645-647 (1993)).
  • the highest Ks for antibody binding are of the order of 10 12 M "1 for lig ⁇ ands such as fluorescein and digoxin.
  • the present invention includes methods of involving cellular receptors located on the plasma mem ⁇ brane or isolated from cytosols and synthetic ligand binders obtained by molecular imprinting.
  • Suitable fluorophore moieties comprise a luminescent substantially planar molecular structure.
  • Preferred central atoms are elements which may form octahedral coordination complexes containing two ligands with a trans or axial orientation, on either side and perpendicular to the planar macrocyclic ligand.
  • the central atom should not have too high atomic number (about 30 or less) so that fluorescence is not diminished through coupling interaction with orbitals of the central atom.
  • Preferred multidentate ligands include nitrogen- containing macrocycles which have conjugated ring systems with pi-electrons. These macrocycles may be optionally substituted, including substitution on bridging carbons or on nitrogens. Suitable macrocycles include derivatives of porphyrins, azaporphyrins, corrins, sapphyrins and por- phycenes and other like macrocycles which contain elec ⁇ trons which are extensively delocalized.
  • an especially preferred class of macrocycles comprise porphyrin derivatives, and azaporphyrin deriva- tives (porphyrin derivatives wherein at least one of the bridging carbons is replaced"by a nitrogen atom) .
  • Azapor ⁇ phyrin derivatives include derivatives of mono-, di- and triazaporphyrin and porphyrazine.
  • These macrocycles may optionally have fused aromatic rings.
  • These azaporphyrin derivatives include phthalocyanine, benzotriazaporphyrin and naphthalocyanine and their derivatives.
  • azaporphyrin derivatives which exhibit a high degree of polarization, that is, those which emit strongly polarized light.
  • macrocycles having lower degrees of symmetry preferably having lower symmetry than D 4h .
  • One preferred group includes macrocycles having at least one fused aromatic ring.
  • preferred macro- cycles include azaporphyrin derivatives having fused aromatic rings at positions which result in decreased symmetry.
  • Preferred classes of azaporphyrin derivatives comprise derivatives of monoazaporphyrin, diazaporphyrin, and triazaporphyrin having lower than D 4h symmetry.
  • Preferred solubilizing polyoxyhydrocarbyl moieties include water soluble carbohydrates such as glucose, sucrose, maltotriose and the like; water soluble carbo ⁇ hydrate derivatives such as gluconic acid and mannitol, and oligosaccharides; polypeptides such as polysine and naturally occurring proteins; and water soluble polymers such as polyvinylpyrrolidone, poly(vinyl alcohol) , poly
  • polyether such as polyoxyalkylenes including poly (ethylene glycol) , or other water soluble mixed oxyalky- lene polymers, and the like.
  • a particularly preferred class of solubilizing poly ⁇ oxyhydrocarbyl moieties comprises poly(ethylene glycol) (PEG) and poly(ethylene glycol) derivatives, such as poly(ethylene glycol) monomethyl ether.
  • PEG derivatives include PEG-silicon derived ethers.
  • Many of these polymers are commercially available in a variety of molecular weights. Others may be conveniently prepared from commercially available materials, such as by coupling of an amino-PEG to a haloalkyl silyl or silane moiety.
  • these polyoxyhydro ⁇ carbyl moieties When linked to a fluorophore moiety, these polyoxyhydro ⁇ carbyl moieties impart particularly advantageous qualities of solubility in aqueous solution with improved measured fluorescence decay time, and improved fluorescence inten ⁇ sity.
  • the resulting marker components are water soluble and show decreased non-specific binding, especi ⁇ ally decreased binding tc serum albumin which has here ⁇ tofore been a problem with certain fluorophores, parti- cularly porphyrin or phthalocyanine dyes which have been functionalized with groups such as sulfonate to impart increased water solubility to the molecule.
  • Non-specific binding of fluorophore or marker component impairs the accuracy of the resulting immunoassay.
  • These marker components which comprise fluorophore linked to PEG may also exhibit improved fluorescence intensity in aqueous solution with decreased quenching.
  • Suitable PEGs may vary in molecular weight from about 200 to about 20,000 or more. Choice of a particular mole- cular weight may depend on the particular fluorophore chosen and its molecular weight and degree of hydropho- bicity, as well as the particular application for which the fluorophore-PEG complex is to be used.
  • marker components which comprise a central atom (for example, silicon) coupled to two PEG moieties may be characterized by measurements of transient state fluores ⁇ cence.
  • the intensity of the two components polarized either parallel or perpendicular to the direction of polarization of the exciting pulse is monitored over a time period equal to about 3 times the decay time of the marker component.
  • Such curves reflect extinction coefficient, quantum yield, decay time and state of polarization and supply sensitive indications on the chemical and physical condition of the marker component. For example, if the excited state is being deacti ⁇ vated or converted to the triplet state the overall intensities are lowered and the decay times shortened. If the rotary brownian motion of the molecule is being altered by an increase in viscosity or by being bound to a large molecule, the ratio of the intensity of the paral ⁇ lel to the perpendicular component is increased.
  • marker components according to the present invention show, within experimental error of about 5%, the same intensities, decay time and polarization in DMF (an organic solvent) as in SAP (saline azide phosphate, an aqueous neutral buffer) . To some extent these properties are shared by other marker component preparations.
  • a distinctive and important property of the marker compo ⁇ nents of the present invention is a insensitivity to (and lack of binding to) the components in serum which is evi ⁇ denced by a lack of any measured effect of serum on the intensities, decay time or relative magnitudes of the polarized components of the fluorescence. This property is crucial for the marker components to be useful for applications such as assays using biological materials. D. Preparation of Preferred Marker Components
  • the appropri ⁇ ate fluorophore moiety having hydroxy or halide groups as axial ligands is reacted with a reactive form of the solu ⁇ bilizing polyoxyhydrocarbyl moiety in a ligand exchange reaction according to the general reaction scheme:
  • Mel denotes the macrocyclic ligand, CA the central atom, X the displaced ligand and SM the solubilizing moiety.
  • This reaction may be carried out neat or, if desired, in solvent. Suitable solvents include quinoline, THF, DMF, imidazole and the like. Suitable reaction tem ⁇ peratures may vary, depending on the nature of the macro- cyclic starting material and the solubilizing group.
  • the reaction is generally complete in about 2 minutes to about 24 hours.
  • the reaction mixture can be conveniently heated under reflux or by means such as a sand bath. For con ⁇ venience, the reaction may be carried out at ambient pressure.
  • these marker components When used as fluorescent labels in fluorescence immunoassays, these marker components may be linked to one member of a specific binding pair ("labelled binding part ⁇ ner") or an analog of such a member.
  • the marker component may be directly attached or conjugated thereto or attached or conjugated via a linker arm.
  • 1,2,4,5-Tetracyanobenzene (Pfaltz & Bauer, 0.5 g, 2.8 mMol) was suspended in methanol (10 ml) in a three-neck round-bottom flask fitted with a reflux condenser and a gas inlet tube. The mixture was stirred at 25°C without external cooling while ammonia gas was rapidly introduced. During the first two minutes of ammonia introduction the temperature of the reaction mixture rose to greater than 50°C and the suspended solid dissolved completely. Within 5 minutes a precipitate began to form. Stirring at 40- 50°C with slow introduction of ammonia was continued for 1 hour. The precipitated solid was collected by filtra ⁇ tion, washed with methanol, and dried. This product exhibited a very low solubility in methanol.
  • dicyano- diiminoisoindoline 350 mg, 1.8 mMol
  • diimino- isoindoline 1.0 g, 6.9 mMol
  • quinoline Fluka, 20 ml
  • the mixture was stirred at 25°C while silicon tetra- chloride (Aldrich, 2.0 ml, 18 mMol) was added dropwise over a period of 1 minute.
  • the flask was then fitted with a reflux condenser (using teflon tape) and a calcium chloride drying tube and stirred for one minute at 25°C.
  • reaction flask was lowered into a large oil bath maintained at 180-185°C and efficient mag ⁇ netic stirring was continued for 30 minutes. The oil bath was then removed and the contents of the flask were allowed to cool to room temperature.
  • step (C) The crude dicyanophthalocyanine from step (C) (1 gram) was placed in a flask with a stir bar and 6 ml of concentrated sulfuric acid and stirred at 50°C overnight. The mixture was then carefully diluted with 4 ml water and heated to 100°C for an additional 20 hours. Cooling and dilution with water (20 ml) gave a blue precipitate which was collected by filtration and washed with water. The solid was then transferred to a flask along with a stir bar and 20 ml of a 1.0 M potassium carbonate solution and stirred and heated at reflux for one hour. The suspension was then slowly and carefully acidified with concentrated HCl and then filtered and the resulting solid was washed with water and acetone and dried in a desiccator. This material (0.7 g) was used without further purification in the next step.
  • the crude dicarboxy silicon phthalocyanine dihydrox- ide from step (D) (85 mg) was placed in a vial along with a stir bar and imidazole (160 mg, 2.3 mMol) and 1 ml of dry DMF. The mixture was stirred for 5 minutes at 25°C andthen3-isocyanatopropyldimethylchlorosilane (Petrarch, 110 ⁇ l, 0.68 mMol) was added to the stirred mixture over a period of 0.5 minutes. The vial was capped in order to exclude moisture and stirring at 25°C was continued for 20-40 hours.
  • step (E) (Compound III) (3 mg, 5 x 10 ⁇ 3 mMol) , which had been obtained in partially purified form by chromatography on silica gel, was dissolved in methanol
  • the resulting deep blue solution was heated to reflux for one hour. Removal of methanol under aspirator pressure at 25°C left a viscous blue oil which was taken up in water (0.5 ml) and applied to a small (10 ml wet volume) DEAE Sepha- dex anion ion exchange column (Pharmacia, 3.5 meq/g, 40- 120 micron, basic form ⁇ 1M K 2 C0 3 ) . The water-soluble blue dye was retained quantitatively by the column. The column was washed with water (15 ml) and the blue dye was then eluted in greater than 70% yield with 10-20 ml of a 15% aqueous acetic acid solution.
  • Fig. 1 is a typical HPLC chromatogram of this prepar ⁇ ation (compound IV) .
  • the yields for a typical dye preparation range from 25-65% of this fraction.
  • the Diatron Analog System is diagramed in Fig. 3.
  • the tunable dye laser used was a PTI model PL2300 nitrogen laser with a dye laser module. By changing the laser dye and adjusting the dye laser grating, 600 picosecond pulses with peak power of near 40 KWatts could be generated at wavelengths from 340 to 900 nm.
  • a beam splitter was used to send a portion of each pulse to a pulse detector which consists of a high speed
  • Hamamatsu photo diode The resulting output of the photo- diode was fed into a pulse shaper which converted the resulting 800 picosecond (ps) pulse into a 100 nanosecond (ns) pulse. This 100 ns pulse was then used as a gate for the Hamamatsu microchannel plate PMT whose gain was changed by 10,000 within a 2 ns time period. The PMT stayed at the high gain until the 100 ns was over.
  • the dye laser module, reaction cell and pulse detector was positioned and connected such that the PMT was gated to its high sensitivity state approximately 2 ns after the laser pulse passed through the reaction cell.
  • a filter was positioned in front of the PMT to guard against high scatter signals when required.
  • a lens was used to image the fluorescence onto the PMT microchannel plate.
  • a rotatable polarizer was positioned in the output optical path to measure the time dependent polari ⁇ zation of the fluorescence.
  • a computer was used to trigger the laser.
  • the laser output was detected by the digitizer via a connection to the pulse detector (not shown) .
  • a programmable sweep on the digitizer set up the time spread to be measured after the laser pulse from 10 ns to as high as several seconds. Typically, the system was operated such that 512 data points were generated over a 20 ns time period.
  • the dye preparations were analyzed for their inter ⁇ action with serum protein.
  • the dye preparations were adjusted to 5 x 10" 9 M/L in FPIA buffer.
  • These dye prep- arations were added to the following solutions to a final dye concentration of 5 x 10" 11 M/L: FPIA buffer, 0.5% bovine gamma globulin, 5.0% bovine serum, 5.0% normal human serum, 5.0% pooled human serum and 5.0% whole blood lysate.
  • FPIA buffer 0.5% bovine gamma globulin
  • 5.0% bovine serum 5.0% normal human serum
  • 5.0% pooled human serum 5.0% whole blood lysate.
  • Fig. 5 Typically, when a dye binds to a protein non-specifically (as can be seen with the "C" fraction) , a significant increase in fluor ⁇ escence polarization occurs. This makes it impossible to distinguish the specific polarization due to antibody binding from the non-specific due to protein-dye inter- action.
  • Example 2 Linkers In certain polarization assays, it is advantageous to use a spacer or linker. These linker or spacer arms are useful when different ligands are terminated by either a carboxyl or amino group. In addition, such compounds are important when the probe needs to be separated (stood off) from the molecule with the antibody binding epitope. This may be necessary to reduce the potential of non-radiated transfer of energy when antibody binds the specific epi ⁇ tope and/or to eliminate stearic hinderance. These linker/spacer arms are generally the same in both the ligand-probe and ligand-protein immunogen used to raise antibodies to the ligand, in order to create a specific binding pair. In polarization immunoassays, it is desir ⁇ able that the spacer create a relatively inflexible linker moiety.
  • caged dicarboxy silicon phthalocyanine is a carboxy-terminated dye
  • Various linkers spacer arms
  • Such compounds include piperazine, ethylenediamine, hex- anediamine, 6-amino hexanoic acid, 5-aminobutanoic acid, 12-aminododecanoic acid, alanine and other amino acids.
  • the following methodology for preparation of the phthalocyanine-12 amino dodecanoic acid compounds is an example of the general reaction for such linkers:
  • HOBT 1-hydroxybenzotriazole
  • the new abducts were purified on reverse phase C-18 columns by HPLC.
  • 12-aminododecanoic acid was used as a linker
  • a 10 nm shift to 690 nm occurred, which matches commercially available 690 nm laser diodes .
  • the change increased the dynamic range of the assay from 0.03 to greater than 0.30 millipolarization in buffer when bound to an antibody molecule.
  • the 10 nm shift increased the signal-to-background ratio by moving away from the excitation maximum of fluor- escing background molecules found in biological fluids .
  • Fig. 6 The absorbance spectrum in methanol for the purified caged dicarboxy silicon phthalocyanine dye-linker is shown in Fig. 6. There is a 10 nm shift from 680 for the frac ⁇ tion "B" dye (Fig. 2) , to 690 nm for the dye-linker.
  • transient state fluorescence polarization was measured on the Diatron Analog System described in Exam ⁇ ple I, in FPIA buffer and glycerol at 680 nm and in glycerol at 690 nm. These data are shown in Figs. 7 and 8.
  • Digoxin is a glycosylated steroid which, when used in patients with congestive heart failure, increases cardiac output, decreases heart size, venous pressure and blood volume, and relieves edema.
  • digoxin has a very narrow therapeutic range (serum levels of 0.5 to 2.5 ng/ml) and is generally toxic at concentrations greater than 2.1 ng/ml. Accordingly, there is a need for a digoxin assay which can accurately and precisely deter ⁇ mine digoxin concentrations at these levels.
  • the digoxin probe was prepared as follows: .2 mg of 3-aminodigoxigenin was placed in a 3.0 ml reaction vial and dissolved with 100 ⁇ l DMF. In a separate vial, 1.0 mg of caged dicarboxy silicon phthalocyanine (Compound IV from Example 1(G)) was dissolved in 400 ml DMF and then transferred to the reaction vial along with 200 ⁇ l of wash DMF for a total of 600 ⁇ l,. 4.2 mg of 1-hydroxybenzotria- zole (HOBT) was added to the reaction vial, which was then dissolved and mixed well.
  • HOBT 1-hydroxybenzotria- zole
  • the digoxin-phthalocyanine probe was purified as fol ⁇ lows: a slurry of 5 gm C-18 was made in acetone and poured into a 1x15 cm glass column. The acetone was removed by the application of light pressure, and the column was equilibrated by the addition of 4 column vol ⁇ umes of 70% methanol/30% water. The reaction mixture containing digoxin-phthalocyanine probe was applied to the column and flushed with 70% methanol/30% water. The probe was eluted with 80% methanol/20% water, concentrated by vacuum and further purified by two subsequent passes on HPLC. After the second chromatograph on HPLC, the probe was brought to dryness in vacuo.
  • Fig. 9 depicts a chromatograph of the HPLC method semi-prep C-18 column with a mobile phase and gradient elution of methanol/water.
  • Fig. 10 depicts the structure of the digoxin-phthalocyanine probe.
  • the probe was analyzed in a Perkin-Elmer spectro- photometer (Lambda 4 c) in two solvents, methanol and
  • Figs. 11 and 12 are repre- sentative spectra. Fluorescence decay time was determined to be 4.7 ns using the Diatron Analog System described in Example 1 (Fig. 13) .
  • R gas constant
  • T temperature (in °K)
  • n solution viscosity volume of molecules
  • the fluorescein probe is only slightly detectable above background in 5% serum and completely non-detectable in 10% urine.
  • the phthalocyanine digoxin probe is detectable at a very sig- nificant level above background in both the same serum and urine samples.
  • Example 4 Competitive Serum Assay for Digoxin: Sequential Binding Procedure
  • Digoxin reacts with serum albumin and other serum proteins at many reaction sites. Probes made with a fluorescent dye and digoxin will also react. "Nonspeci ⁇ fic” binding or serum protein interactions were minimized in this procedure by the action of the cyclodextrin, which has an affinity for digoxin which exceeds digoxin's affi ⁇ nity for constituents in serum. Thus the cyclodextrin interferes with the binding of digoxin with serum consti ⁇ tuents, but allows for binding of digoxin with digoxin antibody. Thus, the assay was designed to allow both the serum digoxin and the digoxin probe to react with the digoxin antibody.
  • Transient state polarization was measured as described in Studholme, et al. , Patent Application entitled “Fluorometer Detection System, " Lyon & Lyon Docket No. 195/129.
  • the transient state optical system was installed in the Diatron "FAST-60 Analyzer, " which contains a laser diode operating at 685 nm was pulsed at a 10 MHz rate. Typically, the laser "on” time was approx ⁇ imately 3 nanoseconds.
  • Photons from the solution were detected using a photomultiplier tube (PMT) operating in a single photon counting mode. The photon event along with the relative time of the photon event as compared with the laser pulse time was determined. By storing the individual photon event times a histogram of frequency of photons as a function of time was generated.
  • PMT photomultiplier tube
  • the Diatron FAST-60 Analyzer includes a transient- state optical system installed in an automated fluores ⁇ cence reader designed to measure fluorescence from immuno ⁇ assay reactions.
  • the reader contains the optical system, motor control for position reaction cuvettes in front of the optical system, thermal control to hold the system at 35°C and a computer link to control the reader, analyze and display results and print those results.
  • the results were formatted into transient-state polarization units or, by using a calibration curve, the results were transformed into concentration units of the analyte being measured.
  • Fig. 16 shows a comparison of digoxin calibration curves by a standard fluorescence polarization procedure (Abbott's TDx ® Fluorescence Polarization Analyzer) and the homogeneous sequential binding assay procedure described in this Example.
  • Fig. 17 displays a correlation plot of 37 serum samples assayed by a commercial digoxin test system manufactured by Abbott Laboratories (TDx ® Digoxin II In Vitro Test, Product #9511-60) and assayed by the digoxin assay procedure described in this example. A correlation of 0.96 and a slope of 0.98 were determined. The slope and y-intercept indicate no systematic bias.
  • the dilution jump procedure described in this Example allows the assay to be performed in the presence of high concentrations of serum, and was designed to reduce "non ⁇ specific" interactions which compete with the antibody for binding to the digoxin and digoxin probe. While not wish ⁇ ing to be bound by any particular theory, applicants believe that when sample, antibody and digoxin probe are incubated in a small reaction volume, the "nonspecific” interactions initially compete with the antibody for binding to digoxin and digoxin probe. When the solution is diluted, the "nonspecific" protein interactions tend to disappear rapidly and only the specific antibody reaction remains.
  • the signal-to- background data is represented as a ratio (signal counts/ background count) .
  • steady state assays can be configured with acceptable signal-to-background ratios using the caged dicarboxy silicon phthalocyanine digoxin probe which is measured at 680 nm. There was an approxi ⁇ mately 10-fold enhancement in this ratio when transient state techniques were used to time-discriminate against fast fluorescers within the background and scattering bands.
  • concentration of fluorescent probe in the fluorescein steady assay system is 2.5 x 10 "10 M/L.
  • the procedure is as follows: 50 ⁇ l of lysing/buffer (see Example 5 above) diluent was mixed with 2.0 ⁇ l serum calibrator or serum, plasma or whole blood sample and 5.0 ⁇ l of rabbit anti-digoxin antibody. This mixture was incubated for 5 minutes and 2.5 ⁇ l of digoxin probe was added and incubated an additional 15 minutes. After this incubation, 1.0 ml FPIA buffer was added as a dilution jump. The transient state polarization measurements were made on the Diatron FAST-60 Analyzer described in Exam ⁇ ple 4.
  • Sample blanks were prepared for each sample or calibrator by performing the same steps, with the excep ⁇ tion that buffer was added in place of the probe. The sample blank was then measured and subtracted from the measurements for the entire reaction mixtures.
  • Fig. 20 displays a calibration curve for commercial serum calibrators containing known concentration of digoxin, which were assayed using the high sensitivity procedure.
  • Example 8 Preparation of Whole Blood Calibrators
  • Whole blood was obtained from two donors by drawing blood into VacutainerTM (Bector Dickinson) tubes containing EDTA anticoagulant. The tubes were mixed thoroughly on a standard laboratory sample rotator. Based on the average specific gravity of blood being 1.056, a series of six 2 ml volumes of whole blood were weighed using standard gravimetric technique. These samples were then spiked using a USP grade digoxin (200 ng/ml) to final concen ⁇ trations of 0, 0.5, 1.0, 2.0, 3.0, and 5.0 ng/ml whole blood. The whole blood calibrators were stored at 4.0 - 8.0 °C and were used within two weeks.
  • Ratio, Serum Versus Whole Blood Applicants determined the signal-to-background ratio of the whole blood preparations which were prepared as described in Example 8 and compared the whole blood (i.e. , blank) intensity to the probe intensity. The resulting values comparing the whole blood and serum signal-to- background ratios are shown in Table 4. These measure ⁇ ments were made at working digoxin probe concentration of 5 X 10 "11 M/L in the transient state system. It can be seen that the net probe intensities remained constant even when the background intensities fluctuated.
  • the average signal-to-noise ratio is 2 to 1 at probe concentrations of 2.5 X 10 "10 M/L, as contrasted with those found by homoge ⁇ neous transient state fluorescence for serum and whole blood of 77.6:1 and 26.7:1, respectively.
  • Example 10 Homogeneous Whole Blood Digoxin Assay Clinical Study Previous whole blood immunoassays have been limited by many factors. For example, separation steps are required in many assay systems, enzymes and other sub ⁇ stances released from red blood cells cause interference in the assays, and the instrumentation is incapable of measuring analytes or reaction products through whole blood hemolysates.
  • Applicants have developed a homoge- neous whole blood assay system which offers the clinical laboratory and other testing facilities significant advan ⁇ tages over currently used methods, including decreased labor cost, and decreased sample manipulation. In addi ⁇ tion, with a homogeneous 5 to 10 minute assay, the pro- cedure can be brought much closer to the patient, for example, to the bedside, emergency care facilities clinics and satellite testing facilities.
  • Digoxin is widely distributed in body tissues. Serum and plasma have been the accepted samples for the assay of digoxin using the current commercially available test kits. Studies have shown a relative constant relationship between heart muscle and serum digoxin levels, thus vali ⁇ dating the use of digoxin serum levels in monitoring patients receiving the drug (Doherty, J.E., et. al.. 1978, "Clinical Pharmacokinetics of Digitalis Glycosides.” Pro ⁇ gress in Cardiovascular Diseases, Vol. XXI, No. 2 (Sept./ Oct.) ) .
  • Each sample pair was ana ⁇ lyzed for serum digoxin levels determined by the Abbott TDx ® , Dade Stratus ® and Diatron FAST-60 Systems. Plasma levels were analyzed by Abbott TDx ® and Diatron FAST-60 Systems. Whole blood levels were analyzed by Abbott TDx ® and Diatron FAST-60 Systems.
  • the Abbott TDx ® System used the TDx ® Digoxin II In Vitro Test, Product #9511-60 (Abbott Laboratories) .
  • the Dade Stratus System used the Dade Stratus ® Digoxin Fluorometric Enzyme Immunoassay (Dade Diagnostics Division of Baxter Healthcare Division, Miami, Florida) .
  • the Diatron FAST-60 System used the methods described in Example 5 and the apparatus described in Example 4 and Studholme, e al. , Lyon & Lyon Docket No. 195/129.
  • the primary concern was whether the whole blood digoxin values were similar to the serum values.
  • the whole blood lysates were clarified by centrifugation before assay.
  • the study subjects were randomly selected patients currently on active digoxin therapy.
  • the Diatron FAST-60 Digoxin Assay System consisted of (1) caged dicarboxy silicon phthalocyanine digoxin probe in FPIA buffer with 1% bovine gamma globulin; (2) rabbit anti-digoxin in FPIA buffer with 0.1% bovine gamma globu ⁇ lin; (3) lysing/buffer diluent; and (4) FPIA buffer (100 mM phosphate buffer with 1% sodium azide and .01% bovine gamma globulin) . Assay procedures were performed as follows:
  • Diatron FAST-60 See Fig. 21.
  • A. Abbott TDx ® Digoxin II In Vitro Test Performed according to manufacturer's instructions, except for the precipitation step. Digoxin extraction from whole blood was accomplished as follows: to 360 ⁇ l whole blood, an equal volume of Abbott -Precipitation Reagent (Digoxin II) was added with immediate vortexing for 30 sec ⁇ onds. The conical tubes were centrifuged at 10,000 RPMs for two minutes. The slightly brownish supernate was removed very carefully with a Pasteur pipette and transferred to the sample cup, to avoid the transfer of small particles.
  • Abbott TDx ® Digoxin II In Vitro Test Performed according to manufacturer's instructions, except for the precipitation step. Digoxin extraction from whole blood was accomplished as follows: to 360 ⁇ l whole blood, an equal volume of Abbott -Precipitation Reagent (Digoxin II) was added with immediate vortexing for 30 sec ⁇ onds. The conical tubes were centrifuged at 10,000 RPMs for two minutes. The slightly brown
  • Red blood cells were lysed prior to assay by addition of lysing buffer (0.001 M/L Tris buffer, pH 8.0 containing 5 x 10 ⁇ 5 M/L stearyl-lysolecithin) . Palmitoyl-lysolecithin and myristoyl-lysolecithin in Tris buffer are equally effective. These reagents are preferred in that they do not interfere with the immunoassay at this concentration, and that red cell ghost particle size is reduced in 30 to 60 seconds, thus reducing any effect of light scatter dur- ing the fluorescence measurements resulting in a homoge ⁇ neous, non-separation whole blood assay.
  • lysing buffer 0.001 M/L Tris buffer, pH 8.0 containing 5 x 10 ⁇ 5 M/L stearyl-lysolecithin. Palmitoyl-lysolecithin and myristoyl-lysolecithin in Tris buffer are equally effective. These reagents are preferred in that they do not interfere with the
  • the results of the testing are tabulated in Table 5.
  • the values from the TDx ® and Stratus ® are the result of single point testing. Because the FAST-60 procedure used "manual pipetting" and automated instrumental analysis, the samples were run in duplicate and the raw values averaged.
  • Figs. 22 through 26 The correlation data are found in Figs. 22 through 26. These include TDx ® serum versus FAST-60 whole blood (Fig. 22) , Stratus ® serum versus FAST-60 whole blood (Fig. 23) FAST-60 serum versus FAST-60 whole blood (Fig. 24) , TDx ® serum versus FAST-60 serum (Fig. 25) and Stratus ® serum versus FAST-60 serum (Fig. 26) .
  • small analytes such as amikacin, gentamicin, netilmicin, tobra- mycin, carbamazepine, ethosuximide, valproic acid, diso- pyramide, lidocaine, procainamide, quinidine, methotrex- ate, amitriptyline, mortripyline, imipramine, desipramine, vancomycin and cyclosporine are particularly suited for the assays described herein due to their size.
  • probes were prepared for digitoxin, theophylline, phenobarbital, thyroxine, N-acetylprocainamide, primidone and phenytoin.
  • caged dicarboxy silicon phthalocyanine probes can be prepared for peptides.
  • such probes were prepared for rubella virus peptide.
  • the digitoxin probe was prepared as follows: 0.8 mg of 3-aminodigitoxigenin was placed in a 3.0 ml reaction vial and dissolved in 100 ⁇ l DMF. Caged dicarboxy silicon phthalocyanine (1.0 mg) was added to the reaction vial. Also added to the reaction vial were 0.5 mg HOBT and 2.0 mg EDAC and the resulting mixture was thoroughly mixed. The reaction mixture was stored overnight at about 4 to 8°C.
  • the digitoxin probe was purified by procedures similar to those described in Example 3 for the digoxin probe.
  • the structure of the caged dicarboxy silicon phthalocyanine-digitoxin probe is shown in Fig. 27.
  • the theophylline probe was prepared as follows: 1.2 mg theophylline-8- (N-2-aminoethyl)butyramide was placed in a 3.0 ml reaction vial and dissolved in 100 ⁇ l DMF. In a separate vial, caged dicarboxy silicon phthalo ⁇ cyanine (1.0 mg) was dissolved in 400 ⁇ l DMF and then transferred to the reaction vial along with 200 ⁇ l of wash DMF (for a total of 600 ⁇ l DMF) . To the reaction vial was added 6.1 mg of HOBT, dissolved and mixed well. To make the final reaction mixture, 7.0 mg EDAC was added and the resulting mixture mixed thoroughly. The reaction mixture was stored overnight at about 4 to 8°C.
  • the theophylline probe was purified using procedures similar to those described in Example 3 for the digoxin probe.
  • the structure of the caged dicarboxy silicon phthalocyanine-digoxigenin probe is shown in Fig. 28.
  • Phenobarbital 663 mg, was dissolved in 2.7 ml con- centrated sulfuric acid cooled in ice. With stirring, a cold solution of 0.16 ml concentrated nitric acid in 0.65 ml concentrated sulfuric acid was added dropwise over a period of 4 minutes. After 1/2 hour in the cold, the reaction mixture was poured into ice water. The precipi- tate was collected, washed with water, and dried in vacuo affording 0.03 g white solid.
  • the phenobarbital probe was prepared as follows: 1.2 mg of 5-ethyl-5- (aminophenyl)barbituric acid (P-amino- phenobarbital) was placed in a 3.0 ml reaction vial and dissolved with 200 ⁇ l DMF. In a separate vial the caged dicarboxy silicon phthalocyanine (1.0 mg) was dissolved in 200 ⁇ l DMF and then transferred to the reaction vial. To the reaction vial was added 2.4 mg HOBT, dissolved and mixed well. To make the final reaction mixture, 2.8 mg of EDAC was added and mixed thoroughly. The reaction mixture was stored overnight at about 4 to 8°C.
  • the phenobarbital probe was purified using procedures similar to those described in Example 3 for the digoxin probe.
  • the structure of the caged dicarboxy silicon phthalocyanine probe is shown in Fig. 29.
  • the thyroxine probe was prepared as follows: 1.0 mg of tetraiodothyroacetic acid-ethylenediamine (Tetrac-EDA) was placed in a 3.0 ml reaction vial and dissolved in 100 ⁇ l DMF. In a separate vial, caged dicarboxy silicon phthalocyanine (1.0 mg) was dissolved in 400 ⁇ l DMF and then transferred to the reaction vial along with 200 ⁇ l of wash DMF for a total of 600 ⁇ l. To the reaction vial was added 1.8 mg HOBT, dissolved and mixed well. To the final reaction mixture, 1.5 mg EDAC was added and the resulting mixture mixed thoroughly. The reaction mixture was stored overnight at about 4 to 8°C. C. Purification of Probe
  • the thyroxine probe was purified using procedures similar to those described in Example 3 for the digoxin probe.
  • the structure of the caged dicarboxy silicon phthalocyanine-thyroxine probe is shown Fig. 30.
  • Example 15 Synthesis of Caged Dicarboxy Silicon Phthalocyanine-N-Acetylprocainamide A. Preparation of Desethyl-N-Acetylprocainamide Desethyl-N-acetylprocainamide was prepared by dis ⁇ solving 1.0 g of p-acetamidobenzoic acid and 0.7 g N-hydroxysuccinimide in 20 ml pyridine. To this solution was added 1.4 g of N, N'-dicyclohexylcarbodiimide. The reaction mixture was placed at 4°C for 18 hours, at which time the crystals were removed by filtration.
  • the filtra ⁇ tion was brought to room temperature and with stirring, 0.51 g N-ethylethylenediamine was added. Stirring con ⁇ tinued for 3 hours, the solution was cooled to 4°C and allowed to react for an addition 24 hours at 4°C.
  • the second crop of crystals was removed by filtration, dis ⁇ solved in 25 ml of distilled water.
  • the pH was adjusted to 10 with sodium hydroxide to form a white precipitate of desethyl-N-acetylprocainamide .
  • the resultant precipitate was dried in vacuo and stored at -20°C in a desiccator.
  • the N-Acetylprocainamide probe was prepared as fol ⁇ lows: 1.0 mg desethyl- ⁇ -Acetylprocainamide was placed in a 3.0 ml reaction vial and dissolved with 100 ⁇ l DMF. In a separate vial, caged dicarboxy silicon phthalocyanine (1.0 mg) was dissolved in 400 ⁇ l DMF and then transferred to the reaction vial along with 200 ⁇ l of wash DMF for a total of 600 ⁇ l. To the reaction vial was added 4.2 mg HOBT, dissolved and mixed well. To make the final reac ⁇ tion mixture, 10.5 mg EDAC was added and mixed thoroughly. The reaction mixture was stirred overnight at about 4 to 8°C.
  • the N-Acetylprocainamide probe was purified using procedures similar to those described in Example 4 for the digoxin probe.
  • the structure of the caged dicarboxy sili ⁇ con phthalocyanine-N-acetylprocainamide probe is shown in Fig. 31.
  • the primidone probe was prepared as follows: 0.8 mg 5-ethyl-5- (4-aminophenyl) hexahydropyrimidine-4, 6-dione (p-aminoprimidone) was placed in 100 ⁇ l DMF in a 3.0 ⁇ l reaction vial. To the reaction vial was added 1.0 mg caged dicarboxy silicon phthalocyanine (1.0 mg) and 3.1 mg HOBT. To make the final reaction mixture, 3.9 mg EDAC was added along with 150 ml DMF and the resulting mixture mixed thoroughly. The reaction mixture was stored over- night at about 4-8°C.
  • the primidone probe was purified using procedures similar to those described in- Example 3 for the digoxin probe.
  • the structure of the caged dicarboxy silicon phthalocyanine-primidone probe is shown in Fig. 37.
  • Example 17 Synthesis of Caged Dicarboxy Silicon Phthalocyanine-Phenvtoin A. Preparation of Probe
  • the phenytoin probe was prepared as follows: 1.2 mg of diphenylglycine was placed in a 3.0 ml reaction vial and dissolved with 100 ⁇ l DMF. In a separate vial, dicar- boxyphthalocyanine (3.0 mg) was dissolved in 400 ml DMF and then transferred to the reaction vial along with 200 ⁇ l of wash DMF for a total of 600 ⁇ l. To the reaction vial was added 6.1 mg of HOBT, dissolved and mixed well. To make the final reaction mixture, 7.0 mg of EDAC was added and mixed thoroughly. The reaction was stored at 4.0-8.0°C overnight.
  • the phenytoin-phthalocyanine probe was purified using procedures similar to those described in Example 3 for the digoxin probe.
  • the structure of the caged dicarboxy sili ⁇ con phthalocyanine-phenytoin probe is shown in Fig. 33.
  • Caged dicarboxy silicon phthalocyanine dye (12 ⁇ moles) prepared according to Example 1 and purified by DEAE Sephadex chromatography was mixed with 1 ml of pyridine-pyridinium chloride buffer made by mixing 5 ml 1 M HCl with 0.5 ml pyridine. The solution was taken to dryness in a sublimation apparatus and the excess pyridine and pyridinium chloride was removed, thus assuring that all acetate ion present would be removed. The dry resi ⁇ dual dye was dissolved in anhydrous dichloromethane to make a 3.5 mM solution.
  • the carboxylic acid groups of the dye were converted to the imidazolide by mixing 1 ml of 3.5 mM dye with 760 ⁇ l of 0.46 M carbonyl diimidazole and allowing 1.5 hour at room temperature for reaction, after which the solvent was removed .in vacuo.
  • DMF was scavenged free of water and reactive amines by adding carbonyl diimidazole to a final concentration of 0.1 M.
  • a portion of the reac ⁇ tion mixture was equilibrated with 10 mM phosphate, pH 7.6 by two treatments in a Minicon concentrator (Amicon Cor ⁇ poration, Danvers, MA, USA) and passed through a hydroxy- lapatite column (Bio Rad Laboratories, Richmond, CA, USA) equilibrated with 10 mM phosphate, pH 7.6. Free dye eluted at this stage and the labeled antibody was recov- ered by elution with 100 mM phosphate, pH 7.6.
  • speci- ficity is defined as the proportion of individuals with negative test results for the disease that the test is intended to reveal, i.e.. true negative results as a proportion of the total number of true negative and false positive results.
  • this assay demonstrated 100% specificity.
  • sensitivity of a procedure can be defined as that pro ⁇ portion of individuals with a positive test result for the disease that the test intended to reveal , i.e. , true posi ⁇ tive results as a proportion of the total true positive and false negative results.
  • Probe A synthetic rubella peptide for example, a portion of the E-. protein of the rubella virus (Therien strain) , can be synthesized by standard peptide synthesis proce ⁇ dure.
  • the coupling of caged dicarboxy silicon phthalo ⁇ cyanine (prepared according to Example 1) to the synthetic rubella peptide was a four step process:
  • Example 20 Immunological Evaluation of Phthalocyanine-Rubella Probe Two assay procedures were performed in order to evaluate the phthalocyanine-Rubella probe.
  • the phthalo ⁇ cyanine-Rubella Probe was diluted in a 0.01 Tris buffer pH 8.0 containing 0.1 % bovine serum and 0.025% Tween 20.
  • the probe concentration was determined to be 1.1 x 10" 11 M/L.
  • Rubella peptide calibrators were made by diluting in the Tris buffer to the following concentrations: 0.0, 1.0 x 10' 12 , 2.7 x 10 "12 , and 5.4 x 10 "12 , 2.7 x 10 -11 , and 5.4 x 10 "11 .
  • the antibody was made by hyper-immunizing a rabbit with the Rubella peptide. Dilutions were made in the Tris buffer described above.
  • Tris buffer 25 ⁇ l Tris buffer, 20 ⁇ l antibody solution and 10 ⁇ l antibody solution and 10 ⁇ l of each peptide calibrator. The tubes were incubated at 35°C for 10 minutes. At this time, 15 ⁇ l of probe was added to each tube and incubated an additional 20 minutes at 35°C. After incubation 1.0 ml of Tris buffer was added and transient state polarization measurements were made.
  • the typical inhibition curve is shown in Fig 35.
  • probes can be made for peptide hormones such as luteiniz- ing hormone, follicular stimulating hormone, human chorio- gonadotropin, thyroid stimulating hormone.
  • Angiotensin I Angiotensin II
  • prolactin prolactin
  • Probes can be made for peptides such as tumor markers (for example, carcino- embryonic antigen) as well.
  • tumor markers for example, carcino- embryonic antigen

Abstract

L'invention concerne des procédés d'immuno-essais par fluorescence dans lesquels on utilise des matrices fluorescentes exemptes d'agrégation et de liant de sérum. Ces procédés d'immuno-essai sont particulièrement utiles pour l'essai de fluides biologiques tels que du sérum, le plasma, le sang entier et l'urine.
PCT/US1993/002470 1990-05-15 1993-03-23 Immuno-essais par fluorescence au moyen de matrices fluorescentes exemptes d'agregat et de liant de serum WO1993019366A1 (fr)

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EP93908390A EP0632893A4 (fr) 1992-03-23 1993-03-23 Immuno-essais par fluorescence au moyen de matrices fluorescentes exemptes d'agregat et de liant de serum.
CA002132708A CA2132708C (fr) 1992-03-23 1993-03-23 Immunoessais de fluorescence, utilisant des colorants fluorescents sans agregation ni liaison serique
US08/035,633 US5846703A (en) 1990-05-15 1993-03-23 Fluorescence immunoassays using fluorescent dyes free of aggregation and serum binding

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US07/856,176 1992-03-23

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EP0632893A1 (fr) 1995-01-11
EP0632893A4 (fr) 1997-01-02
CA2132708C (fr) 2005-05-10

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