WO2005054854A1 - Instruments et procedes de polarisation de fluorescence pour la detection d'exposition aux matieres vivantes par le dosage immunologique par la polarisation de fluorescence de la salive, de liquides buccaux ou organiques - Google Patents

Instruments et procedes de polarisation de fluorescence pour la detection d'exposition aux matieres vivantes par le dosage immunologique par la polarisation de fluorescence de la salive, de liquides buccaux ou organiques Download PDF

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WO2005054854A1
WO2005054854A1 PCT/US2003/032736 US0332736W WO2005054854A1 WO 2005054854 A1 WO2005054854 A1 WO 2005054854A1 US 0332736 W US0332736 W US 0332736W WO 2005054854 A1 WO2005054854 A1 WO 2005054854A1
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fluorescence
antibody
virus
fluorescence polarization
ligand
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PCT/US2003/032736
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English (en)
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Malford E. Cullum
Lloyd G. Simonson
Sylvia Z. Schade
Linda Z. Lininger
Ernest Pederson
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The United States Of America As Represented By The Secretary Of The Navy Naval Medical Research Center
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Priority to AU2003287153A priority Critical patent/AU2003287153A1/en
Priority to PCT/US2003/032736 priority patent/WO2005054854A1/fr
Publication of WO2005054854A1 publication Critical patent/WO2005054854A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6445—Measuring fluorescence polarisation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • 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
    • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
    • G01N33/542—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with steric inhibition or signal modification, e.g. fluorescent quenching
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms

Definitions

  • the present inventive subject matter relates to novel methods for detecting the presence of a biological substance of interest in a test sample of saliva, oral or other bodily fluids. Also provided is a miniaturized, portable, hand-held apparatus for measuring the fluorescence polarization of a liquid sample .
  • Immunoassays Infectious disease rates and immunization strategies continue to evolve in the United States and worldwide in response to societal needs, national defense, and evolutionary changes in the organisms producing disease. Immunizations are performed to prevent many infections, while prophylactic population screening is utilized for infections lacking effective vaccines and for those diseases having a low enough incidence that mass immunization is not deemed most efficacious.
  • the current method for diagnosis of disease, determining exposure to biological materials such as pathogens, or monitoring immunization status varies depending on the specific assay.
  • Some methods employ an in vivo assay. Others require a biological sample, such as blood or serum, to be obtained and tested. Tests performed usually are one of the non-homogeneous type diagnostic methods such as enzyme-linked immunosorbant assay (hereinafter "ELISA”) , radioimmunoassay (hereinafter "RIA”) , or agglutination. All are surface- binding, heterogeneous assays and require the antigen of interest to interact with a surface to achieve success, often at the expense of high non-specific binding and loss of specificity. Fluorescence Polarization.
  • Fluorescence polarization (hereinafter FP") is the process in which visible or ultraviolet light is polarized with a filter and shines on part of a molecule, the fluorochrome, that in turn fluoresces, emitting light of longer wavelength whose signal is captured and recorded. The emitted light recorded stays more polarized in solutions when there are slower turning, large molecule-fluorochrome complexes than when there are smaller labeled molecules.
  • Different fluorochromes can be chosen to accommodate molecules of different sizes up to 10 7 kDa molecular weight .
  • August 13, 2002 discloses a fluorescence polarization method for analyzing an assay-object in a sample, comprising the steps of: (a) providing a fluorescent- labeled protein in which a protein is covalently bound to a fluorochrome (s) , wherein the protein is capable of specifically binding to the assay-object; (b) allowing the fluorescent-labeled protein to bind to the assay- object; and (c) measuring a change in the degree of fluorescence polarization which has taken place in the fluorescent-labeled protein by its binding to the assay- object .
  • a fluorescence polarization method for analyzing an assay-object in a sample comprising the steps of: (a) providing a fluorescent- labeled protein in which a protein is covalently bound to a fluorochrome (s) , wherein the protein is capable of specifically binding to the assay-object; (b) allowing the fluorescent-labeled protein to bind to the assay- object; and (c) measuring a
  • Saliva and oral fluids are biochemically distinct and have been increasingly recognized as acceptable alternatives to serum for use in diagnostic tests for certain hormones, drugs, antibodies and antigens. Oral fluids are collected without pain, needle sticks, or religious and social prohibitions, and their use involves minimal risk or exempt protocols for the use of human subjects.
  • diagnostic assays utilizing saliva and other oral fluids appear as entries in the National Library of Medicine MEDLARS database with a frequency of only 1 in 30 and 1 in 100, respectively, when compared to entries in which blood serum is reported.
  • Knowledge of the epidemiology of diseases is important in health care planning and treatment . Both are dependent upon accurate and rapid diagnosis.
  • FP salivary diagnostics for accurate and rapid detection and diagnosis of antibodies to pathogens producing diseases of military and commercial interest.
  • the technology is homogeneous and can accurately estimate concentrations of diagnostic markers, drugs and chemicals, or bio-hazardous agents in oral fluids and environmental samples within a few seconds to several minutes.
  • inventive oral immunodiagnostic assay systems are robust and relatively unaffected by use with non-homogeneous samples, such as whole blood or saliva. Specificity of FP assays is generally very high, approximately 98%, with good sensitivity.
  • the applications of FP salivary diagnostics to determination of tuberculosis exposure and of anthrax immunization status are provided as examples .
  • Fluorescence polarization instruments and their use for clinical applications are described, for example, in "Design, Construction, and Two Applications for an Automated Flow-Cell Polarization Fluorometer with Digital Read Out”; R. D. Spencer, F. D. Toledo, B. T. Williams, and N. L. Yoss; Clinical Chemistry, 19/8, pages 838-344 (1973) .
  • Such instruments can rapidly analyze body fluid samples labeled with a fluorescent material.
  • Early instruments of such type required relatively high wattage, intense light sources, such as 200-250 watt mercury or xenon gas discharge lamps, in order to obtain the desired emission radiation from the sample at a signal to noise level ratio sufficient for detection and amplification by a low-noise photomultiplier tube and associated electronics.
  • Popelka discloses an optical system for a fluorescent polarization instrument including a low wattage, low intensity focused light source and a polarizer/liquid crystal combination in the excitation path focusing excitation light of alternate planes of perpendicular polarization onto a fluorescent liquid sample, wherein emitted light from the fluorescent sample is filtered, polarized and focused onto a photomultiplier for processing, and a series of non-reflective baffles are placed around the sample to reduce reflections; monitoring means monitor the excitation light and maintain a substantially constant intensity level focused on the sample, while the low wattage, low intensity light source is provided by a 50 watt tungsten halogen projector lamp.
  • An exemplary prior art instrument schematic is shown in Figure 1.
  • the inventive apparatus satisfies this need by providing a miniaturized, portable apparatus for measuring the fluorescence polarization of a liquid sample.
  • the present inventive subject matter relates to a method for detecting the presence of a biological substance in a test sample, comprising the steps of: (a) collecting a test sample selected from the group consisting of saliva, oral fluid and a bodily fluid from a subject, (b) combining said test sample with a fluorescence-labeled ligand to said biological substance, to produce an assay solution, and (c) measuring the change in fluorescence polarization of said assay solution.
  • the inventive subject matter relates to a method for detecting the presence of an antibody in a test sample selected from the group consisting of saliva, oral fluid and a bodily fluid from a subject, comprising the steps of: (a) combining said test sample with a fluorescence-labeled antigen to said antibody, to produce an assay solution, (b) measuring the fluorescence polarization of a negative control solution of said fluorescence-labeled ligand, a positive control solution of said fluorescence-labeled ligand bound to a known target molecule of said ligand, or both, (c) measuring the fluorescence polarization of said assay solution, and (d) comparing the measured fluorescence polarization of said assay solution with the measured fluorescence polarization of said negative control solution, said positive control solution, or both.
  • the inventive subject matter further relates to an apparatus for measuring the fluorescence polarization of a liquid sample, comprising: (A) a sample compartment (10) for containing said liquid sample; (B) a light emitting diode light source (12) for irradiating said sample compartment (10) with excitation light; (C) a liquid crystal (14) between said light source (12) and said sample compartment (10) , wherein said liquid crystal (14) is switchable to produce different light polarization angles to said sample compartment (10) ; (D) a low power primary detector (16) for detecting a polarized emission light from said sample compartment (10) ; (E) a low power reference detector (18) for detecting an emission light from said sample compartment (10) ; (F) a first emission filter (20) , between said sample compartment (10) and said primary detector (16) ; (G) a second emission filter (22) , between said sample compartment (10) and said reference detector (18) ; (H) a first polarizer (24) between said first emission filter (20) and said primary detector (16)
  • Figure 1 is a drawing which depicts the layout and components of a prior art fluorescence polarization instrument .
  • Figure 2 is a schematic drawing which depicts a fluorescence polarization instrument according to one aspect of the inventive subject matter, with the elements labeled in text form.
  • Figure 3 is a drawing which depicts a fluorescence polarization instrument according to one aspect of the inventive subject matter, with the elements labeled with reference numbers .
  • the term "bodily fluid” as used herein refers to oral rinse expectorant, including oral mucosal transudate and gingival crevicular fluid, urine, sweat, tears, blood, serum, stool, gastric fluid, synovial fluid and phlegm.
  • ELISA as used herein refers to an enzyme- linked immunosorbant assay.
  • RIA as used herein refers to a radioimmunoassay.
  • GCF gingival crevicular fluid
  • oral mucosal transudate refers to a fluid obtained from the buccal mucosal surface juxtaposed between the parotid duct and the gingival crest.
  • oral fluid refers to the oral fluids GCF and OMT, which arise due to hydrostatic pressure of the capillaries and venules associated with the lingual or buccal epithelium, and contain antibodies.
  • salivary gland-produced and other locally produced antibodies refers to a fluid obtained from the buccal mucosal surface juxtaposed between the parotid duct and the gingival crest.
  • oral fluid refers to the oral fluids GCF and OMT, which arise due to hydrostatic pressure of the capillaries and venules associated with the lingual or buccal epithelium, and contain antibodies.
  • saliva refers to the composite of all oral fluids, and which contains a diluted mixture of antibodies from the GCF and OMT, as well as salivary gland-produced and other locally produced antibodies.
  • fluorescence-labeled refers to a molecule or other substance which has an associated fluorochrome, whether linked by chemical conjugation, co- produced (such as a chimeric protein translated from RNA with an integrated fluorescent protein sequence such as Green Fluorescent Protein (“GFP”), for example), or by other means known in the art.
  • GFP Green Fluorescent Protein
  • a reasonably skilled artisan will understand that there are many ways to associate a fluorochrome with a substance. Exemplary fluorochromes are discussed herein.
  • fluorescence polarization refers to the effect in which visible or ultraviolet light is polarized with a filter and shines on part of a molecule, the fluorochrome, which in turn fluoresces, emitting light of a longer wavelength than the original light. FP is discussed in detail herein.
  • anisotropy value refers to the measured optical property of a particle, wherein its scattering behavior varies as it is rotated around its axis of symmetry.
  • anisotropic as used herein refers to properties which vary depending on the direction of measurement .
  • microorganism as used herein refers to any noncellular or unicellular organism, including colonial organisms, such as bacteria, protozoa, viruses, microfungi, rickettsia, and other similar organisms.
  • microorganism as used herein specifically includes dormant forms of microorganisms, such as spores and other reproductive bodies and resting states.
  • handheld as used herein refers to an instrument which is compact enough to be used or operated while being held in the hand or hands .
  • low power as used herein refers to an electronic part or component which has sufficiently low power requirements to be used or operated when powered by a DC battery pack or other portable power source.
  • Immunizations are performed to prevent infections. However, there are exceptions to the general strategy of routine immunization, including tuberculosis and HIV, for which population screening is most often employed. Overlapping strategies apply to some infections, such as Hepatitis B virus for example, for which both screening and vaccination are performed.
  • ELISA, RIA, and immunoprecipitation assays are the previously accepted methods to measure antibody levels. There is a need for rapid and accurate diagnostics, as ELISA, RIA, and immunoprecipitation are non-homogeneous assays, requiring surfaces or centrifugation steps to make the assays work.
  • FP is a homogeneous, solution-based technology that can accurately estimate concentrations of diagnostic markers, drugs and other chemicals, and infectious or biohazardous agents in body fluids or environmental samples within a few seconds to several minutes with single point standardization. FP tests currently used in clinical laboratories measure the levels of drugs, hormones, or antibiotics in blood plasma.
  • FP assays are used to measure different types of binding reactions, to follow proteolytic reactions with and without their inhibitors, and to measure various other enzymatic or receptor binding reactions (See, for example, Nasir M, Jolley M, Fluorescence polarization : An analytical tool for i unoassay and drug discovery, Comb Chem High Throughput Screen, 2:177-90 (1999)).
  • FP is both rapid and accurate, and has no counterpart in ELISA or RIA technology.
  • Current tests also require blood or serum samples, which are diluted to begin serum based assays. In contrast, oral fluids do not generally require dilution.
  • fluorescence polarization methods as diagnostic tools to assay antibodies, proteases, sugars, and nucleic acids in saliva and other oral fluids as well as bodily fluids.
  • Fluorescence polarization provides quantitation of molecular interaction, such as antigen-antibody binding, enzymatic activity, or ribozymic activity of a single, small-volume sample, in real-time, and without prior separation or dilution of components.
  • FP diagnostic procedures are not well known. Applicants expect the widespread use of these assays as non-invasive tests, especially as more compact, simplified fluorescence polarimeters become available.
  • FP technology has already been miniaturized from a 27 cubic foot size, used by hospital laboratories to process nearly 1000 samples per hour, to the present 2 cubic foot commercially available portable instrument, which uses a portable single test tube, or a 96-well or 384-well benchtop instrument .
  • FP based assays are expected to become further miniaturized in both civilian and military sectors. To our knowledge, we are the first to design FP assays for use on oral fluid samples.
  • the inventive subject matter relates to a method for detecting the presence of a biological substance in a test sample, comprising the steps of: (a) collecting a test sample selected from the group consisting of saliva, oral fluid and a bodily fluid from a subject, (b) combining said test sample with a fluorescence-labeled ligand to said biological substance, to produce an assay solution, and (c) measuring the fluorescence polarization of said assay solution.
  • said method comprises the additional steps of: (d) measuring the fluorescence polarization of a negative control solution of said fluorescence- labeled ligand, a positive control solution of said fluorescence-labeled ligand bound to a known target molecule of said ligand, or both, and (e) comparing the measured fluorescence polarization of said assay solution with the measured fluorescence polarization of said negative control solution, said positive control solution, or both.
  • said method comprises alternatively the additional steps of: (d) measuring the background fluorescence polarization reading of said test sample; and (e) measuring the change in the fluorescence polarization of said assay solution.
  • said biological substance is selected from the group consisting of a microorganism, a naturally-occurring or synthetic peptide, a protein, a lipid, a saccharide, a nucleic acid, an RNAase, a DNAase, a ribozyme, a prion, a pharmaceutical, an antigen, an environmental pollutant, a toxin, an abused drug, ' an antibody, and another catalytic entity. It is expected that methods and reagents for the preparation and/or isolation of ligand (s) to a biological substance are known to one of ordinary skill in the art .
  • fluorescence-labeled ligands are also prepared using standard methods and reagents.
  • a prion is a small proteinaceous infectious particle which resists inactivation by procedures that modify nucleic acids, and alone can transmit an infectious disease.
  • Prion diseases are often called spongiform encephalopathies because of the post mortem appearance of the brain with large vacuoles in the cortex and cerebellum.
  • Exemplary prion-caused or prion-associated diseases which may be detected include scrapie in sheep, transmissible mink encephalopathy in mink, chronic wasting disease in muledeer and elk, and bovine spongiform encephalopathy in cows.
  • exemplary environmental pollutants which may be detected include acetone, arsenic, asbestos, barium, benzene, cadmium, carbon monoxide, chlorinated solvents, chlorofluorocarbons, chloroform, cyanide, dichloroethylene, dioxins, endocrine disruptors, ether, ethylbenzene, furans, halons, heavy metals, hydrochlorofluorocarbons, inorganic cyanides, ketones, lead, mercury, methyl tertiary butyl ether, methyl bromide, methyl chloride, nitrogen oxides, organic cyanides, perchloroethylene, phthalates, polychlorinated biphenyls, radionuclides, radon, sulfur oxides, tetrachloroethylene, to
  • Exemplary abused drugs which may be detected include marijuana, cocaine, opiates, amphetamines, phencyclidine, flunitrazepam, hallucinogenic mushrooms, gamma hydroxybutyrate, inhalants, ketamine, lysergic acid diethylamide, 3,4-methylenedioxymethamphetamine, methcathinone, methylphenidate, nicotine, caffeine, and steroids.
  • said biological substance is an antibody to a microorganism selected from the group consisting of a bacteria and a virus.
  • said bacteria is selected from the group consisting of Bacillaceae, Mycobacteriaceae, Yersiniaceae, Rhodospirillaceae,
  • Exemplary bacteria which may be particularly relevant for public health, military, and bioterrorism testing include Bacillus anthracis, Mycobacterium tuberculosis, Yersinia pestis, Clostridium botulinum, Vibrio cholerae, Escherichia coli , Salmonella Typhi , Rancisella tularensis, and Rickettsia prowazekii .
  • said bacteria is selected from the group consisting of Bacillus anthracis and Mycobacterium tuberculosis .
  • said virus is selected from the group consisting of Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis D virus, Hepatitis E virus, human immunodeficiency virus, variola major, Enterovirus, Cardiovirus, Rhinovirus, Aphthovirus, Calicivirus, Orbivirus, Reovirus, Rotavirus, Abibirnavirus, Piscibirnavirus, Entomobirnavirus, Rubivirus, Pestivirus, Flavivirus, Influenzavirus, Pneumovirus, Paramyxovirus, Morbillivirus, Vesiculovirus, Lyssavirus, Coronavirus, Bunyavirus, Herpesvirus, Hantavirus, Alphavirus, Filovirus, and Arenavirus .
  • said virus is selected from the group consisting of Hepatitis A virus,
  • said fluorescence-labeled ligand is selected from the group consisting of an antigen, an antibody, a receptor, and an inhibitor, wherein said ligand is capable of specifically binding to said biological substance.
  • ELISA, RIA, and immunoprecipitation assays are the previously accepted methods to measure antibody levels. There is a need for rapid and accurate diagnostics, as both ELISA and RIA are non-homogeneous assays, requiring surfaces or centrifugation steps to make the assays work.
  • FP has proven very useful for quantifying antigens or antibodies present in relatively large concentrations (micrograms per ml) , while radioisotope-based assays or chemiluminescence-based assays have been more sensitive.
  • recent advances in single-molecule fluorescence polarization will extend the range of FP assays. Therefore, its ruggedness in field settings, low cost, long reagent shelf life, few hazards, and rapidly quantitated results make FP ideal as a diagnostic tool.
  • Applicants have developed a non-invasive oral fluid test in place of drawn blood serum.
  • Oral Fluids have been increasingly recognized as acceptable alternatives to serum for use in diagnostic tests for certain hormones, drugs, antibodies and antigens.
  • diagnostic assays utilizing saliva and other oral fluids appear as entries in the National Library of Medicine MEDLARS database with a frequency of only 1 in
  • oral cavity as an immunological entity has been reviewed with respect to oral diseases and microbiology.
  • the use of oral fluids in diagnostic immunology includes detection of HIV; measles, mumps and rubella; hepatitis A; hepatitis B; and hepatitis C; Helicobacter pylori ; dengue; and Chagas' disease.
  • the US Army Medical Research Development Command supported a contract which published one of the first reports of human salivary antibodies to indigenous bacteria. More recently, the United States Department of Agriculture has sponsored a symposium to advance non- or minimally-invasive technologies to monitor health and nutritional status in the Special Supplemental Nutrition Program for Women Infants and Children using saliva as a diagnostic tool.
  • saliva and oral fluids are a diagnostic media of choice for the future, particularly in those instances were rapid detection in field situations is required.
  • Oral fluids are collected without pain, needle sticks, or religious and social prohibitions, and use of these involves minimal risk or exempt protocols for the use of human subjects.
  • the Department of Defense has made salivary diagnostics a Future Naval Capability, and both the Office of Naval Research and the Military Infectious Disease Research Program of the US Army have funded projects to investigate the use of oral fluids or saliva to diagnose disease or monitor immunization status.
  • Saliva and oral fluid are distinct biochemically. They generally reflect the serum pool, but neither is a passive ultrafiltrate of serum as previously thought .
  • GCF gingival crevicular fluid
  • OHT Oral Mucosal Transudate
  • GCF is obtained by inserting an absorbent paper into the pocket or sulcus of a tooth, between the tooth and gingiva, after clearing the supragingival plaque.
  • OMT is 3-4 fold higher in protein concentration than saliva based on IgG content and is collected, for example, by placing a thick pad against the buccal mucosal surface juxtaposed between the parotid duct and the gingival crest.
  • a current OMT device design uses a salt-impregnated pad that is subsequently treated to release the antibody-containing fluid and retain the glycoproteins on the pad yielding "oral fluid".
  • oral fluid One such oral fluid collection device is commercially available and eliminates the necessity for venipuncture .
  • the GCF concentrations of antibodies are about one- third to one-fourth those found in blood.
  • Oral fluid contains IgG concentrations about two-fold to four-fold that of whole saliva.
  • Oral fluid IgG concentration varies with host factors such as HIV status, tobacco use, time after meals, and bleeding gums.
  • Whole saliva contains a diluted mixture of these serum-derived antibodies, as well as salivary gland-produced and other locally produced antibodies.
  • the levels of immunoglobulins in various fluid pools are shown in Table 2.
  • said fluorescence-labeled ligand is an antigen selected from the group consisting of a ligand to a monoclonal antibody, a ligand to a polyclonal antibody, a ligand to a chimeric antibody, a ligand to a Fab antibody fragment, and a ligand to a (Fab) 2 antibody fragment.
  • the inventive subject matter is more specifically directed to the use of fluorescence polarization-based tests to detect antibodies to anthrax vaccine and to tuberculosis exposure, using saliva, GCF, or OMT as a test fluid.
  • said fluorescence-labeled ligand is an antigen to an antibody to Bacillus anthracis or Mycobacterium tuberculosis .
  • said antibody to Bacillus anthracis is a monoclonal antibody to Bacillus anthracis protective antigen protein.
  • the method is analogous to that for anthrax immunization. We expect that our FP test will detect human antibodies to m. tuberculosis antigens such as CFP-10, ESAT- 6, and MPT-63.
  • said antibody to Mycobacterium tuberculosis is selected from the group specific antigens consisting of CFP-10, ESAT-6, and MPT-63.
  • the complete genome of Mycobacterium tuberculosis has been sequenced, and approximately 4000 genes have been identified in its genome (Cole, et al . , Deciphering the Biology of Mycobacterium tuberculosis from the Complete Genome Sequence, Nature 393:537-544 (1998)). It is expected that an antibody to any one of the gene products of the genes of Mycobacterium tuberculosis may be utilized to produce an antibody within the scope of the inventive subject matter.
  • FP assays offer rational approaches for the detection of antibodies, for example for assays for tuberculosis exposure or infection, or anthrax vaccination status, using saliva, gingival crevicular fluid, or oral fluid.
  • TST Tuberculosis Skin Test
  • Anthrax In an anthrax infection, protective antigen protein, along with two other proteins, lethal factor and edema factor, are released by the bacterium Bacillus anthracis . Protective antigen protein interacts with these other two components to produce the active anthrax toxin.
  • a concise summary of the details of this toxin formation and action is given in a recent paper, Sellman B, Mourez M, Collier R, Dominant -negative mutants of a toxin subuni t : An approach to therapy of anthrax, Science 292:695-7 (2001).
  • the complete DNA and protein sequence of protective antigen protein and its three-dimensional structure from x- ray crystallography are available.
  • AZA Anthrax Vaccine Adsorbed
  • Tuberculosis Applicants have developed a rapid, 1-2 minute, non-invasive diagnostic tool for tuberculosis screening. FP tests are designed to detect tuberculosis- specific antibodies in saliva, oral or bodily fluids, which serve as an indicator of infection with M. tuberculosis . Specific epitopes are known to exist for several immunodominant antigens, defined by monoclonal antibodies used in human serological studies.
  • Small recombinant proteins or peptides containing specific epitopes of early infection stage antigenic tuberculosis proteins form the basis for rapid FP assays to capture patient antibodies.
  • Numerous tuberculosis antigens have been purified from cultures of virulent and non-virulent strains of both Mycobacterium tuberculosis and M. bovis .
  • Useful recombinant proteins have been produced for ELISA tests.
  • Affinity labeled M. tuberculosis proteins suitable for FP, and that react with specific monoclonal antibodies used to titer sera from human tuberculosis patients, have been developed.
  • the total DNA sequence of M. tuberculosis is published, for which an index and references are available.
  • said fluorescence-labeled ligand has a molecular weight less than about 30,000 Daltons . In a more preferred embodiment, said fluorescence- labeled ligand has a molecular weight less than about 20,000 Daltons. In a most preferred embodiment, said fluorescence- labeled ligand has a molecular weight less than about 10,000 Daltons .
  • said fluorescence-labeled ligand comprises a fluorochrome selected from the non-limiting group consisting of 7-AAD, Acridine Orange, Alexa 488, Alexa 532, Alexa 546, Alexa 568, Alexa 594, Aminonapthalene, Benzoxadiazole, BODIPY 493/504, BODIPY 505/515, BODIPY 576/589, BODIPY FL, BODIPY TMR, BODIPY TR, Carboxytetramethylrhodamine, Cascade Blue, a Coumarin, Cy 2 , CY 3 , CY 5 , CY 9 , Dansyl Chloride, DAPI, Eosin, Erythrosin, Ethidium Homodimer II, Ethidium Bromide, Fluorescamine, Fluorescein, FTC, GFP (yellow shifted mutants T203Y, T203F, S65G/S72A) , Ho
  • fluorochromes contemplated within the scope of the inventive subject matter include vitamin B ⁇ , flavin-adenine dinucleotide, and nicotinamide- adenine dinucleotide.
  • said fluorescence-labeled ligand comprises a fluorochrome having a fluorescence emission spectra lifetime greater than about 3 nanoseconds.
  • said fluorescence- labeled ligand comprises a fluorochrome having a fluorescence emission spectra lifetime greater than about 4 nanoseconds .
  • said fluorescence- labeled ligand comprises a fluorochrome having a fluorescence emission spectra lifetime greater than about 5 nanoseconds.
  • the inventive subject matter relates to a method for detecting the presence of an antibody in a test sample selected from the group consisting of saliva, oral fluid and a bodily fluid from a subject, comprising the steps of: (a) combining said test sample with a fluorescence-labeled antigen to said antibody, to produce an assay solution, (b) measuring the fluorescence polarization of a negative control solution of said fluorescence- labeled ligand, a positive control solution of said fluorescence-labeled ligand bound to a known target molecule of said ligand, or both, (c) measuring the fluorescence polarization of said assay solution, and (d) comparing the measured fluorescence polarization of said assay solution with the measured fluorescence polarization of
  • the sample radiates an emission light from which the polarization, P, is determined in accordance with the expression: I (Z) -I (Y) /I (Z) +1 (Y) , where I (Z) is the measured intensity of one polarization component of the emitted light at a first polarization angle when the liquid sample is irradiated with polarized excitation light at the first polarization angle, and where I (Y) is the measured intensity of the polarization component of emitted light at the first polarization angle when the liquid sample is irradiated with polarized excitation light at a second polarization angle perpendicular with respect to said first polarization angle.
  • the inventive subject matter further relates to an apparatus for measuring the fluorescence polarization of a liquid sample, comprising: (A) a sample compartment (10) for containing said liquid sample; (B) a light emitting diode light source (12) for irradiating said sample compartment (10) with excitation light; (C) a liquid crystal (14) between said light source (12) and said sample compartment (10) , wherein said liquid crystal (14) is switchable to produce different light polarization angles to said sample compartment (10) ; (D) a low power primary detector (16) for detecting a polarized emission light from said sample compartment (10) ; (E) a low power reference detector (18) for detecting an emission light from said sample compartment (10) ; (F) a first emission filter (20) , between said
  • said polarizer (24) is a vertical polarizer.
  • said apparatus further comprises: (i) one or more excitation light focusing lense(s) (32) between said light source (12) and said sample compartment (10) , for focusing excitation light to said sample compartment (10) ; (ii) one or more emission light focusing lense(s) (34) between said sample compartment (10) and said reference detector (18) , for focusing emitted light to said reference detector (18) ; and (iii) one or more emission light focusing lense(s) (34) between said sample compartment (10) and said primary detector (16) , for focusing emitted light to said primary detector (16) .
  • said light emitting diode emits red-shifted excitation light.
  • the inventive apparatus represents several improvements over the prior art exemplified by U.S. Patent No. 4,516,856 to Popelka, et al . : 1) in a preferred embodiment, it is miniaturized so that its preferred overall dimensions are not larger that about 7 inches long, about 4 inches wide, and about 2 inches thick.
  • the inventive subject matter is optionally roughly 50 times smaller than the prior art instruments, is easily portable for field use, and is considered to be a handheld instrument.
  • said apparatus is handheld.
  • its light source is a light emitting diode that uses very little power compared to the prior art tungsten halogen lamp, and the light source is red-shifted to avoid the intrinsic background fluorescence contributed by fluorescent biological molecules such as NAD, NADH, NADP and NADPH.
  • it uses a low power consumption silicone photodiode or avalanche diode detector that avoids the high voltage and high power usage of the prior art photomultiplier type detector.
  • it uses a low power consumption controller chip that avoids the high power consumption of the prior art CPU.
  • it uses a battery pack to isolate it from line voltage noise and its design is intrinsically "quiet" compared to the prior art AC line attached machines.
  • the optics block design is simplified by use of a single light polarizing liquid crystal that operates on the off/on mode to give either vertical polarization or no light to the sample excitation light, and which in a preferred embodiment is vertical polarization.
  • the prior art excitation filter is omitted for maximum light throughput.
  • a single horizontal polarizer reading at 90 degrees to the excitation path allows reading of horizontal polarization by the primary detector when the excitation liquid crystal polarizer is off, i.e. the LCD is passing vertically polarized light.
  • a single vertical polarizer is placed ahead of the reference detector at 90 degrees to the excitation path directly across from the primary detector, which collects horizontal fluorescence intensity when the LCD is off, i.e.
  • the sample tube used with said apparatus optionally has a rectangular or substantially circular peripheral cross- sectional configuration, as may be convenient when optimizing the design of the instrument. Further, it is preferred, but not required, that the excitation light be focused substantially at the center of the sample. In addition, it is preferred, but not required, that the emission filter pass emitted light corresponding substantially to the emission band of the fluorescent sample. Optimization of these and other parameters of the inventive apparatus are known to one of ordinary skill in the art .
  • EXAMPLE 1 Fluorescence Polarization Assays in Saliva and Oral Fluids
  • the following example illustrates an exemplary fluorescence polarization assay in saliva or oral fluid, provided according to the present inventive subject matter.
  • FP based assays visible or ultraviolet light is polarized with a filter and shines on part of a molecule, the fluorochrome, that in turn fluoresces, emitting light of longer wavelength whose signal is captured and recorded.
  • FP antigen-antibody binding assays require only the mixing of fluorescent reagent, containing antigen, with a sample containing antibody, in a liquid buffer.
  • FP assays In a rapid diagnostic format, essentially two FP readings are necessary: a base-line, negative control reading and a reading after a specified time.
  • the FP value increases over time as binding of antigen and antibody occurs, until limited by the exhaustion of free antibody or antigen in a test sample.
  • a distinct advantage to FP assays is that they can accommodate somewhat cloudy solutions, such as bacterial suspensions, and tolerate a variation in total fluorescence that may be found in oral fluids .
  • the pH of saliva often drops to pH 4.
  • fluorescein intensity drops dramatically to an extent that would affect FP measurements.
  • other fluorescent dyes that are considerably brighter, more photo-stable, and largely independent of pH can be substituted.
  • fluorescent dyes have relaxation times and absorption and emission spectral characteristics similar to fluorescein, and are available in various chemical forms for labeling.
  • a preferred fluorescent dye has a fluorescence emission spectra lifetime greater than about 3 nanoseconds. In a more preferred embodiment, said fluorescent dye has a fluorescence emission spectra lifetime greater than about 4 nanoseconds. In a most preferred embodiment, said fluorescent dye has a fluorescence emission spectra lifetime greater than about 5 nanoseconds .
  • a fluorescent label, used in the inventive subject matter is any environment-sensitive probe whose fluorescence lifetime changes upon binding to another molecule.
  • Exemplary fluorescent labels include 7-AAD, Acridine Orange, Alexa 488, Alexa 532, Alexa 546, Alexa 568, Alexa 594, Aminonapthalene, Benzoxadiazole, BODIPY 493/504, BODIPY 505/515, BODIPY 576/589, BODIPY FL, BODIPY TMR, BODIPY TR, Carboxytetramethylrhodamine, Cascade Blue, a Coumarin, Cy 2 , CY 3 , CY 5 , CY 9 , Dansyl Chloride, DAPI, Eosin, Erythrosin, Ethidium Homodimer II, Ethidium Bromide, Fluorescamine, Fluorescein, FTC, GFP (yellow shifted mutants T203Y, T203F, S65G/S72A) , Hoechst 33242, Hoechst 33258, IAEDANS, an Indopyras Dye, a Lanthan
  • fluorochromes contemplated within the scope of the inventive subject matter include vitamin B 12 , flavin-adenine dinucleotide, and nicotinamide-adenine dinucleotide.
  • the characteristics of fluorochromes are known to persons of ordinary skill in the art. See, for example, Brismar, et al . , Spectra and Fluorescence Lifetimes of Lissamine Rhodamine, Tetramethylrhodamine Isothiocyanate, Texas Red, and Cyanine 3 . 18 Fluorophores, J. Histochem. Cytochem. 43:699 (1995); Darzynkiewicz, et al .
  • GFP yellow shifted mutants T203Y, T203F, S65G/S72A
  • GFP yellow shifted mutants T203Y, T203F, S65G/S72A
  • Hoechst 33258 3.50
  • Hoechst 33242 2.30 nsec IAEDANS 21.19 nsec
  • Oregon Green 488 4.20 nsec Oregon Green 500 4.10 nsec Oregon Green 514 4.20 nsec Propidium Iodide (DNA) 13.20 nsec pyrene butyrate 115.00 nsec pyrene maleimide 100.00 nsec Rhodamine Green 4.30 nsec Rhodamine 123 4.20 nsec Rhodamine 6G 4.09 nsec SPQ 25.30 nsec
  • said fluorescence-labeled ligand comprises a fluorochrome having a fluorescence emission spectra lifetime greater than about 3 nanoseconds. In a more preferred embodiment, said fluorescence- labeled ligand comprises a fluorochrome having a fluorescence emission spectra lifetime greater than about 4 nanoseconds . In a most preferred embodiment, said fluorescence- labeled ligand comprises a fluorochrome having a fluorescence emission spectra lifetime greater than about 5 nanoseconds. Selection of a fluorescent label for use in particular circumstances is known to the ordinarily skilled artisan, using no more than routine experimentation for the purposes of optimization.
  • EXAMPLE 2 Fluorescent Polarization Assay for Tuberculosis Exposure
  • Vaccination for tuberculosis occurs in most of the world using Bacille Calmette-Guerin (hereinafter "BCG") vaccine, with the exception of the United States.
  • BCG Bacille Calmette-Guerin
  • the TST used for tuberculosis screening requires two visits to the clinic 48-72 hours apart, the first for administration of the test material, and a second visit where the TST is read and recorded by a trained technician or medical professional .
  • an immune system stimulating part of the vaccine will be used to screen immunized individuals.
  • a small antigen preferably less than 10,000 Da molecular weight, is labeled with a fluorescent dye while retaining its antigenic determinant.
  • a small antigen is also the target epitope of immunodominant antibody (s) in the host.
  • an exemplary target epitope is the highly antigenic region in the protective antigen protein molecule identified in the mouse model .
  • suitable protein or peptide antigens of less than about 10 kDa are chosen for use in FP assays. The heterogeneity of the human response to different M.
  • tuberculosis proteins requires the use of immunodominant epitopes from several antigens, for example CFP-10, ESAT-6, MPT-63 and others known to those of ordinary skill in the art.
  • CFP-10, ESAT-6, MPT-63 immunodominant epitopes from several antigens
  • a patient is suspected to have been exposed to tuberculosis.
  • a saliva, oral or a bodily fluid test sample is collected, the test sample is combined with a fluorescence-labeled ligand to tuberculosis bacteria antibodies, the fluorescence polarization of a negative control solution of said fluorescence-labeled ligand and the test sample are measured, and the presence or absence of tuberculosis antibodies is determined.
  • EXAMPLE 3 Fluorescence Polarization Assay for Anthrax Vaccination The following example illustrates an exemplary fluorescence polarization assay for anthrax vaccination, provided according to the present inventive subject matter. Vaccination for anthrax occurs for all Department of
  • the United States Armed Forces anthrax vaccination program currently uses the AVA vaccine. As a result, some subjects have greater than 750 micrograms/ml anti-AVA IgG per ml of serum after five or six subcutaneous doses of vaccine and a titer up to 1:4000 after three doses. Titers of this magnitude mean anti-AVA antibody can be detected in saliva.
  • the serum anthrax enzyme-linked immunosorbant assay is the current gold standard diagnostic test, but requires a laboratory setting, trained personnel, specialized equipment, and up to 8 hours to quantitate the results.
  • Applicants have developed an FP assay for anthrax immunization which is straightforward and based on the fact that individuals are immunized with a prescribed quantity of antigen, at defined intervals, and in the same manner, by injection with the same antigen.
  • the sequence, x-ray structure, and biological functions of protective antigen protein have been published, which provides relevant peptides to label for FP assays. Thus, it is necessary to determine the immunization status of patient in need of immunity.
  • a saliva, oral or bodily fluid test sample is collected, the test sample is combined with a fluorescence-labeled ligand to anthrax bacteria antibodies, the fluorescence polarization of a negative control solution of said fluorescence-labeled ligand and the test sample are measured, and the presence or absence of anthrax antibodies is determined.
  • Homocysteine Metabolites are involved in the transsulfuration and transmethylation pathways, and are believed to serve as markers for cardiovascular diseases, vascular stroke, tissue damage resulting from cell damage or cell death due to necrosis or apoptosis, neuronal mediated tissue damage or diseases, neural tissue damage resulting from ischemia and reperfusion injury, neurological disorders and neurodegenerative diseases, age-related macular degeneration, AIDS and other immune diseases, arthritis, atherosclerosis, cachexia, cancer, degenerative diseases of skeletal muscle involving replicative senescence, diabetes, head trauma, immune senescence, inflammatory bowel disorders, muscular dystrophy, osteoarthritis, osteoporosis, chronic pain, acute pain, neuropathic pain, nervous insult, peripheral nerve injury, renal failure, retinal ischemia, s
  • a patient is suspected to suffer from a disorder marked by the presence or absence of a homocysteine metabolite.
  • a saliva, oral or bodily fluid sample is collected, the test sample is combined with a fluorescence- labeled homocysteine metabolite and a high affinity antibody to the homocysteine metabolite, the fluorescence polarization of a negative control solution of said fluorescence-labeled ligand and the test sample are measured, and the presence or absence of the homocysteine metabolite is determined in a competition assay.
  • Test Time An instrumental assay that will give a quantitative measurement, and simple yes/no indication, of a specific marker antibody in saliva or oral fluids for detection of Mtb and for evaluation of immune response to the anthrax vaccine. Results will develop under 5 minutes compared to the 1-2 days for Enzyme-Linked Immunosorbent Assay (ELISA) testing or the 3 -day turnaround time for the current tuberculin skin test (TST) .
  • Power Source Hand-held instrument may be operated with batteries or with portable electrical sources if available.
  • This feature sets FP apart from real-time PCR or other fluorescence-based micro- array systems .
  • d. Required Training; Test may be administered by minimally trained personnel .
  • Scope of Use May be used in far-forward field settings to monitor exposure to infectious diseases or biological warfare agents, and also in general clinical settings to screen for disease exposure.
  • Anthrax antibody test can be used to monitor vaccine efficacy in immunized DOD personnel and emergency first responders in this and other countries .
  • a tuberculosis (TB) assay will be used to screen all DOD personnel, civilian medical and laboratory workers, others at risk and major immigration points of entry. Third-world countries are sorely in need of a rapid, inexpensive TB screening field test . Further development could lead to a rapid, hand-held detection device for biological warfare agents and other militarily relevant diseases.
  • Prototype II is to be designed and built to perform at least as well as existing FP instruments. Final specifications are to be achieved in later versions of the hand-held instrument, and under separate contract.

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Abstract

La présente invention a trait à un procédé pour la détection de la présence d'une substance biologique d'intérêt dans un échantillon d'essai de la salive ou de liquide buccal, comprenant la combinaison dudit échantillon d'essai avec un ligand marqué par fluorescence à ladite substance biologique et la détection d'une modification dans la polarisation de fluorescence dudit échantillon d'essai produite par la liaison dudit ligand marqué par fluorescence à ladite substance biologique. Dans un aspect de l'invention, ledit procédé comprend des étapes additionnelles pour la comparaison de la polarisation de fluorescence dudit échantillon d'essai avec la polarisation de fluorescence d'une solution témoin. L'invention a également trait à un appareil portatif miniaturisé pour la mesure de la polarisation de fluorescence d'un échantillon liquide.
PCT/US2003/032736 2003-11-05 2003-11-05 Instruments et procedes de polarisation de fluorescence pour la detection d'exposition aux matieres vivantes par le dosage immunologique par la polarisation de fluorescence de la salive, de liquides buccaux ou organiques WO2005054854A1 (fr)

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CN102519926A (zh) * 2011-12-09 2012-06-27 厦门大学 罗丹明b衍生物在亚硝酸根离子检测中的应用
CN102778568A (zh) * 2012-04-24 2012-11-14 万里明 一种检测发热伴血小板减少综合征病毒总抗体elisa试剂盒的制备和应用
CN102809653A (zh) * 2012-04-24 2012-12-05 万里明 一种检测新布尼亚病毒抗原的elisa试剂盒的制备和应用
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WO2014059281A1 (fr) * 2012-10-11 2014-04-17 The Regents Of The University Of Michigan Dosage de polarisation à fluorescence permettant de détecter des molécules de poids moléculaire élevé dans des fluides biologiques
CN110261607A (zh) * 2019-05-28 2019-09-20 北京市动物疫病预防控制中心 用于检测禽白血病毒的荧光偏振免疫分析试剂盒及方法
CN113295656A (zh) * 2020-02-22 2021-08-24 青岛科技大学 一种细胞内As3+、Pb2+和Hg2+的同时荧光成像方法

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WO2012059786A1 (fr) 2010-11-03 2012-05-10 Reametrix Inc. Système de mesure pour la détection de fluorescence, et procédé associé
CN102519926A (zh) * 2011-12-09 2012-06-27 厦门大学 罗丹明b衍生物在亚硝酸根离子检测中的应用
CN102778568A (zh) * 2012-04-24 2012-11-14 万里明 一种检测发热伴血小板减少综合征病毒总抗体elisa试剂盒的制备和应用
CN102809653A (zh) * 2012-04-24 2012-12-05 万里明 一种检测新布尼亚病毒抗原的elisa试剂盒的制备和应用
CN102809649A (zh) * 2012-04-24 2012-12-05 万里明 一种检测发热伴血小板减少综合征病毒抗体IgM的ELISA试剂盒的制备和应用
WO2014059281A1 (fr) * 2012-10-11 2014-04-17 The Regents Of The University Of Michigan Dosage de polarisation à fluorescence permettant de détecter des molécules de poids moléculaire élevé dans des fluides biologiques
CN110261607A (zh) * 2019-05-28 2019-09-20 北京市动物疫病预防控制中心 用于检测禽白血病毒的荧光偏振免疫分析试剂盒及方法
CN113295656A (zh) * 2020-02-22 2021-08-24 青岛科技大学 一种细胞内As3+、Pb2+和Hg2+的同时荧光成像方法

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