WO2016191507A1 - Use of fluorescence for the quick and easy determination of s-adenosylmethionine, s-adenosylhomocysteine and homocysteine - Google Patents
Use of fluorescence for the quick and easy determination of s-adenosylmethionine, s-adenosylhomocysteine and homocysteine Download PDFInfo
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- WO2016191507A1 WO2016191507A1 PCT/US2016/034202 US2016034202W WO2016191507A1 WO 2016191507 A1 WO2016191507 A1 WO 2016191507A1 US 2016034202 W US2016034202 W US 2016034202W WO 2016191507 A1 WO2016191507 A1 WO 2016191507A1
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- 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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54366—Apparatus specially adapted for solid-phase testing
- G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
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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
- A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
- A61K49/0058—Antibodies
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
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- 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
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
- G01N2333/4701—Details
- G01N2333/4737—C-reactive protein
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/50—Determining the risk of developing a disease
Definitions
- the present invention relates to the use of fluorescent materials such as quantum dots, fluorescent lanthanide metal chelate complexes, and colloidal microspheres in the immunological determination of S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), and C-reaction protein (CRP).
- SAM S-adenosylmethionine
- SAH S-adenosylhomocysteine
- CRP C-reaction protein
- the invention further relates to the use of a photochemical method for the determination of homocysteine (HCy) in a dry strip and the combinations of both methods.
- the invention further relates to the quantitative measurement of SAM, SAH and HCy simultaneously using fluorescence-optical density devices that read immunological fluorescence and photochemical colors simultaneously for quick and convenient reporting.
- the present invention further relates to assays of clinical samples.
- This invention also relates to fluorescent compounds useful as indicator molecules for detecting the presence or concentration of an analyte in a medium, such as a liquid, and to methods for achieving such detection. More particularly, the invention relates to fluorescent lanthanide metal chelate complexes and their use as indicator molecules for detecting the presence or concentration of an analyte such as SAM, SAH and CRP in a medium, including a liquid medium such as a biological fluid or other biological samples.
- the invention additionally relates to the development of an assay system capable of discriminating mixtures of cardiovascular risk factor analytes for the prediction of coronary heart disease and stroke.
- the invention is also directed to the determination of SAM and SAH, HCy and CRP to determine cardiac care and cardiac prognosis.
- the instant invention is also particularly useful in the field of in vitro diagnosis (IVD) and point-of-care
- organic dye fluorophores have been the favored materials and have the capability to be modified with a range of materials, enabling targeted binding to a wide range of biological structures based on known affinities and chemistries.
- an activating light of a given wavelength is used to excite the dye, from which it responds by fluorescently emitting a characteristic light radiation specific to the properties of the organic dye employed.
- traditional organic dyes have numerous limitations when used to tag biological materials.
- Quantum dots are nanometer sized semiconductor, light-emitting crystals, spherical in shape and have superior fluorescent properties to organic dyes. Quantum dots are generally synthesized with Type II- VI (e.g. CdSe, CdTe, CdS and ZnSe) or Type III-V (e.g. InP and InAs) column elements from the periodic table and can be capped with numerous shells, layers or molecules to modify their physical properties, such as for surface functionalization.
- Type II- VI e.g. CdSe, CdTe, CdS and ZnSe
- Type III-V e.g. InP and InAs
- Quantum dots are emerging as the new biological label with applications and properties superior to traditional fluorescent proteins and organic dyes.
- the first shortcoming is that the peak emission of organic dyes cannot be altered—each dye corresponds to a different molecule with a different pre-set emission wavelength, or fluorescent color, that is set by nature.
- the second shortcoming is the narrow absorption pattern of organic dyes— dyes tend to display absorption peaks that are not always in convenient regions of the spectrum, making the excitation of various organic dyes challenging and costly.
- the third shortcoming is that of uneven absorption and emission peaks— organic dyes have a tendency to produce "shoulders" in the geometry of their emission and absorption peaks, which is a major disadvantage in applications that require Gaussian type emission patterns to work correctly.
- quantum dots can be made to emit light at any wavelength in the visible and infrared ranges, and can be inserted almost anywhere, including in liquid solutions, dyes, paints, epoxies, and sol- gels. Furthermore, quantum dots can be attached to a variety of surface ligands, and inserted into a variety of organisms in vivo or in vitro.
- bioconjugate a bio-molecular conjugates
- functional quantum dot which are used in labeling, detection and imaging applications to attach or bind a quantum dot to a biological material based on specific chemical or biological affinity.
- bioconjugate employ a variety of chemistries to water-soluble quantum dots from which several cross-linker molecules can be coupled to enable the attachment of the primary functional biomaterial.
- Other examples of bioconjugate techniques enabling the attachment of various materials to quantum dots are known to those skilled in the art.
- bioconjugation methods are classified into mechanisms using: (1) Biofunctional linkages, (2) Electrostatic attraction, (3) Hydrophobic attraction, (4) Silanization, and (5) Nanobead linkages.
- methods employing bioconjugative techniques are polyethylglycol modification of the underlying carboxyl quantum dots, and optimization of the surface loading of amino groups for high conjugation efficiency and specificity.
- Another example is modifying the quantum dots with peptides through the amino or carboxyl groups at the terminus, or using other residues, small molecules, proteins, or nucleic acids, and other methods known to those skilled in the art.
- schemes used for the conjugation of antibodies to quantum dots are based on well-known chemistries using the fast and efficient coupling of thiols to maleimide groups, with reactive groups such as primary amines, alcohols, carboxylic acids and thiols used to link the antibodies to the quantum dots.
- Quantum dots represent a marked increase in performance over standard organic dyes, because they can be tuned to absorb or emit at any visible or infrared wavelength and can be fabricated into a great variety of forms and media, eliminating completely the shortcomings of dyes. These unique abilities are due to their very small sizes (typically between 1-10 nm in diameter). The small size and its direct relationship to fluorescence also allows for enormous versatility and flexibility of form, letting phosphors match whatever shape their underlying light- emitting diode (LED) needs to assume.
- LED light- emitting diode
- quantum dot bands When light impinges on quantum dots, it encounters discretized energy bands specific to the quantum dot.
- the discretized nature of quantum dot bands means that the energy separation between the valence and conduction bands (the bandgap) can be altered with the addition or the subtraction of just one atom—making for a size dependent bandgap.
- Pre-determining the size of the quantum dots fixes the emitted photon wavelength at the appropriate customer-specified color, even if it is not naturally occurring— an ability limited only of quantum dots.
- certain rare-earth metal chelates emit visible light upon irradiation with UV light and different forms of visible light (e.g., violet or blue light), an emission which is characterized by the chelated cation.
- Some lanthanide ions such as those of europium (Eu ), Samarium (Sm ), terbium (Tb ), and to a lesser extent dysprosium (Dy ) and neodymium (Nd 3+ ), exhibit typical fluorescence characterized by the ion, especially when chelated to suitable excitation energy mediating organic ligands.
- the fluorescent properties of these compounds— long Stokes' shift, narrow band-type emission lines, and unusually long fluorescence lifetimes— have made them attractive candidates for fluorescent immunoassays and time-resolved fluorometric techniques.
- the major emission lines of these fluorescent lanthanide chelates are formed from a transition called hypersensitive transition and are around 613-615 nm with Eu 3+ , 545 (and 490) nm with Tb 3+ , 590 and 643 nm with Sm 3+ , and 573 with Dy 3+ .
- Radiation is typically absorbed by the chelates at a wavelength characteristic of the organic ligand and emitted as a line spectrum characteristic of the metal ion because of an intramolecular energy transfer from the ligand to the central metal ion.
- the organic ligand absorbs energy and is raised or excited from its singlet ground state, So, to any one of the vibrational multiplets of the first singlet excited state, Si, where it rapidly loses its excess vibrational energy.
- Si first singlet excited state
- Fluorescent europium chelates are known to exhibit large Stokes shifts ( ⁇ 29 nm) with no overlap between the excitation and emission spectra and very narrow (10-nm bandwidth) emission spectra at 615 nm.
- the long fluorescence lifetimes (measurable in microseconds instead of the nanosecond lifetimes measurable for conventional fluorophores) of the chelates help filter out noise and other interference having a low fluorescent lifetime.
- the long fluorescent lifetimes thus permit use of the chelates for microsecond time-resolved fluorescence measurements, which further reduce the observed background signals. Additional advantages of using europium chelates include that europium chelates are not quenched by oxygen.
- Radioimmunoassay sensitivity limits the assay to measurements of concentration of 10 "12 M, and more often only in the 10 "8 to 10 "10 M range.
- radiolabels suffer from the drawbacks of short half life and handling hazards.
- a sample containing a fluorescent species to be analyzed is irradiated with light of known spectral distribution within the excitation spectrum of the target fluorescent species.
- the intensity of the resulting characteristic emission spectrum of the fluorescent target molecules is determined and is related to the number of target molecules.
- Time resolution offers an independent means of isolating the specific fluorescent signal of interest from nonspecific background fluorescence. Time resolution is possible if the label has much longer-lived fluorescence than the background, and if the system is illuminated by an intermittent light source such that the long-lived label is measurable during the dark period subsequent to the decay of the short-lived background.
- Certain fluorescent molecules have been commonly used as tags for detecting an analyte of interest.
- Organic fluorescent dyes are typically used in this context.
- there are chemical and physical limitations to the use of such dyes One of these limitations is the variation of excitation wavelengths of different colored dyes. As a result, the simultaneous use of two or more fluorescent tags with different excitation wavelengths requires multiple excitation light sources.
- a drawback of organic dyes is the deterioration of fluorescence intensity upon prolonged and/or repeated exposure to excitation light. This fading, called photobleaching, is dependent on the intensity of the excitation light and the duration of the illumination. In addition, conversion of the dye into a nonfluorescent species is irreversible. Furthermore, the degradation products of dyes are organic compounds which may interfere with the biological processes being examined.
- spectral overlap exists from one dye to another. This is due, in part, to the relatively wide emission spectra of organic dyes and the overlap of the spectra near the tailing region. Few low molecular weight dyes have a combination of a large Stokes shift, which is defined as the separation of the absorption and emission maxima, and high fluorescence output. In addition, low molecular weight dyes may be impractical for some applications because they do not provide a bright enough fluorescent signal.
- Figure 1 illustrates two embodiments of the lateral flow immunochromatographic test strips of the invention.
- Figure 2 shows the standard curve for the SAM fluorescent immunochromatographic test strip of example 1 invention.
- Figure 3 shows the standard curve for the SAH fluorescent immunochromatographic test strip of example 2.
- Figure 4 shows the standard curve for a CRP fluorescent immunochromatographic strip of example 4.
- FIG. 5 illustrates the flow cytometry (FCM) results from cells double stained with
- Alexa Fluor 647 conjugated anti-SAM 1 18-6 antibody (Cat# MAF00201, Arthus Biosystems, VA) at 4 ⁇ g/ml.
- Figure 6 shows FCM results from cells double stained with Alexa Fluor 488 conjugated anti-SAH antibody 301-3 (Cat# MAF00301, Arthus Biosystems, VA) at 45 ⁇ ⁇ .
- FIG. 7 illustrates the Laser Scan Confocal Microscopy (LSCM) results of L02 and
- HepG2 cells that were cultured for 40h and then stained with the SAMe fluorescence labelled anti-SAM and anti-SAH antibodies of the invention.
- Figure 8 shows simple diagrams illustrating how the two formats of TR-FRET technology may be used to quantitatively measure SAM and SAH using the bio-conjugates described in this invention.
- the present invention provides quantum dots having attached thereto an antibody selected from the group consisting of anti-SAM, anti-SAH, anti-HCy and anti-CRP antibodies.
- the invention also provides an immunochromatographic strip having incorporated therein quantum dots covalently bonded to anti-SAM, anti-SAH, anti-HCy and anti-CRP antibodies.
- the present invention is also directed to the use of quantum dots based immunoassays in combination with chemical methods to measure three closely related bio-molecules in a metabolic pathway simultaneously.
- the invention is also a method of determining risk of experiencing a major adverse cardiac event in a patient, within one year from presentation of at least one symptom of acute coronary syndrome comprising the steps of: (a) obtaining a test sample from said patient; (b) determining the amount of at SAM, SAH, HCy and optionally C reactive protein using a quantum dot based assay; (c) calculating the MI in said test sample; and c) comparing the amount of said four biomarkers to biomarker reference standards, wherein said risk is determined by results of said comparison.
- the invention further provides a method for assaying homocysteine in a sample, said method comprising the steps of: (i) contacting said sample with a homocysteine-converting enzyme that produces SAH and (ii) then measuring SAH using an immunochromatographic strip as described above.
- the invention also provides a lateral flow immunoassay test strip for detecting and quantifying the presence of SAM, and SAH alone or simultaneously in a fluid sample, comprising a membrane strip coated with a SAM or SAH-protein conjugate on a test line, and particles conjugated with their antibodies respectively.
- the invention is also directed to a fluorescent lanthanide chelate conjugated to an antibody selected from the group consisting of anti-SAM, anti-SAH, and anti-CRP antibodies and use of the conjugates in making immunochromatographic strips.
- the invention further provides a method of detecting and quantifying SAM and SAH in a sample, comprising: (a) providing a sample containing or suspected of containing SAM and SAH on a solid support; (b) combining said sample with a semiconductor nanocrystal anti SAM antibody and anti SAH antibody conjugate, wherein said combining is performed under conditions that allow formation of a complex comprising said conjugate and said SAM and SAH, when present; (c) removing any unbound conjugate; and (d) detecting the presence of the complex, if present, by monitoring a spectral emission mediated by the semiconductor nanocrystal in the complex, wherein the emission indicates the presence and quantity of SAM and SAH in the sample.
- the invention further relates to the use of a SAM immuno-chromatographic strip to determine and monitor the levels of SAM in patients afflicted with a disease selected from the group consisting of depression, osteoarthritis, liver and gall bladder diseases and then proposing a therapeutic regimes for administering S-Adenosyl-methionine.
- the invention also provides a method for determining the effectiveness of a diet program for administration to a patient having at least one diet-responsive condition comprising the steps of: (a) selecting a plurality of patients, each having at least one diet-responsive condition; (b) identifying in said patient the body mass index and at least one other quantifiable indicator selected from methylation index and SAM levels for each of said diet-responsive conditions and measuring said at least one indicator for each of said patients during a baseline period; (c) monitoring each of said patients during said baseline period to determine a baseline quality of life; (d) dividing said plurality of patients randomly between a first group and a second group; (e) administering said diet program to each of said patients in said first group during an intervention period; (f) maintaining each of said patients in said second group on a control diet with known beneficial effects on said at least one diet-responsive condition during said intervention period; and (g) monitoring said at least one indicator of each of said conditions for each of said patients after said intervention period.
- semiconductor nanocrystal and “quantum dot” are used interchangeably herein and refer to an inorganic crystallite between about 1 nm and about 1000 nm in diameter or any integer or fraction of an integer therebetween, preferably between about 2 nm and about 50 nm or any integer or fraction of an integer therebetween, more preferably about 2 nm to about 20 nm (such as about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nm).
- a semiconductor nanocrystal is capable of emitting electromagnetic radiation upon excitation (i.e., the semiconductor nanocrystal is luminescent) and includes a "core" of one or more first semiconductor materials, and may be surrounded by a “shell” of a second semiconductor material.
- a semiconductor nanocrystal core surrounded by a semiconductor shell is referred to as a "core/shell” semiconductor nanocrystal.
- the surrounding "shell” material will preferably have a bandgap energy that is larger than the bandgap energy of the core material and may be chosen to have an atomic spacing close to that of the "core” substrate.
- the core and/or the shell can be a semiconductor material including, but not limited to, those of the group II- VI (ZnS, ZnSe, ZnTe, CdS, CdSe, CdTe, HgS, HgSe, HgTe, MgS, MgSe, MgTe, CaS, CaSe, CaTe, SrS, SrSe, SrTe, BaS, BaSe, BaTe, and the like) and III-V (GaN, GaP, GaAs, GaSb, InN, InP, InAs, InSb, and the like) and IV (Ge, Si, and the like) materials, and an alloy or a mixture thereof.
- group II- VI ZnS, ZnSe, ZnTe, CdS, CdSe, CdTe, HgS, HgSe, HgTe, MgS, MgSe, MgTe, CaS, CaSe,
- a semiconductor nanocrystal is, optionally, surrounded by a "coat" of an organic capping agent.
- the organic capping agent may be any number of materials, but has an affinity for the semiconductor nanocrystal surface.
- the capping agent can be an isolated organic molecule, a polymer (or a monomer for a polymerization reaction), an inorganic complex, and an extended crystalline structure.
- the coat is used to convey solubility, e.g., the ability to disperse a coated semiconductor nanocrystal homogeneously into a chosen solvent, functionality, binding properties, or the like.
- the coat can be used to tailor the optical properties of the semiconductor nanocrystal. Methods for producing capped semiconductor nanocrystals are discussed further below.
- antibody as used herein includes antibodies obtained from both polyclonal and monoclonal preparations, as well as, hybrid (chimeric) antibody and, any functional fragments obtained from such molecules, wherein such fragments retain specific-binding properties of the parent antibody molecule.
- the term "monoclonal antibody” refers to an antibody composition having a homogeneous antibody population.
- the term is not limited regarding the species or source of the antibody, nor is it intended to be limited by the manner in which it is made.
- the term encompasses antibodies obtained from murine hybridomas, as well as human monoclonal antibodies obtained using human rather than murine hybridomas.
- a semiconductor nanocrystal is "linked” or “conjugated” to, or “associated” with, a specific-binding molecule or member of a binding pair when the semiconductor nanocrystal is chemically coupled to, or associated with the specific-binding molecule.
- these terms intend that the semiconductor nanocrystal may either be directly linked to the specific-binding molecule or may be linked via a linker moiety, such as via a chemical linker described below.
- the terms indicate items that are physically linked by, for example, covalent chemical bonds, physical forces such van der Waals or hydrophobic interactions, encapsulation, embedding, or the like.
- nanocrystals can be conjugated to molecules that can interact physically with biological compounds such as cells, proteins, nucleic acids, subcellular organelles and other subcellular components.
- nanocrystals can be associated with biotin which can bind to the proteins, avidin and streptavidin
- a biological sample refers to a sample of isolated cells, tissue or fluid, including but not limited to, for example, plasma, serum, spinal fluid, semen, lymph fluid, the external sections of the skin, respiratory, intestinal, and genitourinary tracts, tears, saliva, milk, blood cells, tumors, organs, and also samples of in vitro cell culture constituents (including but not limited to conditioned medium resulting from the growth of cells in cell culture medium, putatively virally infected cells, recombinant cells, and cell components).
- a "small molecule” is defined as including an organic or inorganic compound either synthesized in the laboratory or found in nature. Typically, a small molecule is characterized in that it contains several carbon-carbon bonds, and has a molecular weight of less than 1500 grams/Mol.
- the present invention provides a composition that can provide information about a biological state or event associated with S-adenosylmethionine, S- adenosylhomocysteine and homocysteine and C-reactive protein.
- the composition by way of example can detect the presence or amounts of the above molecules.
- the composition is comprised of a fluorescent semiconductor nanocrystal (also known as a Quantum Dot) having a characteristic spectral emission, which is tunable to a desired energy by selection of the particle size, size distribution and composition of the semiconductor nanocrystal.
- the composition further comprises a compound i.e., an antibody against SAM or SAH associated with the semiconductor nanocrystal that has an affinity for the biological target.
- the composition interacts or associates with a biological target due to the affinity of the compound with the target. Location and nature of the association can be detected by monitoring the emission of the semiconductor nanocrystal.
- the composition is introduced into an environment containing a biological target and the composition associates with the target.
- the compositiomtarget complex may be spectroscopically view or otherwise detected, for example, by irradiation of the complex with an excitation light source.
- the semiconductor nanocrystal emits a characteristic emission spectrum which can be observed and measured, for example, spectroscopically.
- the emission spectra of a population of semiconductor nanocrystals have linewidths as narrow as 25-30 nm, depending on the size distribution heterogeniety of the sample population, and lineshapes that are symmetric, gaussian or nearly gaussian with an absence of a tailing region.
- the combination of tunability, narrow linewidths, and symmetric emission spectra without a tailing region provides for high resolution of multiply-sized nanocrystals, e.g., populations of monodisperse semiconductor nanocrystals having multiple distinct size distributions, within a system and enables researchers to examine simultaneously a variety of biological moieties, e.g., target analytes, tagged with nanocrystals.
- the range of excitation wavelengths of the nanocrystals is broad and can be higher in energy than the emission wavelengths of all available semiconductor nanocrystals. Consequently, this allows the simultaneous excitation of all populations of semiconductor nanocrystals in a system having distinct emission spectra with a single light source, usually in the ultraviolet or blue region of the spectrum.
- Semiconductor nanocrystals are also more robust than conventional organic fluorescent dyes and are more resistant to photobleaching than the organic dyes. The robustness of the nanocrystal also alleviates the problem of contamination of the degradation products of the organic dyes in the system being examined. Therefore, the present invention provides uniquely valuable tags for detection of biological molecules and the interactions they undergo.
- the composition comprises semiconductor nanocrystals associated with molecules that can physically interact with biological compounds.
- molecules include ones that can bind to proteins, nucleic acids, cells, subcellular organelles, and other biological molecules.
- the compound used in the composition of the present invention preferably has an affinity for a biological target.
- the compound has a specific affinity for a biological target. The affinity may be based upon any inherent properties of the compound, such as without limitation, van der Waals attraction, hydrophilic attractions, ionic, covalent, electrostatic or magnetic attraction of the compound to a biological target.
- biological target is meant any moiety, compound, cellular or sub-cellular component which is associated with biological functions.
- the biological target includes without limitation proteins, nucleic acids, cells, subcellular organelles and other biological moieties.
- semiconductor nanocrystals have radii that are smaller than the bulk exciton Bohr radius and constitute a class of materials intermediate between molecular and bulk forms of matter. Quantum confinement of both the electron and hole in all three dimensions leads to an increase in the effective band gap of the material with decreasing crystallite size. Consequently, both the optical absorption and emission of semiconductor nanocrystals shift to the blue (higher energies).
- the optical properties of quantum dots are primarily dictated by their physical size and chemistry.
- electromagnetic radiation having a wavelength within the visible light and infrared portions of the spectrum will excite quantum dots.
- the absorption spectrum of a quantum dot appears as a series of overlapping peaks that become increasingly larger at decreasingly shorter wavelengths. Each peak corresponds to an energy transition between discrete electron-hole energy states (exciton) within the quantum dot.
- the size of a quantum dot and the difference between its energy states are inversely proportional. Thus, the difference between energy states of larger quantum dots is smaller than the difference between energy states of smaller quantum dots.
- the size of the quantum dots of the invention are in the range of 2-10 nm.
- the wavelength of electromagnetic radiation emitted by a quantum dot may be tailored by selecting the material from which the quantum dot is to be synthesized and the size to which the quantum dot is to be synthesized.
- known quantum dots may emit electromagnetic radiation (e.g., light) having a wavelength from about 490 nm (blue) to about 705 nm (red).
- Quantum dots have high quantum yields and resist photobleaching; their use therefore providing for very sensitive fluorescent biological assays.
- Different types of quantum dots are excited when exposed to different ranges of wavelengths of electromagnetic radiation.
- Currently available quantum dots may be excited by electromagnetic radiation having wavelengths as low as about 300 nm and as high as about 2,300 nm.
- the markers within reagent solution have a Stoke's shift of about 50 nm or greater (e.g., the difference between excitation of the marker at about 658 nm and emission at about 703 nm) or even of about 100 nm or greater (e.g., quantum dots that are excited at about 405 nm may emit radiation having a wavelength of about 530 nm).
- each semiconductor nanocrystal distribution Upon exposure to a primary light source, each semiconductor nanocrystal distribution is capable of emitting energy in narrow spectral linewidths, as narrow as 12 nm to 60 nm, and with a symmetric, nearly Gaussian line shape, thus providing an easy way to identify a particular semiconductor nanocrystal.
- the linewidths are dependent on the size heterogeneity, i.e., monodispersity, of the semiconductor nanocrystals in each preparation.
- semiconductor nanocrystal distributions with larger linewidths in the range of 35 nm to 60 nm can be readily made and have the SAM physical characteristics as semiconductor nanocrystals with narrower linewidths.
- the present invention uses a composition comprising semiconductor nanocrystals associated with a specific-binding molecule or affinity molecule, such that the composition can detect the presence and/or amounts of biological and chemical compounds, detect interactions in biological systems, detect biological processes, detect alterations in biological processes, or detect alterations in the structure of biological compounds.
- semiconductor nanocrystal conjugates comprise any molecule or molecular complex, linked to a semiconductor nanocrystal, that can interact with a biological target, to detect biological processes, or reactions, as well as alter biological molecules or processes.
- the molecules or molecular complexes or conjugates physically interact with a biological compound.
- the interactions are specific.
- the interactions can be, but are not limited to, covalent, noncovalent, hydrophobic, hydrophilic, electrostatic, van der Waals, or magnetic.
- these molecules are small molecules, proteins, or nucleic acids or combinations thereof.
- Semiconductor nanocrystal conjugates can be made using techniques known in the art.
- moieties generally used in the production of semiconductor nanocrystals may be readily displaced and replaced with other functional moieties, including, but not limited to carboxylic acids, amines, aldehydes, and styrene to name a few.
- functional moieties including, but not limited to carboxylic acids, amines, aldehydes, and styrene to name a few.
- factors relevant to the success of a particular displacement reaction include the concentration of the replacement moiety, temperature and reactivity.
- any functional moiety may be utilized that is capable of displacing an existing functional moiety to provide a semiconductor
- nanocrystal with a modified functionality for a specific use is a nanocrystal with a modified functionality for a specific use.
- a preferred embodiment of the present invention utilizes semiconductor nanocrystals that are solubilized in water.
- the outer layer includes a compound having at least one linking moiety that attaches to the surface of the particle and that terminates in at least one hydrophilic moiety.
- the linking and hydrophilic moieties are spanned by a hydrophobic region sufficient to prevent charge transfer across the region.
- the hydrophobic region also provides a "pseudo-hydrophobic" environment for the nanocrystal and thereby shields it from aqueous surroundings.
- the hydrophilic moiety may be a polar or charged (positive or negative) group. The polarity or charge of the group provides the necessary hydrophilic interactions with water to provide stable solutions or suspensions of the semiconductor nanocrystal.
- hydrophilic groups include polar groups such as hydroxides (—OH), amines, polyethers, such as polyethylene glycol and the like, as well as charged groups, such as carboxylates (— C0 2 " ), sulfonates (S0 3 " ), phosphates (-- P0 4 2” and ⁇ P0 3 2” ), nitrates, ammonium salts ( ⁇ NH 4 + ), and the like.
- polar groups such as hydroxides (—OH), amines, polyethers, such as polyethylene glycol and the like, as well as charged groups, such as carboxylates (— C0 2 " ), sulfonates (S0 3 " ), phosphates (-- P0 4 2" and ⁇ P0 3 2” ), nitrates, ammonium salts ( ⁇ NH 4 + ), and the like.
- a water-solubilizing layer is found at the outer surface of the overcoating layer.
- a displacement reaction may be employed to modify the semiconductor nanocrystal to improve the solubility in a particular organic solvent.
- a particular solvent or liquid such as pyridine
- the surface can be specifically modified with pyridine or pyridine-like moieties to ensure solvation.
- the surface layer may also be modified by displacement to render the semiconductor nanocrystal reactive for a particular coupling reaction.
- displacement of certain moieties with a group containing a carboxylic acid moiety enables the reaction of the modified semiconductor nanocrystals with amine containing moieties (commonly found on solid support units) to provide an amide linkage. Additional modifications can also be made such that the semiconductor nanocrystal can be associated with almost any solid support.
- a solid support for the purposes of this invention, is defined as an insoluble material to which compounds are attached during a synthesis sequence, screening, immunoassays, etc. The use of a solid support is particularly advantageous for the synthesis of libraries because the isolation of support-bound reaction products can be accomplished simply by washing away reagents from the support-bound material and therefore the reaction can be driven to completion by the use of excess reagents.
- a solid support can be any material that is an insoluble matrix and can have a rigid or semi-rigid surface.
- Exemplary solid supports include but are not limited to pellets, disks, capillaries, hollow fibers, needles, pins, solid fibers, cellulose beads, pore-glass beads, silica gels, polystyrene beads optionally cross-linked with divinylbenzene, grafted co-poly beads, polyacrylamide beads, latex beads, dimethylacrylamide beads optionally crosslinked with N--N'- bis-acryloylethylenediamine, and glass particles coated with a hydrophobic polymer.
- the semiconductor nanocrystals of the present invention can readily be functionalized to create styrene or acrylate moieties, thus enabling the incorporation of the semiconductor nanocrystals into polystyrene, polyacrylate or other polymers such as polyimide, polyacrylamide, polyethylene, polyvinyl, polydiacetylene, polyphenylene-vinylene, polypeptide, polysaccharide, polysulfone, polypyrrole, polyimidazole, polythiophene, polyether, epoxies, silica glass, silica gel, siloxane, polyphosphate, hydrogel, agarose, cellulose, and the like.
- polyimide polyacrylamide
- polyethylene polyvinyl
- polydiacetylene polyphenylene-vinylene
- polypeptide polysaccharide
- polysulfone polysulfone
- polypyrrole polyimidazole
- polythiophene polyether
- test strips of our invention have the configuration as shown in Figure 1.
- element 1 is a PVC plate incorporating a sample pad 2 for antibody conjugate layer 3.
- the test device further includes an absorption zone 7 which is typically paper and a nitrocellulose membrane 4 which includes a control band 5 and a test band 7.
- test device A in embodiment B of Figure 1, the construction is similar to the test device A however it includes another test band 8 for either SAM or S-Adenosylhomocysteine.
- test strip of embodiment A of figure 1 includes one test band and one control band.
- Thd test strip of embodiment B of figure 1 includes two test bands for SAM and SAH
- MI strip Methylation Index (MI) strip
- the diagram of figure 1 shows how each component is assembled (lateral view). Liquid samples are applied through the left side of the sample pad, and the sample immediately migrates in the sample flow direction as shown in figure 1. The results are ready to be read in about 15 minutes after sample application to the strips. Numerous variations of the strip of figure 1 are possible. But the basic construction of an immunochromatographic strip is as follows and some of the different elements of the strip are optional and used as required depending on the needs of the tests.
- the elements can be placed in various arrangements, according to the assay format intended and the type of assay to be carried out, in general, the characteristics of the elements defined herein do not change between one arrangement and another.
- the elements described can be in any suitable physical form for the purposes of assay devices according to the present invention, such as, but not limited to, membranes, pads, strips, or other physical forms.
- the chromatographic strip can be composed of any suitable material that has a high protein binding capability and supports a lateral flow assay.
- the chromatographic strip is a hydrophilic element and the protein binding is through noncovalent binding.
- Applicants do not intend to be bound by this theory, current theory of binding of proteins to nitrocellulose states that the initial interaction is electrostatic, but subsequently hydrophobic interactions and hydrogen bonds considerably strengthen the binding.
- An example of a chromatographic material is the commonly used nitrocellulose element, which has been treated to make it hydrophilic.
- Another example of a chromatographic element is one made up of particles of a polymer, such as polyethylene, fused together.
- the chromatographic strip is of any size appropriate for the instrument or device used to read the results or for being read visually.
- the protein solution distributes itself throughout the depth of the nitrocellulose element.
- the proteins bind to the pore surfaces. Because of the method of application and the physics of the binding, more protein is bound to the top and center of the line compared to other areas wetted by the solution used to coat the antigens or antibodies onto the chromatographic strip.
- the chromatographic strip as used in assay devices according to the present invention includes a capture band, described further below.
- the chromatographic strip also typically includes one or more control bands, also described further below.
- the chromatographic strip of the present invention contains at least one capture band for capturing the analyte and at least one control band and, optionally, a second control band.
- the capture band, and the control band or bands can be viewed through a testing window.
- the capture band contains materials that are capable of capturing an analyte in a sample if the analyte is present.
- the capture band will contain antibody to SAM immobilized on the chromatographic strip at the capture band.
- the chromatographic strip will additionally contain conjugates or detectable agents at the second end for detecting the captured analyte.
- Assay devices may employ a sample filter (in some cases, two sample filters).
- the location of the sample filter or sample filters can vary, but the sample filter is situated so that fluid present in a sample, when applied onto the sample filter will flow from the sample filter to the chromatographic strip, either directly or indirectly.
- the sample filter is, in one alternative, a hydrophobic element, or alternatively a hydrophilic element or a synthetic composite of such as typically used in lateral flow assays for sample application. Examples of such sample filters include, but are not limited to hydrophobic filters such as glass fiber filters and hydrophilic filters such as cellulose.
- sample pad refers to a hydrophobic element, such as a hydrophobic element, that can be used to receive a sample.
- conjugate pad is used to describe an element that is used in many embodiments of assay devices according to the present invention.
- the conjugate pad is composed of a hydrophobic material, such as glass fiber and contains a conjugate or a detectable agent that can react with an analyte in a sample or with an analyte that is captured on the capture band on the chromatographic strip.
- the detectable agent includes, for example, antibodies or antigens specific for the analyte that are conjugated to a detectable material such as a colored material, a fluorescent material, or a chemiluminescent material or a quntum dot.
- An example of a colored material is colloidal gold.
- the conjugate pad herein is of a size suitable for the chromatographic strip within the parameters described.
- the conjugate pads can be preblocked with a buffer solution containing trehalose and casein, although other buffer solutions can alternatively be used for preblocking. Use of the conjugate pad is not necessarily required in all embodiments of assay devices according to the present invention. In some alternatives, the conjugate pad is omitted, and the conjugate is applied to the chromatographic strip. These alternatives are described further below.
- Fluid Collector is used to describe an element used in some configurations of assay devices according to the present invention.
- the fluid collector is typically a hydrophobic element, just like the hydrophobic element of the conjugate pad.
- the fluid collector does not contain any detectable agents and is used as an intermediate element, typically to transmit fluid, directly or indirectly, to the chromatographic strip.
- the test strip always includes at least one capture band.
- capture band refers to a region or zone on the chromatographic strip that contains at least one analyte binding agent.
- the analyte binding agent is usually immobilized in a band or zone such that after reaction with a detectable agent, the band or zone produces an observable or measurable result reflecting the presence or amount of analyte present in the sample.
- the "capture band” may be comprised of more than one capture zone for capturing more than one analyte in the sample, in which event, more than one analyte binding agent may be used. For example, two assay combinations that are considered to be within the scope of the invention as shown in the examples.
- the chromatographic strip of a device according to the present invention also includes one or more control bands, which contain control agents immobilized in control binding zones.
- Some embodiments of assay devices according to the present invention employ a buffer pad.
- the buffer pad is a hydrophilic element or a synthetic composite.
- the buffer pad is of a size suitable for the chromatographic strip within the parameters described.
- assay devices typically include one or more absorbent pads. These absorbent pads serve to direct fluid flow within the device. The size and location of these absorbent pads largely determines the flow pattern, as described above.
- the absorbent pad is a hydrophilic element that can absorb liquid, such as or a cellulose-glass fiber composite.
- the absorbent pad herein is of a size suitable for the chromatographic strip within the parameters described.
- J. Backing Pad Some assay devices according to the present invention include a backing pad that serves as a backing for the chromatographic strip.
- the backing pad can be made of any inert material that is capable of supporting the chromatographic strip, such as a piece of plastic material The size of the backing pad is suitable for the chromatographic strip within the parameters described.
- Some embodiments of assay devices according to the present invention incorporate a fluid-impermeable barrier interposed between elements such as a sample filter at or near the first end of the chromatographic strip and the chromatographic strip itself.
- immunoassays such as ELISA (Enzyme- linked Immunosorbent Aaasy) for determining qualitatively and quantitatively the concentration of SAM and SAH in a biological sample
- ELISA Enzyme- linked Immunosorbent Aaasy
- semiconductor nanocrystal conjugates are used as the detection reagents.
- the immunosorbent assay of the present invention has several advantages over current immunosorbent assays including, but not limited to, simultaneous multicolor detection and, hence, multiple analyte detection, with no requirement for enzyme development, increased photostability over alternative fluorophores thereby allowing increased detection sensitivity by virtue of the ability to monitor the signal over a long period of time, increased sensitivity over enzyme-based detection systems.
- Semiconductor nanocrystals of varying core sizes (10-150 .ANG.), composition and/or size distribution are conjugated to specific-binding molecules which bind specifically to SAM and SAH.
- Any specific anti-analyte can be used, for example, an antibody, an immunoreactive fragment of an antibody, and the like.
- the anti-analyte is an antibody.
- semiconductor nanocrystal conjugates are used in an immunosorbent assay to detect any analyte for which a specific-binding agent exists.
- the specific-binding molecule may be derived from polyclonal or monoclonal antibody preparations, may be a human antibody, or may be a hybrid or chimeric antibody, such as a humanized antibody, an altered antibody, F(ab').sub.2 fragments, F(ab) fragments, Fv fragments, a single-domain antibody, a dimeric or trimeric antibody fragment construct, a minibody, or functional fragments thereof which bind to the analyte of interest.
- the invention also deals with a new device that facilitates the aforementioned invention for the purpose of POCT uses.
- the spectrometer used in the invention is a combination of a fluorescence spectrometer and an absorbance UV/VIS spectrometer.
- the invention provides a method of determining risk of experiencing a major adverse cardiac event, in a patient, within one year from presentation of at least one symptom of acute coronary syndrome comprising the steps of: (a) obtaining a test sample from said patient; (b) determining the amount of at SAM, SAH, HCy and C reactive protein using a quantum dot based assay employing an immunochromatographic strip; (c) calculating the MI in said test sample; and c) comparing the amount of said four biomarkers to biomarker reference standards, wherein said risk is determined by results of said comparison.
- the preparation and assemblage of the immunoassay test immunochromatographic strip is done as follows. Briefly, goat anti-mouse IgG and BSA-SAH were separately applied to NCM (2.5 x 2.0 cm) with 3.5 ⁇ g in 10 mM phosphate-buffered saline, pH 7.4, to be used as the control zone and the test zone. The distance between the control zone and the test zone was 0.5 cm. The NCM was then dried for 1.5 hours at 37°C to fix the antibody and antigen. The NCM was pasted onto the polyvinyl chloride strip with the adsorption pad on the top end, and the quantum dot-conjugated pad overlapped by the sample pad was adhered to the bottom end of the NCM.
- the quantum dot-conjugated pad had been prepared by adding the anti-SAH MoAb-coated quantum dots (i.e, CdSeNPs) to the glass fiber (2.5 1.0 cm). The resultant conjugated pad was incubated at 37°C for 1.5 hours until fully dried. The sample pad of glass fiber (2.5 ⁇ 2.0 cm) was submerged in 10 mM phosphate-buffered saline, pH 7.4 and containing 0.05% Tween 20, and dried at 37°C for 1.5 hours. Finally, the test device was cut into 5 mm-wide strips and stored at RT before use.
- the anti-SAH MoAb-coated quantum dots i.e, CdSeNPs
- the SAM or SAH binding antibody is conjugated with a fluorescent label such as, without limitation, the rare earth chelates (e.g., europium chelates).
- the fluorescent labels can be conjugated to the antibody using conventional techniques in immunology. Fluorescence can be quantified using a fluorimeter or UV/vis spectrophotometer using the known extinction coefficient of the fluorescent label.
- the fluorescence properties of certain lanthanide chelates are well suited fluorescent markers.
- the absorbance of these chelates is very strong, (more than 10 4 ) and dependent upon the ligands.
- the quantum yield is often smaller than that for organic markers these chelates have other advantages, thus the emission appears at relatively long wavelengths (terbium 544 nm, europium 613 nm) in which wavelength range the serum fluorescence is low and furthermore the excitation maximum is within the short UV-range (Terbium-chelates 270-320 nm, Eu-chelates 320-360 nm) independent of the ligands which makes it possible to excite them with lamps or lasers commercially available and furthermore the Stoke's shift is very long (240-270 nm) and the emission band is sharply limited which enables a small band width.
- the most essential property is however that the fluorescence time is long, about 50-1000 microseconds which makes it possible to use the above mentioned instrumentation. As the fluorescence is measured with a certain delay during which the background fluorescence has decayed, the effect of an unspecific background radiation can be eliminated.
- the chelates of europium and to a certain extent terbium together with different .beta.- diketones are the most used chelates due to their ability to laser in different solutions and at different temperatures.
- the most widely used ⁇ -diketones are benzoyl acetone (BA), dibenzoylmetane (DBM), thenoyltrifluoroacetone (TTA), benzoyl trifluoroacetone (BTA), 1- and 2-naphihoyltrifluoroacetone (1-/2-NTA), acetylaceton (AcA), trifluoroacetylacetone (TFAcA), and hexafluoroacetyl acetone (HFAcA).
- BA benzoyl acetone
- DBM dibenzoylmetane
- TTA thenoyltrifluoroacetone
- BTA benzoyl trifluoroacetone
- the strong fluorescence of the lanthanide chelates is due to the absorption by the ligands of the excitation radiation and of the energy transfer from the triplet state of the ligand which gives rise to a narrow band radiation with a long wavelength characteristic for metals.
- a chelate of the above mentioned type could be used as a fluorescent marker it has to be attached to the antibody/antigen to be investigated. Furthermore, the metal has to give a fluorescent radiation also after the binding and in a water solution. To be stable enough, also in very diluted form (even below 10 9 M) and under conditions where other chelate forming reagents are present as well as an excess of other metal ions, the binding system must be very strong. The stability constant of the chelate must be well above 10 10 and additionally the binding ligand has to leave coordination positions free for another bidentate ligand.
- the fluorescence-labeled anti-SAM and anti-SAH antibodies of the invention that have been proven to be specific, quick and easy measurements of SAM and SAH can be performed at the cellular level via flow cytometry, immunofluorescence microscopy or LSCM.
- the immunofluorescence microscopy has the advantage of studying the levels and locations of SAM and SAH even with a small number of cells, e.g. studying SAM and SAH from cells in their early stages of embryo development with a couple of hundreds of cells or even less.
- the LSCM results from Figure 7 showed that intracellular localizations of SAM and SAH were somewhat similar. SAM and SAH were seen mostly in mitochondria, peri-nuclei and in nucleoli.
- the invention also provides an easy and quick homogeneous immunoassay that does not have special strip preparation as well as no washing and separation steps that can also be used conveniently in the point-of-care test (POCT) setting besides the commonly known dry test strips.
- Figure 8 show simple diagrams illustrating how the two formats of TR-FRET technology may be used in the quantitative measurement of SAM and SAH using the bio-conjugates described in this invention. With format A of figure 8, specific antibodies against SAM or SAH are associated with acceptor dyes directly or indirectly through rabbit or goat anti-mouse IgG that is labeled with acceptor dye. Two tracing methods, SA-biotin and Dig-anti-digoxin antibody specific binding partners, are shown that are conjugated to donor dyes.
- the biotin-conjugated (or Dig-conjugated) SAM or SAH with different linkers brings donor and acceptor dyes together in close proximity, most likely less than 100 angstrom (A), which allows the donors to excite the acceptor dyes.
- A angstrom
- the energy transfer with the donors occurs and a distinguished fluorescence emitted at a specific wave length from acceptor dyes is measured that reflects only the portion of the molecules that are able to connect donors and acceptors together specifically.
- Free SAM or SAH molecules from a sample compete with the bio-conjugates for binding the anti-SAM or anti-SAH antibodies, therefore lead to reduced fluorescent signals.
- Competitive measurement can be established based on the competitive binding characteristics.
- SAM analog or SAH is conjugated (with or without a linker) to an acceptor dye, which will compete with free SAM or SAH from samples for binding to the antibodies against SAM or SAH that are attached to donor indirectly through rabbit or goat anti-mouse IgG.
- acceptor dyes reflects the amounts of SAM or SAH bound to the donor dyes that are not competed by the SAM or SAH in the samples, i.e. donor-specific antibody-antigen-acceptor complex.
- the amount of specific antibodies that bind to un-conjugated SAM or SAH molecules will not have fluorescence to be read, which constitutes one of the competing parties in the competitive assay.
- Free anti-SAM or SAH antibody if any, which is not conjugated with donor dyes, will consume either labeled or unlabeled antigens. Both donor and acceptor fluorescence signals are read with the TR-FRET microplate reader and the acceptor fluorescence/donor fluorescence can be calculated that will be used in quantifying SAM or SAH from a sample.
- BRET Bioluminescence Resonance Energy Transfer
- TR-FRET Fluorescence Resonance Energy Transfer
- FRET Fluorescence Resonance Energy Transfer
- the acceptor dye should be chosen so that it has an optimal spectral overlap between the Luc bioluminescent spectra and the dye excitation spectra and higher quantum yield.
- SAM or SAH antigen
- the fluorescent dye that meets the criteria above is conjugated to the anti-SAM or anti-SAH antibody.
- BRET index acceptor fluorescence/donor luminescence
- the BRET-based method does not require laser excitation of donor dye at the time of detection. Instead it only needs to add the substrate of the luciferase. When enough substrates start to generate luminescence that can be measured, it also excites the acceptor fluorescent materials that are brought to its close proximity by specific antigen-antibody. It does not excite acceptor fluorescent dyes that are not associated with luciferase donor. Therefore, the emission signals measured reflect the part of antigen-antibody complex containing both the donors (bio- conjugates) and acceptors, not the SAM or SAH antigens from samples or standards that are only associated with acceptors via antibodies.
- biological sample is intended to include saliva, urine, blood, serum, plasma, brain fluids, cerebrospinal fluids, tissue samples and cells or anything derived from the body of a mammal including a human.
- the quantum dots (CdTe/CdSe, CdHgTe/ZnS, etc.) with mean diameter of 2 - lOnm were purchased from NN-Labs, LLC (Fayetteville, AR 72701). Fluorescent dye Europium chelates or other lanthanide metals, etc. with mean diameter at 200 nm - 300nm were purchased from Bangslab (Fishers, IN 46038).
- fluorescent tracers fluorescent tracers
- HTRF® Homogeneous Time-Resolved Fluorescence
- competitive method to quantify SAM from samples by using anti-SAM monoclonal antibody and bio-conjugates as exemplified in our application No. 15/091,544 filed April 5, 2016, the entire contents of which are incorporated by reference herein as if they were entirely denoted.
- Rabbit anti-mouse IgG-XL665 and Europium (Eu3+) cryptate labeling kit were purchased from Cisbio Bioassays. Label mouse anti-digoxin or anti-digoxigenin antibody (anti- Dig antibody, PerkinElmer) to Eu3+ cryptate.
- the assay plates are read with a small point-of-care micro-titer strip reader for HTRF assays.
- Time-resolved fluorescence is measured at a 50 ⁇ 8 delay after each excitation pulse.
- Emissions are measured at 665 nm for detection of the FRET signal (A counts), and at 620 nm for detection of the Eu(K) signal (B counts).
- the B counts should be the same for all assay wells, which act as an internal control and indicator of the absorbance of the background.
- the fluorescent signals are measured simultaneously, and the ratio ((A counts - 10,000)/B counts) is reported.
- This ratio is minimally affected by absorbance as both the 665 nm and the 620 nm signals are impacted similarly.
- the ratio and the concentration of the SAM standards are used to plot the standard curve. The more the SAM is from a sample, the lower the A counts and hence the ratio.
- Mouse anti-SAM antibody 118-6 was conjugated to Alexa Fluor 610-x using fluorescent antibody labeling kit (Thermo-Fisher). Optimize the molar ratio of the bio-conjugate to luciferase, molar ratio of mouse anti-SAM antibody to Alexa Fluor 610-x, the working concentrations of Luciferase-6C-aza-SAM (donor Luc-SAM), mouse anti-SAM antibody 118-6 (acceptor FL-Ab) and the competing SAM from a sample or standard in a buffer containing lOOmM PB, pH 7.0, 0.1% protease-free BSA, lOOmM KF, 0.1% Tween 20.
- SAM standard is tested in the range of 0-3000nM.
- the test is performed with a micro-titer strip of 1-10 wells to a final volume of ⁇ /well.
- Three assay components above and the substrate luciferase are combined and incubated for 15-30min at room temperature.
- the assay plates are read with a small point-of-care micro-titer strip reader for BRET assays.
- Time- resolved fluorescence is measured at a 50 ⁇ 8 delay after each excitation pulse. Emissions are measured at 630nm for detection of the BRET signal, and at 550nm for detection of the luciferin signal. Find the proper molar ratio of The BRET index (FL-Ab/Luc-SAM).
- the amount of antibody bound is in linear relationship with BRET index, the BRET index and the concentration of the SAM standards are used to plot the standard curve. The more the SAM is from a sample, the lower the BRET index.
- anti-SAM antibody 84-3 (Cat# MA00202, Arthus Biosystems, VA) at the final concentration of 40 ⁇ g/ml and shaken at room temperature for 2.5h.
- the conjugate was stored in 20mM Tris buffer with 0.5% BSA and EDTA-Na2, applied evenly to the glass fiber after proper dilution at the density of 4ul/cm, followed by drying at 37°C for 12h.
- the standard curve is shown in figure 2, where the x-axis is base 10 logarithm of the concentration of SAM ranging from 0 to 3000nM.
- the y-axis is the base 10 logarithm of the ratio of fluorescent signal of test line (T) to that of control line (C).
- HTRF ® technology Homogeneous Time-Resolved Fluorescence
- competitive method to quantify SAH from samples by using anti-SAH monoclonal antibody and bio-conjugates as exemplified in our application No. 15/091,544 filed April 5, 2016, the entire contents of which are incorporated by reference herein as if they were entirely denoted.
- a mouse anti-SAH antibody 301-3 (Cat# MA00303, Arthus Biosystems, VA) was used at the final concentration of 80 ⁇ g/ml.
- the standard curve for this particular strip is shown in Figure 3, where x-axis is base 10 logarithm of the concentration of SAH ranging from 0 to 3000nM. The y-axis is the base 10 logarithm of the ratio of fluorescent signal of test line (T) to that of control line (C).
- a fluorescent immunochromatographic test strip for measuring methylation index (MI) Using the method of Example 1 as described above but BSA-SAM (or SAM analog) and BSA-SAH were applied to different areas of the NC membrane and dried. Both FT-anti-SAM and FT-anti-SAH were absorbed evenly to the glass fiber, and then assembled as shown in the Figure IB. The fluorescence intensity of the FT was measured separately, which will be converted into actual levels of SAM and SAH based on the preinstalled standard curves for the batch of strips. SAM and SAH test lines will display as two SAMe or different colors depending the type of FTs used to label the anti-SAM and anti-SAH antibodies. The strip allows measuring SAM and SAH at the SAMe time quickly and easily. MI is calculated and displayed on the Dry Immunofluorescence Analyzer.
- EDC 1 -Ethyl -3 -(3- dimethylaminopropyl)-carbodiimide
- NHS N-hydroxysuccinimide
- test strip is illustrated in Figure IB without a sample pad as blood samples will be diluted at about 600 folds before testing.
- the standard curve for this particular strip is shown in Figure 4, where the x-axis is the concentration of CRP ranging from 0 to 130 mg/L.
- the y-axis is the ratio of fluorescent signal of the second test line (T2) to that of control line (C).
- HCy plasma or serum samples underwent some chemical reactions so that all HCys were freed from protein associations and in a reductive form before they were converted to SAH as follows:
- HMT Homocysteine + S-adenosylmethionine— (HMT)— > S-adenosylhomocysteine + Methionine, whereas HMT is homocysteine methyltransferase.
- the reaction product SAH is measured with a qualitative SAH strip with a proper cutoff value that reflects the cutoff value of limiting material HCy in human plasma or serum, i.e. normal subjects have HCy at 10 ⁇ and below; patients with abnormal HCy that is higher than 15 ⁇ . Therefore, we made a colloidal gold SAH test strip that shows test line (T) and control line (C) with the following readout: C line does not have any colloidal gold signal: the strip is invalid.
- Both T and C have the similar colloidal gold signal intensity: HCy level from a sample ⁇ 10 ⁇ ;
- the C has stronger colloidal gold signal than T line and T line is visible: HCy level from a sample is between 10 to 15 ⁇ ;
- the SAH test strip was made according to the following procedure:
- Test strip was assembled on a PVC plate according to the method illustrated in the Figure 1A.
- MTHC1 represents a methylation index and homocysteine triple test strip format 1.
- the unit consists of two test strips, (a) One is an MI strip as in Example 3. (b) The other one is an HCy strip as in Example 5.
- MIHC2 represents a methylation index and homocysteine triple test strip format 2.
- the unit consists of two test strips, (a) One is an MI strip as in Example 3. (b) The other one is an HCy strip as in Example 6.
- the accompanying device is able to read, process and output the results at the SAM time reporting the values of SAM, SAH, MI and HCy from a sample qualitatively or/and quantitatively.
- An immunochromatographic strip for simultaneous measurement of SAM, SAH, Homocysteine (HCy) and CRP MIHCR1 represents a methylation index, homocysteine and C-reactive protein quadruple test strip format 1.
- the unit consists of three test strips, (a) One is an MI strip as in Example 3. (b) The second one is an HCy strip as in Example 5. (c) The third one is the CRP strip as in Example 4.
- MIHCR2 represents a methylation index, homocysteine and C-reactive protein quadruple test strip format 2.
- the unit consists of three test strips, (a) One is an MI strip as in the Example 3. (b) The second one is an HCy strip as in Example 6. (c) The third one is the CRP strip as in Example 4.
- the accompanying device is able to read, process and output the results at the SAM time reporting the values of SAM, SAH, MI, CRP and HCy from a sample qualitatively or/and quantitatively.
- the test unit does not require any device to read results.
- Triple test strips are assembled into one single unit with each strip having detection band at cutoff values of 50nM, 400nM, 800nM respectively.
- the cutoff values can be changed to different values besides 50nM, 400nM and 800nM.
- the value from a blood sample can be read out as the following: ⁇ 50nM; 50- 400nM; 400-800nM; > 800nM.
- Colloidal gold or microspheres were used to label mouse anti- SAM antibody (Cat# MA00201, Arthus Biosystems, VA). Conjugation of antibody was similar to that in Example 6.
- Assembling of the test strip is the SAM as in shown Figure 1A and in the Example 1. Colloidal gold or microsphere results can be seen with naked eyes. Therefore, this method is quick, easy and cost-effective without having to use any additional device.
- the test unit does not require any device to read results.
- Triple test strips are assembled into one single unit with each strip having a detection band with cutoff values of 200nM, 600nM, 1200nM respectively.
- the cutoff values can be changed to different values besides 50nM, 600nM and 1200nM.
- the value from a blood sample can be read out as the following: ⁇ 200nM; 200-600nM; 600-1200nM; > 1200nM.
- Colloidal gold or microspheres were used to label mouse anti-SAH antibody 839-6 (Cat# MA00307, Arthus Biosystems, VA). Conjugation of antibody was similar to that in Example 6.
- Assembling of the test strip is the SAM as in shown Figure 1 A and in the Example 1. Colloidal gold or microsphere results can be seen with naked eyes. Therefore, this method is quick, easy and cost-effective without having to use any additional device.
- FTs can also be used in other aqueous systems as tracers in a list of potential measurements below.
- FT-anti-SAM and FT-anti-SAH antibodies can be used in cell-base technologies such as flow cytometry and immunofluorescence microscopy to investigate the metabolism, dynamics, distribution and levels of SAM and SAH within cells, tissues and organs under different scenarios.
- MAT activity was stimulated by Met in cells using FCM.
- SAM and SAH SAA
- nuclear SAM constitutes 80-85% of the total SAM and methylation indices are similar too.
- SAM In normal mouse liver cells, about 4.6% of SAM is located in nucleus. With ImM Met-stimulation for 24h, nuclear SAM level is increased by 4 folds, constitutes about 22.5% of the total SAM.
- Met-stimulated MAT causes nuclear SAM to increase whereas cytoplasm SAM is decreased within ImM Met dosage.
- Primary liver cells were cultured in Met-free medium for 20h, MAT activity was induced and SAM was increased in nucleus but was reduced in cytoplasm (Figure 6). This indicated critical roles that SAM needs to play are in nucleus in response to Met hunger/deficiency (regulated expressions of certain genes).
- MI total cell methylation index
- fixation/permeabilization buffers Two different types were tested for all cell types, i.e. nuclear fixation/permeabilization buffer (Cat# 00-5523 FoxP3_TF Staining Buffer Set, eBioscience, San Diego, CA) by which both cytoplasm and nucleus targets were stained and intracellular fixation/permeabilization buffer was used (Cat# 00-8824, eBioscience, San Diego, CA) by which only cytoplasm targets were measured.
- nuclear fixation/permeabilization buffer Cat# 00-5523 FoxP3_TF Staining Buffer Set, eBioscience, San Diego, CA
- intracellular fixation/permeabilization buffer was used (Cat# 00-8824, eBioscience, San Diego, CA) by which only cytoplasm targets were measured.
- FIG. 5 shows the flow cytometry (FCM) results from cells double stained with Alexa Fluor 647 conjugated anti-SAM 118-6 antibody (Cat# MAF00201, Arthus Biosystems, VA) at 4 ⁇ g/ml while Figure 6 shows FCM results from cells double stained with Alexa Fluor
- 488 conjugated anti-SAH antibody 301-3 (Cat# MAF00301, Arthus Biosystems, VA) at 45 ⁇ g/ml. Both SAM and SAH levels from cytoplasm and nucleus compartments are shown. Normal liver cell line L02 and hepatocellular carcinoma cell line HepG2 were treated with 0, 0.5mM and ImM methionine (Met) for 24 h. Mouse primary liver cells were isolated and treated with 0, 0.5mM, ImM Met for 24 h and cultured in Met-free MEM medium for 20 h. Figure 5 shows SAM levels while Figure 6 shows SAH levels.
- FIG. 7 illustrates the Laser Scan Confocal Microscopy (LSCM) results of L02 and HepG2 cells that were cultured for 40h and then stained with the SAMe fluorescence labelled anti-SAM and anti-SAH antibodies of the invention.
- A are the L02 cells stained with anti-SAM antibody
- B are the HepG2 cells stained with anti-SAM antibody
- C are L02 cells stained with anti-SAH antibody
- D HepG2 cells stained with anti-SAH antibody.
- the photos were taken by Zeiss LSM 780 under 630-fold magnification.
- C Fluorescence immunology in connection with streptavidin (SA) and biotin system
- FTs can be labeled onto SA.
- SAM and SAH are conjugated with biotin through various linkers (as exemplified in our application No. 15/091,544 filed April 5, 2016, the entire contents of which are incorporated by reference herein as if they were entirely denoted).
- Different FTs are labeled onto SA.
- SA Through the specific and strong binding between SA and biotin, small molecule antigen SAM and SAH can be therefore labeled to different FTs separately, i.e. FT-SAM and FT-SAH are obtained.
- FT-SAM and FT-SAH are obtained.
- SAM and SAH Other indirect methods of tracing SAM and SAH include (1) conjugating SAM or/and SAH to digoxigen or digoxingenin through various linkers (as exemplified in our provisional application No. 15/091,544 filed April 5, 2016, the entire contents of which are incorporated by reference herein as if they were entirely denoted). (2) Different FTs are labeled onto mouse anti- digoxigenin or mouse anti-digoxin antibodies. (3) Mix products from step (1) and step (2), so SAM and SAH are indirectly labeled to different colored FTs. (4) Uses of FT-SAM and FT-SAH are as described above as in Example 11C.
- EXAMPLE 12 Using the test strips to measure SAM and SAH levels from healthy human blood samples and monitoring progress in weight reduction
- BMI Body Mass Index
- the average MI from the high BMI group is only 30.76% of the MI from the low BMI group in females and about 63.49% in male subjects. This implied that high BMI had more impacts on (reduced) Mis of females than on males. BMI less than 24 is considered ideal for health reason. Therefore, abnormal BMI is related to SAM levels in both genders. Low SAM might be the reason for the abnormal and unfavorable BMI that usually underlies a series of health concerns including cardiovascular and renal diseases, diabetes, obesity and other metabolic disorders, etc. (Lydi M. J. W. van Driel reported the relationship between BMI and methylation in young females (Body Mass Index Is an Important Determinant of Methylation Biomarkers in Women of Reproductive Ages, J. Nutr. 139: 2315-2321, 2009.). The results indicated SAM, SAH and MI are good indicators or biomarkers for health issues caused by abnormal BMI, such as cardiovascular diseases.
- Table 1 becomes Table 3 below.
- the averages of SAM, SAH and MI from 12 normal BMI females were higher than (SAM 33.41%, SAH 97.73%, MI 25%) higher respectively) those corresponding values from 13 male subjects.
- the differences of SAM, SAH and MI values between females and males were even more obvious, i.e. the average female SAM level is 33.41% (instead of 25.51%) if subjects with all BMI values were considered) higher than that of male if only looking at the normal BMI subjects in each gender.
- Table 4 showed abnormal BMI may blur (decrease) the differences in SAM, SAH and MI values between females and males. This indicates BMI is a factor that complicates the values of SAM, SAH and MI, which is consistent with the fact that the levels of SAM and SAH vary according to race, gender, body weight, age and general healthy conditions.
- Table 3 Levels of SAM, SAH and MI in healthy plasma samples by gender (all BMI ⁇ 24)
- cTnl Cardiac Troponin-I
- CRP Creactive protein
- CK-MB Creatine-kinase-MB
- Myo Myoglobin
- CVD cardiovascular disease
- the data from Table 5 showed 9 cases without acute myocardial injury (AMI) as determined by clinical lab's negative cTnl, CRP, CK-MB and Myo results had the average SAM value as 164 nM, SAH as 232 nM, MI as 1.75 and 44.44% of them with HCy higher than 15 ⁇ , and CRP values measured using test strip as described in the Example 4 showed an average of 0.8mg/ml (normal). Whereas, in the 7 heart attack cases that were diagnosed with much increased cTnl, CRP, CK-MB and Myo the average SAM value was 94 nM, SAH as 558 nM, MI as 0.2 and 85.71% of them with HCy higher than 15 ⁇ .
- AMI acute myocardial injury
- the average CRP for the 7 cases with heart attack or AMI is 4.37mg/l, which was higher than normal but not related to inflammation reaction as it is less than lOmg/1.
- CRP levels were not high and just about to increase.
- CRP levels were much elevated. This indicated that CRP elevation happened after about a day or so.
- SAM and MI increased SAH and HCy are also good biomarkers for heart diseases.
- SAM, SAH, HCy and CRP values measured for all samples using the immunochromatographic test strips described in the Example 4 in this invention may help identify and sort out certain groups of patients that may be overlooked by merely checking cTnl, CK-MB, Myo and HCy alone with photochemical methods that are currently often checked.
- SAM, SAH, HCy and CRP are useful biomarkers that will add to the current cardiac panel in order to timely diagnose, differentiate, predict the prognosis and help direct treatment of CVDs.
- Table 5 Measurement of biomarkers in clinical samples (n 16)
- quantum dot probes and fluorescent chelates provide higher fluorescence than that provided by other probes that have been labeled with organic fluorescent molecules; and their longer lasting fluorescence allow for stable and reliable systems to be built; Therefore, it is believed that quantum dot and fluorescent chelate based assays for determining SAM, SAH and HCy provide series of advantages over assays that employ traditional organic fluorescent molecules.
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JP2017561249A JP6974179B2 (en) | 2015-05-25 | 2016-05-25 | Use of fluorescence to measure S-adenosylmethionine, S-adenosyl homocysteine and homocysteine quickly and easily |
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EP16800682.3A EP3308167A4 (en) | 2015-05-25 | 2016-05-25 | Use of fluorescence for the quick and easy determination of s-adenosylmethionine, s-adenosylhomocysteine and homocysteine |
CN201680032653.5A CN108291908A (en) | 2015-05-25 | 2016-05-25 | With immune and the fast and convenient measurement s-adenosylmethionine of chemical method, AdoHcy and homocysteine |
AU2020202395A AU2020202395B2 (en) | 2015-05-25 | 2020-04-05 | Use of fluorescence for the quick and easy determination of s-adenosylmethionine, s-adenosylhomocysteine and homocysteine |
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