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WO2002005698A2 - Extraction de fluorophores a partir d'echantillons biologiques - Google Patents

Extraction de fluorophores a partir d'echantillons biologiques Download PDF

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Publication number
WO2002005698A2
WO2002005698A2 PCT/US2001/021981 US0121981W WO0205698A2 WO 2002005698 A2 WO2002005698 A2 WO 2002005698A2 US 0121981 W US0121981 W US 0121981W WO 0205698 A2 WO0205698 A2 WO 0205698A2
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WO
WIPO (PCT)
Prior art keywords
mixture
diseased
sample
hepatitis
volume
Prior art date
Application number
PCT/US2001/021981
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English (en)
Other versions
WO2002005698A3 (fr
Inventor
Omanand Koul
Victor S. Sapirstein
Yongwu Yang
Original Assignee
Seroptix, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Seroptix, Inc. filed Critical Seroptix, Inc.
Priority to AU2001273404A priority Critical patent/AU2001273404A1/en
Publication of WO2002005698A2 publication Critical patent/WO2002005698A2/fr
Publication of WO2002005698A3 publication Critical patent/WO2002005698A3/fr

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G01N33/56988—HIV or HTLV
    • 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
    • G01N33/576—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis

Definitions

  • the present invention relates to the detection of the presence of a diseased or infected state and more particularly to a sample preparation technique to enhance such detection in a sample.
  • BACKGROUND ART Spectroscopy has been used to determine the chemical and physical composition of matter. For example, quantitative or qualitative measurement of the absorption, emission, or scattering of radiation upon irradiating a sample with radiation allows the abundance and identity of a sample's constituents to be determined. (Hereinafter, absorption, emission, and/or scattering of radiation of any wavelength or bandwidth are referred to collectively as spectroscopic signals unless otherwise stated.) These determinations are based on the fact that the sample constituents give rise to unique spectroscopic signals or fingerprints, which can generally be represented as a spectrum or . plot of the absolute or relative intensity of the spectroscopic signal at various wavelengths.
  • biological samples in a diseased or infected state comprise compounds that are indicative of the diseased or infectious state. Therefore, the presence, e.g., abundance of the compounds can be used to discriminate a diseased or infected sample from a disease-free or non-infected sample.
  • U.S. Patent Application Nos. 09/224,141 filed December 31, 1998 and 09/436,207 filed November 8, 1999 which are hereby incorporated by reference in their entireties, describe the identification of fluorescence wavelength ranges wherein the fluorescence spectrum of a sample will yield information that may be used to detect and identify the presence of infection or disease.
  • spectral overlap is particularly important in biological samples, which, in addition to the compound of interest, often include a complex matrix of concomitant species. Morever, when the spectral overlap is intrinsic to the shape and characteristics of the spectroscopic signals being measured, even high resolution spectroscopic instruments are insufficient to completely discriminate the desired spectroscopic signals from the spurious spectroscopic signals of the concomitant species. Problems caused by spectral overlap are further aggravated when the quantity of the compounds of interest is low, such as at early stages of a disease or infection. Thus, conventional analyzers require that the disease or infection progress for a period of time before detection. Obviously, such a progression time undesirably delays the onset of treatment for an infected individual.
  • the 09/224,141 and 09/436,207 patent applications disclose preparing a biological sample, such as plasma, by passing the sample over adsorbants such as C-M Affi Gel Blue or activated charcoal to reduce spurious signals and improve the ability to discriminate a diseased or infected state from and a non-diseased • or non-infectious state.
  • adsorbants such as C-M Affi Gel Blue or activated charcoal to reduce spurious signals and improve the ability to discriminate a diseased or infected state from and a non-diseased • or non-infectious state.
  • the present invention relates to a different method of preparing a biological sample prior to detection of a diseased state.
  • the present invention relates to a method for preparing a biological sample for detection of a diseased or infected state, such as, for example, HTV, Hepatitis A, Hepatitis B, Hepatitis C, or a combination thereof.
  • the method comprises combining the biological sample with a fluid to obtain a first mixture and heating the first mixture to a temperature and for a time sufficient to obtain a second mixture comprising a supernatant having a detectable amount of free compounds and wherein the presence or abundance of the free compounds may be used to determine the presence of the diseased or infected state.
  • free compounds are preferably dissociated from proteins to enhance a spectroscopic property of the free compounds such as, for example, a fluorescence intensity or a fluorescence yield.
  • a preferred embodiment of the present method further comprises irradiating the supernatant with radiation, measuring quantitatively or qualitatively a fluorescence emission or absorbance of the free compounds, and determining the presence of a diseased state by analyzing the fluorescence emission or absorbance.
  • the fluorescence emission or the absorbance is preferably compared to a signal characteristic of a non-diseased state.
  • the supernatant is irradiated with radiation that has at least one wavelength of from about 270 to 750 nm.
  • the fluorescence emission is preferably detected over at least a portion of the range from about 400 to 850 nm.
  • the biological sample comprises plasma.
  • the first mixture comprises from about 4 to 8% by volume plasma, 0.9 to 1.5% by volume buffer, 0.3 to 0.9% by volume acid, and 88 to 96% by volume organic liquid.
  • the buffer is a phosphate buffer having pH of from about 3 to about 5.
  • the acid is preferably trifluoroacetic acid.
  • the organic liquid is preferably acetonitrile.
  • the first mixture may further comprise water.
  • FIG. 1 shows fluorescence spectra of an untreated Hepatitis C negative plasma sample and an untreated Hepatitis C positive plasma sample acquired with an excitation wavelength of 355 nm;
  • FIG. 2 shows fluorescence spectra of the samples from FIG. 1, which have been treated according to the method of the present invention.
  • a method for preparing a biological sample for detection of the presence of a diseased or infected state
  • the method is suited for preparing biological samples for spectroscopic determination of a diseased or infectious state comprising, for example, HF/, Hepatitis A, Hepatitis B, or Hepatitis C.
  • the spectroscopic determination generally involves measuring the presence or abundance of compounds that are associated with the diseased or infected state to discriminate a diseased or infected sample from a disease-free or non-infected sample.
  • the compounds may be fluorescent or may exhibit an absorbance, which can be detected by spectroscopic techniques. In untreated samples, however, spectroscopic signals from the compounds may be overwhelmed by background or spurious signals from concomitants in the biological sample.
  • spectroscopic information can be used to provide information regarding the environment of a compound, such as, for example, whether or not the compound is bound to a protein.
  • Such techniques include but are not limited to, for example, determining the absolute or relative amplitude of a spectrum at one or more predetermined wavelengths or determining the absolute or relative area underneath all of or a portion of a spectrum. The wavelength corresponding to the peak or minimum spectroscopic signal and/or the relative shift in either of these values can also be used in this regard.
  • the emission lifetime can be used to help identify a compound.
  • spectroscopic signals are interpreted as a function of their frequency content
  • Multivariate techniques such as, for example, neural nets or principal components analysis, wherein information from more than one wavelength or spectrum is utilized in a comprehensive analysis, can also be used to interpret overlapped spectra. Any of the above mentioned techniques or similar techniques that provide information regarding the presence, abundance, or environment of a compound within a sample are applicable to the presently recited method.
  • the present method greatly enhances the ability of a spectroscopic technique to discriminate biological samples infected with a disease or pathogen (e.g., HTV viruses) from samples not infected with such diseases or pathogens.
  • a disease or pathogen e.g., HTV viruses
  • the method of the ⁇ invention preferably provides a supernatant liquid, comprising compounds, which have been dissociated from proteins in the biological sample, as described below.
  • the dissociated compounds in the supernatant liquid may exhibit enhanced spectroscopic properties such as, for example, a fluorescence yield and or intensity compared to compounds associated with the protein molecules, as found in an untreated sample.
  • the term "compounds” as used herein includes, but is not limited to, molecules of any size or complexity, fragments of molecules, such as, for example, a DNA fragment or a denatured protein, or complexes comprising more than one chemically and/or physically associated molecule.
  • fluorophores or “fluorescent compounds” refer to compounds which emit detectable fluorescence upon irradiation with a suitable light source.
  • the method comprises the steps of: (a) obtaining a biological sample;
  • the biological specimen or sample used in the present invention may be any biological material such as plasma, serum, blood particles, blood, bodily fluid, or tissue.
  • the term "specimen” is often taken to mean a biological material that has been subjected to essentially no processing, whereas, the term “sample” is taken to mean a product that results from even a basic level of processing a specimen.
  • plasma is a sample that results after removing cellular material from a blood specimen.
  • the method of the present invention may be applied to a substantially unprocessed specimen such as tissue or to a partially processed sample such as plasma, the terms specimen and sample are used interchangeably hereinafter.
  • Biological samples suitable for use in the present invention may be obtained from an individual, animal, or other organism using methods that are known in the art.
  • Plasma or other blood fluids are particularly convenient samples to use for identifying a diseased or infectious state of an individual.
  • Human plasma for example, contains as many as 100 to 125 proteins, many of which, such as albumin and lipoproteins, serve as carriers for smaller metabolically important compounds, as well as their metabolites.
  • at least some of the compounds associated with the proteins emit fluorescence or have an absorbance, which is useful for identifying a diseased or infected state.
  • features of the fluorescence spectrum of plasma between about 380 nm and about 600 nm are a function of the presence or absence of a diseased state such as, for example, HIV. Treating a sample according to the method of the present invention accentuates differences in these features, thereby improving the ability to discriminate between a spectrum obtained from an infected sample and from a non-infected sample.
  • the biological sample is combined with a buffer to form a first mixture.
  • a buffer any buffer composition that provides adequate buffering capacity at a desired pH may be used in the present method.
  • the buffer is a phosphate buffer, such as, for example, an aqueous solution comprising potassium dihydrogenphosphate, anhydrous monosodium phosphate, trisodium phosphate dodecahydrate, or any combination thereof.
  • the buffer may also comprise organic liquids.
  • the biological sample is a liquid such as for example, plasma
  • the combining step can be achieved by mixing the buffer and plasma to obtain a substantially homogeneous solution.
  • the combining step may further require homogenizing the buffer and tissue, as by blending, digestion, or other methods known in the art. It may also be useful to chemically lyse cells in the biological sample.
  • the buffer should maintain the pH of the sample within an appropriate range for dissociating the compounds carried by or otherwise associated with the proteins in the sample. However, the pH should not be so high or low as to inhibit or perturb the fluorescent properties of the dissociated molecules.
  • the pH of the buffer is between about 2.5 and 5.5, such as, for example, between about 3.5 and 4.5, and most preferably about 4.
  • Compounds released from the proteins at about pH 4 include, for example, pterins.
  • One of ordinary skill in the art understands how to prepare a buffer according to the desired pH and buffer capacity.
  • the buffer should be present in an amount sufficient to provide adequate buffering capacity.
  • the buffer solution comprises from about 0.6 to 1.3 molar buffer, for example, between about 0.9 and 1.1 molar and most preferably around 1 molar buffer.
  • the volume of buffer solution is preferably from about 10 to 50% as large as the volume of biological sample, preferably from about 15 to 25% as large.
  • the exact volume and buffer concentration can be determined by one of ordinary skill in the art and depends, for example, on the particular biological sample chosen for analysis, the diseased or infectious state in question, and the amount of acid used, as described below.
  • An organic acid preferably trifluoroacetic acid
  • a spectrophotometric grade of acid is well suited for use in the present invention.
  • spectrophotometric grade trifluoroacetic acid has minimal absorbance at ultraviolet and visible wavelengths greater than about 270 nm and has minimal fluorescent impurities.
  • the first mixture and acid may be combined by mixing, although further homogenization or digestion may also be desirable in order to achieve a substantially homogeneous mixture.
  • the acid is preferably added to the first mixture in the form of a liquid comprising more than about 60% by volume acid, for example, more than about 90%, or most preferably, more than about 99% acid by volume.
  • the volume of organic acid added to the first mixture is preferably from about 20% to 75% as large as the volume of buffer used, preferably from about 40 to 60% as large.
  • the amount of acid should be sufficient to separate the fluorescent compounds from their associated proteins. Although some of the bound compounds may be dissociated from the proteins immediately upon adding the acid, a substantial portion are dissociated during the subsequent heating step, which is described below.
  • the exact amount and strength of acid can be determined by one of ordinary skill in the art and depends, for example, on the buffer used, the particular biological sample chosen for analysis and the diseased or infectious state in question.
  • An organic liquid is then added to the second mixture to obtain a third mixture. Although a variety of organic liquids may be used, it is important that detectable amounts or concentrations of compounds dissociated from the proteins are soluble in the chosen liquid.
  • Acetonitrile is a particularly useful liquid in the method of the present invention. Spectrophotometric grades of acetonitrile, which have minimal fluorescent and absorbing impurities, are also suited for use in the present method.
  • the volume of the organic liquid should not be so large as to overly dilute compounds extracted from the biological sample.
  • the volume of organic liquid such as, for example, acetonitrile
  • the volume of organic liquid is preferably about 5 to 25 times as large as the volume of plasma, more preferably about 12 to 18 times as large.
  • the exact amount of liquid can be determined by one of ordinary skill in the art and depends, for example, on the particular biological sample chosen for analysis and the diseased or infectious state in question.
  • the third mixture is heated to a temperature and for a period of time sufficient to dissociate compounds from the proteins or other biological molecules in biological sample.
  • the mixture may be heated to from about 65 to 115 °C, preferably to from about 90 to 110 °C.
  • the mixture is heated for more than about one-half hour, preferably more than about 1 hour.
  • a substantially clear supernatant is formed over solids, which are partially or wholly insoluble in the supernatant.
  • the solids comprise proteins which have been precipitated from the third mixture.
  • Compounds, previously associated with the proteins or other biological molecules within the biological sample are substantially dissociated from the proteins and extracted into the supernatant liquid.
  • the supernatant and solids can be separated using techniques known in the art, such as filtration, centrifugation, selective adsorption, or combination thereof.
  • NADH reduced nicotinamide adenine dinucleotide
  • thiochrome reduced nicotinamide adenine dinucleotide
  • holo-lipoproteins holo-lipoproteins
  • holo-albumin contribute to the fluorescence spectrum of human plasma.
  • the oxidized form of nicotinamide adenine dinucleotide (NAD) exhibits no fluorescence in the 380-600 nm wavelength range. It has been documented, however, that the presence of HTV viruses results in a reduction of niacin and NADH through the effects of interferon gamma's induction of the catabolism of tryptophan, the precursor to niacin.
  • the fluorescent compounds extracted into the supernatant phase comprise NADH, which has been dissociated from proteins in the sample and is indicative of the presence of HTV.
  • the supernatant obtained from the heating step is better suited to spectroscopic analysis than untreated biological samples such as plasma. Removing large molecules such as proteins from the sample improves the spectroscopic quality of the sample by reducing spurious scattering and fluorescence signals associated with these molecules. Additionally, dissociating the compounds from the proteins may increase the fluorescence yield of these compounds, thereby, improving the ability to discriminate between a sample infected with a disease and a sample free of disease.
  • Acquisition of a fluorescence sample from the presently prepared samples comprises irradiating the sample with radiation having a sufficient energy for inducing a fluorescence emission from a predetermined compound, e.g., molecule.
  • the sample is irradiated with at least one wavelength of from about 270 to 750 nm, more preferably from about 270 to 400 nm.
  • Lasers such as, for example, Nd:YAG lasers that emit a fundamental or harmonic line in the ultraviolet are ideal excitation sources.
  • the resulting fluorescence emission may be measured quantitatively or qualitatively using a light sensitive detector.
  • a particularly useful fluorescence instrument comprises a dispersion element and a charge coupled device detector (CCD) for measuring the intensity of the emitted fluorescence as a function of the wavelength of the emitted light.
  • the detected fluorescence comprises radiation with at least one wavelength of from about 400 to 850 nm, more preferably of from about 400 to 600 nm.
  • Algorithms based on characteristics of the observed spectra may be used to further discriminate partially overlapped spectra.
  • a particularly powerful class of algorithms comprise multidimensional or multivariate techniques, which simultaneously utilize multiple parameters from one or more spectra. Combining multiple parameters into a single analysis or algorithm increases the predictive accuracy of the technique.
  • Related techniques such as neural network algorithms can be "taught" to discriminate a diseased or infectious sample from disease free or infection free samples. Each of these techniques is suitable for use with the sample preparation method of the present invention.
  • the supernatant of the present invention may be treated to a chromatographic separation, such as, for example, liquid chromatography or electrophoresis, in order to further enhance the difference between a diseased or disease-free sample.
  • a chromatographic separation such as, for example, liquid chromatography or electrophoresis
  • microfabricated devices such as those described in U.S. Patent No. 5,858,195 to Ramsey discloses systems and methods for performing fluid manipulations in channels etched in a microfabricated substrate. Within such devices, electrokinetic pumping allows rapid mixing, reaction, and separation of minute quantities of samples, reagents, and products.
  • the method of the present invention may be adapted for use on such devices and, following formation of the supernatant, spectroscopic detection of the compounds may be accomplished with or without a separation step.
  • a sample of plasma from a Hepatitis C infected individual and a control sample from an individual free of Hepatitis C were obtained.
  • a fluorescence instrument comprising an ultra-violet excitation source and a multiwavelength detector was used to obtain a Hepatitis C fluorescence spectrum 1 and a control fluorescence spectrum 2 from the Hepatitis C infected sample and Hepatitis C free control sample, respectively.
  • Spectra 1,2 were obtained prior to treatment with the method of the present invention. The spectra are shown in Figure 1. Although Hepatitis C spectrum 1 exhibits a somewhat larger intensity than control spectrum 2, the spectra are not substantially resolved and it is difficult to discriminate between the infected and non-infected samples on the basis of these spectra. Subsequently, a portion of each plasma sample was treated separately according to the following steps:
  • the clear supernatant obtained from the Hepatitis C plasma and the control plasma were subjected to fluorescence analysis as described above to obtain a treated Hepatitis C spectrum 3 and a treated control spectrum 4, respectively, as shown in Figure 2.
  • a peak amplitude and area of treated Hepatitis C spectrum 3 are both significantly greater than a peak amplitude and area of treated control spectrum 4.
  • the treated Hepatitis C spectrum exhibits a red shift (shift to longer wavelengths) compared to the treated control spectrum.
  • the treating the samples according to the present invention provides the ability to more selectively distinguish between positive and negative samples. Additionally, the present invention provides a more sensitive detection of
  • HTV infection and other diseases particularly at the early stage of the infection when the infection is undetectable by conventional methods.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Hematology (AREA)
  • Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Urology & Nephrology (AREA)
  • Food Science & Technology (AREA)
  • Analytical Chemistry (AREA)
  • Microbiology (AREA)
  • Pathology (AREA)
  • Biotechnology (AREA)
  • Virology (AREA)
  • General Physics & Mathematics (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Cell Biology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • AIDS & HIV (AREA)
  • Communicable Diseases (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

Cette invention concerne un procédé d'obtention d'un échantillon biologique pour la détection d'un état pathologique ou d'une infection tel que le VIH, l'hépatite A, l'hépatite B, l'hépatite C, seuls ou combinés. Ce procédé fait intervenir un liquide surnageant renfermant une dose détectable de composés libres, la présence desdits composés pouvant indiquer l'existence d'un état pathologique ou une infection.
PCT/US2001/021981 2000-07-13 2001-07-12 Extraction de fluorophores a partir d'echantillons biologiques WO2002005698A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2001273404A AU2001273404A1 (en) 2000-07-13 2001-07-12 Method for extracting fluorophores from biological samples

Applications Claiming Priority (2)

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US61636300A 2000-07-13 2000-07-13
US09/616,363 2000-07-13

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WO2002005698A2 true WO2002005698A2 (fr) 2002-01-24
WO2002005698A3 WO2002005698A3 (fr) 2003-09-25

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8240513B2 (en) 2008-03-24 2012-08-14 Fluid Management Operations Llc Fluid dispenser with nested displacement members

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5695756A (en) * 1990-08-31 1997-12-09 University Of Maryland At Baltimore Methods for treating hypertension
AU1075197A (en) * 1995-11-14 1997-06-05 Mark A. Goodwin Efficient method of detecting an infectious agent in blood

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8240513B2 (en) 2008-03-24 2012-08-14 Fluid Management Operations Llc Fluid dispenser with nested displacement members

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AU2001273404A1 (en) 2002-01-30
WO2002005698A3 (fr) 2003-09-25

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