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US20020072590A1 - Methods and kits for separation and detection of proteins in biological samples - Google Patents

Methods and kits for separation and detection of proteins in biological samples Download PDF

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US20020072590A1
US20020072590A1 US09/934,297 US93429701A US2002072590A1 US 20020072590 A1 US20020072590 A1 US 20020072590A1 US 93429701 A US93429701 A US 93429701A US 2002072590 A1 US2002072590 A1 US 2002072590A1
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proteins
serum
subject
biological sample
protein
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Jennifer Van Eyk
Ralf Labugger
Irena Neverova
David Colantonio
Jeremy Simpson
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Queens University at Kingston
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Assigned to QUEEN'S UNIVERSITY AT KINGSTON reassignment QUEEN'S UNIVERSITY AT KINGSTON ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: COLANTONIO, DAVID A., EYK, JENNIFER E. VAN, LABUGGER, RALF, NEVEROVA, IRENA, SIMPSON, JEREMY A.
Publication of US20020072590A1 publication Critical patent/US20020072590A1/en
Priority to US11/138,184 priority patent/US7709193B2/en
Priority to US12/771,351 priority patent/US8481274B2/en
Abandoned legal-status Critical Current

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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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • 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
    • G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6887—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids from muscle, cartilage or connective tissue
    • 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
    • G01N2333/4712—Muscle proteins, e.g. myosin, actin, protein
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2550/00—Electrophoretic profiling, e.g. for proteome analysis

Definitions

  • the present invention relates to methods and kits for separating a mixture of proteins in a biological sample by mixing the biological sample with a solution which substantially denatures proteins in the biological sample and subjecting the resulting mixture to a protein separation technique. Following separation, proteins of interest are preferably subjected to characterization.
  • the method of the present invention is useful in the development of highly sensitive analytical and diagnostic assays and kits for assessing cellular damage and diagnosing cellular injury in biological samples.
  • the presence and quantity of particular proteins can be used to detect and determine the extent or severity of a disease or other abnormality in an individual.
  • the blood of an animal i.e., a wild type or a transgenic animal
  • a protein of interest e.g., the product of a transgene
  • protein separation and characterization can be carried out using any convenient method(s), such as, for example, electrophoresis (e.g., sodium dodecyl sulfate-polyacrylamide gel electrophoresis or SDS-PAGE) and subsequent immunoblotting (e.g., Western blotting).
  • electrophoresis e.g., sodium dodecyl sulfate-polyacrylamide gel electrophoresis or SDS-PAGE
  • immunoblotting e.g., Western blotting
  • albumin blood's primary carrier protein
  • FIG. 1 The effectiveness of analysis of serum is also limited because such serum proteins often bind to marker proteins, and hence interfere with the migration of the marker proteins on a gel. For an effective analysis, therefore, a marker protein must be separated from any other serum protein that may interfere with its migration on the gel.
  • ELISAs enzyme linked immunosorbent assays
  • binding of antibody to a target epitope on the marker protein can be inhibited by the target epitope being hidden by the binding of other proteins to the marker protein.
  • the binding by other serum proteins may reduce access to target epitope enough to cause false negative results.
  • the myofilament proteins cardiac troponin I (cTnI) and cardiac troponin T (cTnT) are biochemical cardiac markers frequently used in the assessment of acute coronary syndrome (ACS) and other myocardial injuries.
  • cTnI and cTnT are not present in the blood of normal, healthy individuals.
  • elevated marker levels have been found in the blood in cases of congestive heart failure (Missov et al. Circ. 1997 96:2953-2958; Missov and Mair Am. Heart J. 1999 138:95-99), unstable angina (Ottani et al. Am. Heart J.
  • kits may or may not detect all of the cTnI depending upon the combination of antibodies provided.
  • commercial kits can not differentiate whether or not modified forms of cTnI are present.
  • these kits identify which forms of cTnI are in the sample.
  • Another problem is that proteins in serum are bound to each other as well as to other proteins which can mask or hide epitopes, thus rendering the proteins undetectable by the antibodies of the kit.
  • AMD acute myocardial infarction
  • the current cTnI kits do not detect all of the intact cTnI and modification products thereof that are released into the serum.
  • cTnI kits are not always capable of detecting intact cTnI and modification products thereof in cardiac patients who are not experiencing an AMI, as the commercially available assays appear to have low analytical and diagnostic sensitivity. These patients can be discharged from emergency with a diagnosis of chest pain/not yet diagnosed.
  • An object of the present invention is to provide a method of separating a mixture of proteins in a biological sample comprising mixing the biological sample with a solution comprising a sulfhydryl reducing agent, a anionic detergent, and at least one detergent selected from the group consisting of an ionic detergent, a non-ionic detergent and a zwitterionic detergent, at concentrations sufficient to substantially denature proteins in the biological sample, and subjecting the mixture to a separation technique.
  • the proteins are further subjected to characterization.
  • kits of the present invention comprise a solution containing a sulfhydryl reducing agent, an anionic detergent, and at least one detergent selected from the group consisting of another ionic detergent, a non-ionic detergent and a zwitterionic detergent, and instructions for separating proteins in said biological sample.
  • kits of the present invention further comprise a means for characterization of a separated protein or proteins.
  • Another object of the present invention is to provide methods for producing a profile of proteins in a biological sample which comprises mixing the biological sample with a solution comprising a sulfhydryl reducing agent, an anionic detergent, and at least one detergent selected from the group consisting of an ionic detergent, a non-ionic detergent and a zwitterionic detergent, at concentrations sufficient to substantially denature proteins in the biological sample, and subjecting the mixture to separation and characterization so as to produce a profile of proteins in said biological sample.
  • kits for producing a profile of proteins in a biological sample comprise a solution containing a sulfhydryl reducing agent, an anionic detergent, and at least one detergent selected from the group consisting of another ionic detergent, a non-ionic detergent and a zwitterionic detergent, and instructions for separating proteins in said biological sample.
  • Kits of the present invention further comprise a means for characterization of the separated proteins so that a profile of proteins in the biological sample can be generated.
  • a biological sample such as serum containing a mixture of proteins from a subject is mixed with a solution comprising a sulfhydryl reducing agent, an anionic detergent, and at least one detergent selected from the group consisting of an ionic detergent, a non-ionic detergent and a zwitterionic detergent, at concentrations sufficient to substantially denature proteins in the biological sample.
  • a solution comprising a sulfhydryl reducing agent, an anionic detergent, and at least one detergent selected from the group consisting of an ionic detergent, a non-ionic detergent and a zwitterionic detergent, at concentrations sufficient to substantially denature proteins in the biological sample.
  • the mixture is then subjected to separation and the separated proteins are characterized so that proteins indicative of damage to the cell or cells in the subject can be detected.
  • a profile can now be generated from a serum sample of a subject which is indicative of a distinct cardiovascular condition or elapsed time after onset of an AMI or severity of an infarct or reinfarction.
  • the methods and kits of the present invention can also be used for detection of a single myofilament protein in a biological sample such as serum for early clinical assessment, ongoing monitoring of chronic conditions and/or diagnosis of myocardial damage in subjects, particularly subjects with non-diagnostic electrocardiograms and/or where routine clinical testing shows non-significant elevations of biochemical cardiac markers.
  • these methods and kits can be used to monitor the state of the myocardium in a subject by monitoring myofilament protein levels, preferably myofilament protein modifications, in serum.
  • the methods and kits can be used to detect and monitor skeletal muscle damage in a biological sample of a subject.
  • myofilament proteins were detectable in serum of a subject following skeletal muscle damage, e.g. respiratory muscle injuries.
  • damage to skeletal muscle, as well as healing and regeneration of the skeletal muscle following muscle damage can be monitored.
  • the ratio of two different isoforms of a skeletal myofilament protein such as TnI or TnT, are monitored to assess the severity of a disease involving skeletal muscle.
  • the ratio of the fast form of TnI versus the slow form of TnI can be measured to diagnosis and monitor the state and type of skeletal muscle damage in a subject.
  • the methods and kits can also be used to differentiate between various isoforms of a protein such as between a cardiac and skeletal myofilament protein.
  • the method is performed on serum samples and the characterization following separation is performed by western-blot.
  • This preferred method is referred to herein as Western Blot-Direct Serum Analysis or WB-DSA.
  • FIG. 1 provides a comparison of a human serum sample separated in accordance with the method of the present invention prior to loading (lane A) and the same human serum sample treated with SDS as the only detergent in the loading buffer (lane B). Both samples were subjected to SDS-PAGE and stained with Coomassie blue.
  • FIG. 2 shows a spectrum of cTnI and cTnT modifications found in AMI patient serum.
  • cTnI and its modification products in AMI patient serum were detected by the anti-cTnI mAb 8I-7.
  • WB-DSA of serial serum samples obtained from 5 patients is shown in panels a, b, c, d and e.
  • the samples from patient 1 were also probed for cTnT and its degradation products (cTnT Degn) with an anti-cTnT pAb (panel a, lower).
  • FIG. 3 shows the characterization of cTnI modification products present in AMI patient serum.
  • WB-DSA using 4 anti-cTnI antibodies to different epitopes on cTnI are shown for native patient serum (N) and for native serum following dephosphorylation (D).
  • Antibodies used are listed beneath their corresponding blots with their epitope in subscript. The relative positions of molecular weight markers are indicated to the left.
  • FIG. 4 shows results from a stability study with human recombinant cTnI (rcTnI).
  • FIG. 5 shows human serum from a patient with respiratory muscle (diaphragm) injury (Patient 1) and human serum from a patient with a limb injury (Patient 2).
  • Samples were pre-treated according to separation method of the present invention, subjected to SDS-PAGE and visualized by western blotting with anti-skeletal TnI antibody.
  • FIG. 6 provides a western blot from serum analyzed by WB-DSA from a patient undergoing thrombolytic therapy (TPA) for a blocked coronary artery. Only intact cTnI was observed in the patient's serum prior to treatment. Upon reperfusion, however, degradation products were also observed.
  • TPA thrombolytic therapy
  • FIG. 7 provides a western blot from serum analyzed by WB-DSA of serial time points (initial sample time point designated time zero) of a patient with the respiratory muscle dysfunction chronic obstructive pulmonary disorder (COPD) (FIG. 7A and FIG. 7C) and a patient with rhabdomyolysis (FIG. 7B and FIG. 7D).
  • COPD chronic obstructive pulmonary disorder
  • skeletal troponin I (skTnI) was detected in serum at all time points in the patient with COPD and the patient with rhabdomyolysis, respectively.
  • mAb F-32 did not reveal skTnI in the first time point of the patient with COPD (FIG. 7A) and mAb 3I-35 did not reveal skTnI in any time point of the patient with rhabdomyolysis (FIG. 7D). Blots depicted in FIGS.
  • FIGS. 7A and 7D were prepared using longer exposures (overnight versus 1 hour) than those depicted in FIGS. 7B and 7C, to increase the ability to detect any cTnI.
  • the blot depicted in FIG. 7D shows cross reactivity with IgG.
  • FIG. 8 provides a western blot of serum analyzed by WB-DSA of serial time points (initial sample time point designated time zero) of a patient with respiratory muscle dysfunction (COPD) (FIGS. 8A and 8C) and a patient with rhabdomyolysis (FIGS. 8B and 8D).
  • Blots depicted in FIGS. 8A and 8B were prepared with mAb F-32 specific for the fast isoform of skTnI (Spectral Diagnostics Inc., Toronto);
  • blots depicted in FIGS. 8C and 8D were prepared with a mAb specific for the slow isoform of skTnI (Matsumoto et al. Biotech. Histochem. 1997 72(4):191-7).
  • the fast, but not the slow, isoform of skTnI was detected in the patient with COPD while both isoforms were detected in the patient with rhabdomyolysis.
  • FIG. 9 provides a western blot of serum analyzed by WB-DSA of serial time points (initial sample time point designated time zero) of a patient with rhabdomyolysis.
  • FIG. 9A is a blot wherein the antibody is an anti-skeletal troponin I mAb F-32 (Spectral Diagnostics Inc., Toronto) specific for the fast isoform.
  • FIG. 9B is a blot show prolonged exposures of three time points from FIG. 9A.
  • FIG. 9C is a blot wherein the antibody is anti-skeletal troponin I mAb specific for the slow isoform (Matsumoto et al. Biotech. Histochem. 1997 72(4):191-7). Both the fast and slow isoforms of TnI were detected in this patient. As shown in FIG. 9B, some modification products were present at specific time points during the progression of the disease (proteolytic fragment designated by arrow). Changes in the total amounts of the protein, as well as changes in the ratio of the fast and slow isoforms, were also detected.
  • the present invention provides methods for separating a mixture of proteins in a biological sample by mixing the biological sample with a solution which substantially denatures proteins in the biological sample and subjecting the resulting mixture to a protein separation technique. These methods are useful in the detection of single proteins in a biological sample as well as in profiling a number of proteins in the sample.
  • biological sample it is meant to include, but is not limited to, serum, plasma, urine, milk, lymph, amniotic fluid, semen and cerebrospinal fluid.
  • biological sample is serum.
  • the present invention is based, at least in part, on the discovery that the confounding effect of other serum proteins binding to protein(s) of interest can be overcome by subjecting the serum to strong denaturing and sulfhydryl reducing conditions. Accordingly, the present invention provides methods for separating proteins of a biological sample such as serum by mixing the serum with a solution comprising a sulfhydryl reducing agent, an anionic detergent (preferably sodium dodecyl sulfate, SDS), and at least one detergent selected from the group consisting of an ionic detergent, a non-ionic detergent and a zwitterionic detergent.
  • the solution comprises the sulfhydryl reducing agent and the detergents at concentrations sufficient to substantially denature proteins in the mixture.
  • the mixture of serum and solution comprising the thus-denatured proteins is then subjected to separation, and optionally further characterization.
  • the serum and solution mixture is heated after the mixing step and prior to the separation step, and the concentrations of the sulfhydryl reducing agent and the selected detergents in the solution are sufficient to substantially denature proteins in the serum and solution mixture when heated. Still more preferably, the serum and solution mixture is boiled, and the concentrations of the sulfhydryl reducing agent and the selected detergents are sufficient to substantially denature proteins when the serum and solution mixture is boiled.
  • Separation may be performed using any convenient means.
  • SDS-PAGE is used.
  • other separation techniques including, but not limited to, capillary electrophoresis, fast system mini-gels, affinity chromatography, ion exchange chromatography and reverse phase chromatography (conventional or high performance) can also be used.
  • gel filtration, capillary electrophoresis or some other separation technique care must be taken to maintain the proteins of the mixture in a state where they do not become complexed with undesirable proteins such as albumin. That is, the proteins should preferably be maintained in a substantially denatured state. This may be achieved by employing buffer containing detergent and/or denaturing and/or reducing agents.
  • the protein of interest having been separated from other (sticky) proteins present in the serum starting material, may be renatured, e.g., by dialyzing into buffer that does not contain a denaturing agent.
  • the solution used in the separation method of the present invention comprises a sulfhydryl reducing agent, an anionic detergent, and at least one of an ionic detergent, a non-ionic detergent or a zwitterionic detergent.
  • the solution comprises a sulfhydryl reducing agent, an anionic detergent, a non-ionic detergent and a zwitterionic detergent.
  • the solution further comprises urea or thiourea.
  • Concentrations of the components of the solution must be sufficient to achieve substantial, or complete denaturation.
  • 1 to 2% SDS in conjunction with a reducing agent such as BME, is commonly employed in the art in SDS-PAGE loading buffer.
  • these concentrations are insufficient to completely denature serum proteins such as albumin so that proteins in the sample are well resolved by SDS-PAGE. Rather, the proteins appear as a huge smear on the resulting gel (see FIG. 1).
  • simply increasing the SDS concentration of standard loading buffer to 4% fails to produce any improvement in resolution.
  • the addition of one or more detergents as set forth in the present invention provides a substantial increase in resolution of proteins.
  • protein is intended to mean any protein, polypeptide, peptide, or fragment thereof, as well as protein-modification products, such as, for example, phosphorylated proteins, glycosylated proteins, radio-iodinated proteins, and the like. Further, by use of the term protein it is meant to be inclusive of one or more proteins. By protein it is also meant to be inclusive of post-translationally modified proteins including not only phosphorylation of amino acid residues, but also of other chemical adducts.
  • Chemical adducts known in the art relating to post-translational modification of proteins which can be separated using the present invention include, but are not limited to, phosphorylation, glycosylation, myristylation, phenylation, acetylation, nitrosylation, s-glutathiolation, amidation, biotinylation, c-mannosylation, flavinylation, farnesylation, formylation, geranyl-geranylation, hydroxylation, lipoylation, methylation, palmitoylation, sulphation, gamma-carboxyglutamic acids, N-acyl diglyceride (tripalmitate), O-GlcNAc, pyridoxal phosphate, phospho-pantetheine, and pyrrolidone carboxylic acid.
  • Preferred chemical adducts are phosphorylation, glycosylation, myristylation, phenylation, acetylation, nitrosylation, and sulphation.
  • modifications it is meant to be inclusive of both naturally occurring modifications and artificially induced modifications.
  • substantially denatured is intended to mean that at least 90% of the protein or proteins in the biological sample is denatured.
  • the term “characterizing” or “characterization” is intended to encompass detecting, identifying, profiling, and quantifying one or more proteins in a biological sample, by any means known in the art. The presence or absence of a chosen protein, or its level relative to the level of another protein or proteins, or a change in the level of a protein over time, may be determined. Accordingly, the invention provides profiles that are characteristic of certain physiological conditions, wherein the levels of one, two, three or more proteins or protein-modification products present in a biological sample such as serum or plasma are determined relative to each other. Any convenient technique for characterization may be employed. In a preferred embodiment, gel electrophoresis following by further analysis by immunoblotting, preferably Western blotting is used. This preferred method of the present invention is referred to herein as Western Blot-Direct Serum Analysis or WB-DSA.
  • WB Western blotting
  • SDS-PAGE SDS-polyacrylamide gel
  • the resolved proteins in the gel are transferred (by electroblotting) to a paper-thin nitrocellulose membrane which binds most proteins.
  • the protein or proteins of interest is detected on the protein-studded membrane.
  • the protein-studded membrane is soaked in a solution of antibodies that are specific for the protein or proteins of interest. In one embodiment, antibodies in this solution can be labeled for easy detection of protein-bound antibodies.
  • the protein-bound antibodies can be detected using a second antibody that is specific for the first.
  • This second antibody may be bound to a fluorescing enzyme that is detected using radiographic film or a calorimetrically detectable enzyme such as horse radish peroxidase or alkaline phosphatase.
  • Modified proteins may also be detected by P32 labeling or lectin binding to carbohydratess. More recently, a technique has been developed referred to as UNIBLOT (Pierce Chemicals) which allows the western blot to be performed directly on the gel without the need for transfer of the resolved proteins to a nitrocellulose or PVDF membrane.
  • the WB-DSA procedure used in the present invention overcomes problems limiting the application of SDS-PAGE to serum. Specifically, large quantities of albumin and IgG in serum hamper migration within a polyacrylamide gel and limit the sample volume that can be applied to the gel. These large quantities of proteins overwhelm the small amounts of proteins which may be present in early clinical diagnosis of diseases such as myocardial damage. Using the method of present invention to separate proteins of a biological sample, however, only 1 to 3 ⁇ l, preferably 2 ⁇ l of serum is required for reliable detection of proteins such as myofilament proteins in serum.
  • the gel electrophoresis be performed under denaturing and reducing conditions.
  • a sample buffer containing 0.33% SDS, 0.33% CHAPS, 0.33% NP-40, 0.1 M DTT, 4 M urea, and 50 mM Tris-HCl, pH 6.8 in 50% glycerol is used.
  • Serum is diluted, preferably about 12.5 times, in the sample buffer to prevent precipitation of serum proteins during boiling. Diluted samples are then preferably boiled for 10 minutes to assure separation of the myofilament proteins from serum proteins and to break-up binary and ternary complexes.
  • nitrocellulose 45 Micron, Micton Separation Inc., Westborough, Mass.
  • PVDF membranes such as Immobilon-P (Millipore) can be used and the transfer performed in a buffer containing 25 mM Tris-HCl, 192 mM glycine, and 20% methanol for 1.5 hours at 200 mA. Thereafter, membranes are blocked overnight at 4° C. in 10% blocking reagent (Boehringer Mannheim, Mannheim, Germany).
  • kits for separating a mixture of proteins in a biological sample are useful in the detection of one or more proteins in a biological sample and in the profiling a more than one protein in a biological sample.
  • kits of the present invention comprise a solution containing a sulfhydryl reducing agent, an anionic detergent, and at least one detergent selected from the group consisting of an ionic detergent, a non-ionic detergent and a zwitterionic detergent, and instructions for separating proteins in the biological sample.
  • Kits of the present invention preferably further comprise a means for detecting one or more proteins in the sample or a means for profiling various proteins in the sample.
  • Suitable sulfhydryl reducing agent which can be used in the solution of the methods and kits of the present invention include, but are not limited to, dithiothreitol (DTT), dithioerythritol (DTE) and ⁇ -mercaptoethanol (BME).
  • DTT dithiothreitol
  • DTE dithioerythritol
  • BME ⁇ -mercaptoethanol
  • a suitable zwitterionic detergent that can be used in the methods and kits of the present invention is 3[(3-chlolamidopropyl)dimethyl-ammonio]-1-propane sulfonate (CHAPS), and a suitable non-ionic detergent is ethylphenolpoly(ethylene-glycolether) n (Igepal CA-630, formerly known as Nonidet P-40 or NP-40). Additional suitable zwitterionic detergents include, without limitation, N-alkyl-N,N-dimethylammonio-1-propanesulfonates.
  • non-ionic detergents include, without limitation, Triton X-100, Triton X-114, n-octyl-glucoside, digitonin, Tween, Tween 20, Tween 80, and saponin.
  • the detergents can be combined in equal portions, or the portions can be optimized for a specific application through routine experimentation.
  • the detergents can be combined in equal portions, or the portions can be optimized for a specific application through routine experimentation.
  • a preferred solution comprises about 70 mM SDS and about 100 mM DTT. More preferred is a solution comprising the components as set forth in Table 1.
  • TABLE 1 Serum Protein Separating Solution Preferred Concentration Range Component for 12.5X dilution SDS about 5 mM to about 150 mM; preferably about 25 mM to about 100 mM CHAPS about 5 mM to about 50 mM; preferably about 5 mM to about 25 mM Igepal CA-630 about 0.2% to about 4%; preferably about 1% to about 2% DTT about 5 mM to about 150 mM; preferably about 50 mM to about 120 mM Urea about 0.2M to about 8M; preferably about 1M to about 2M buffer solution low salt, inorganic, neutral pH; e.g., 50 mM Tris-HCl, pH 6.8
  • FIG. 1 shows a Coomassie blue-stained gel of human serum prepared for SDS-PAGE according to a preferred embodiment of the invention (i.e., with SDS, CHAPS, Igepal CA-630, urea, and DTT), there is distinct separation (resolution) of the proteins (compare with FIG. 1 (lane B), described above).
  • the method of the present invention has been used to characterize myofilament proteins as well as their modification products in a serum sample to assess muscle damage in a subject.
  • a serum sample from the subject is first obtained.
  • the serum sample is then mixed with a solution comprising a sulfhydryl reducing agent, an anionic detergent, and at least one detergent selected from the group consisting of an ionic detergent, a non-ionic detergent and a zwitterionic detergent, at concentrations sufficient to substantially denature albumin in the mixture.
  • Myofilament proteins which can be characterized in serum include, but are not limited to, troponin I, troponin T, myosin light chain 1, myosin light chain 2, actin, actinin, desmin, caldesmin, titin, protein C and calponin, as well as modification products thereof. Damage may be assessed in both cardiac and skeletal muscle as evidenced by FIGS. 2, 3 and 5 , and the characterized proteins (profile) provide an indication of damage or potential damage, the disease or condition causing the damage, and the state or condition of the subject with the disease or condition.
  • FIG. 2 provides western blots from serum for 5 representative AMI patients subjected to WB-DSA.
  • WB-DSA In addition to intact cTnI, as many as 8 truncated degradation products and 3 products of higher molecular weight were observed.
  • the number and extent of cTnI modifications in each patient changed throughout the time course following infarction.
  • This profile of the visually detectable cTnI modification products, as well as their intensity, as indicated by WB-DSA corresponded with the time profiles of serum CK, CKMB and cTnI, as determined by CX7 and Immunol.
  • the method of the present invention allows for the direct detection of cTnI and cTnT in serum from patients with diagnosed AMI.
  • the method of the present invention provides means to obtain characteristic profiles of proteins such as troponin modification products or a distinct pattern of products over time, which can indicate a distinct cardiovascular condition or a specific elapsed time after onset of an AMI or potentially the severity of an infarct or reinfarction.
  • this invention provides a new immunological diagnostic tool for measuring the variety of forms of troponin in a patient's blood which is useful not only to detect myocardial damage, but also to provide more information about the condition of the diseased myocardium and its viability. This provides for therapeutic applications, and a more differentiated risk stratification of patients with acute coronary syndromes.
  • the methods and kits of the present invention can also be used to monitor damage to skeletal muscle, as well as healing and regeneration of the damaged skeletal muscle.
  • damage to skeletal muscle it is meant to be inclusive of disease, injury and/or fatigue.
  • Examples of skeletal muscle damage which can be diagnosed, monitored and differentially diagnosed from other cell injury via the present invention include, but are not limited to, exercise, traumatic injury including surgery and injury to a limb, muscle wasting during atrophy, sepsis, ischemia, asthma, fatigue, COPD, rhabdomyolosis, and acute respiratory distress syndrome (ARDS).
  • ARDS acute respiratory distress syndrome
  • the ratio of isoforms of a skeletal myofilament protein such as TnI or TnT, are monitored to diagnose and/or assess the severity of a disease involving skeletal muscle.
  • the ratio of two different isoforms of a myofilament protein can also be used to distinguish between different disease types, also referred to herein as differential diagnosis.
  • serum skeletal TnI (skTnI) levels were measured using WB-DSA in two patients with rhabdomyolysis (one patient, FIGS. 7 and 8; a second patient, FIG. 9), and in a patient with the respiratory disorder COPD (FIGS. 7 to 9 ).
  • the ratio of detectable isoforms of skTnI or shTnT may differ between certain types of skeletal muscle damage as well as during progression and/or healing of the damage as demonstrated in FIGS. 8 and 9.
  • the respiratory condition COPD only fast TnI was detefcted.
  • the ratio of the fast form of TnI versus the slow form of TnI can be measured to diagnosis and monitor the state or type of skeletal muscle damage in a subject.
  • Ratios of other isoforms of myofilament proteins which can be measured to diagnosed and monitor the state or type of muscle damage in a subject include, but are not limited to, cardiac troponins versus skeletal troponins, fetal cTnT versus adult cTnT, and ventricle MLC1 versus atrial MLC1.
  • the invention further provides means to distinquish between different disease types.
  • panels A & C are western blots from a respiratory patient, probed respectively with mAb F-32 and mAb 3I-35; panels B & D are similar Western blots from a rhabdomyolysis patient.
  • F-32 detects fast skeletal TnI in both patients
  • 3I-35 detects this protein only in the respiratory patient.
  • possibly binding of the antibody in this patient is inhibited by a rhabdomyolysis-specific post-translational modification.
  • absence of signal could be indicative of level or severity of disease.
  • differential diagnosis is conveniently provided according to the invention. Such differential diagnosis may also be provided for a variety of other disease states or conditions, potentially exercise, traumatic injury including surgery (e.g., muscle cut during surgery), muscle wasting during atrophy.
  • kits of the present invention have also been demonstrated to be useful for early clinical assessment and/or diagnosis of myocardial damage in subjects by detection of low levels of serum myofilament proteins and for monitoring the state of the myocardium in a patient by monitoring myofilament protein levels, preferably myofilament protein modifications, in serum.
  • myocardial damage for purposes of the present invention, it is meant to be inclusive of any and all types of acute and chronic injury to the heart muscle tissue.
  • myocardial damage include, but are in no way limited to, damage resulting from early and/or end stage heart failure, hypertension, arteriosclerosis, congestive heart failure, viral attack of the heart muscle, stunning, unstable angina, stable angina, thrombolytic treatment, heart transplant, drug toxicity and sepsis.
  • ERTAIN clinical assessment and/or diagnosis it is meant that myocardial damage is detectable in a patient before the damage results in, causes, or is detectable by ECG and/or where routine clinical testing shows non-significant elevations of biochemical cardiac markers, or when the damage is only minor, such as that resulting from some drug toxicities or sepsis.
  • routine clinical testing it is meant to be inclusive of commercially available assays such as those for CK-MB, CK, Troponin I (TnI) and Troponin T (TnT).
  • WB-DSA Western Blot-Direct Serum Analysis
  • the analytical sensitivity (the lowest detectable limit) and specificity for detection of myofilament proteins such as cTnI in serum has implications for earlier detection of cardiac injury associated with acute myocardial infarction, better risk stratification for patients with ACS, and earlier disposition of patients with chest pain of non-cardiac origins.
  • the increased analytical sensitivity of this method also allows for the development of screening assays to monitor for the presence of myofilament proteins in the serum of patients with coronary artery disease and hypertension, permitting risk stratification and/or customization of treatment strategies.
  • detecting the presence of myofilament proteins such as cTnI and/or cTnT and/or myosin light chain 1 and/or modification products thereof by the method of the present invention in a patient undergoing anti-thrombolytic therapy is useful in assessing and/or monitoring for clearance of a clot from the coronary arteries.
  • antibodies useful in the detection of myofilament proteins in serum via WB-DSA include, but are not limited to, anti-cTnI antibodies such as mAb 8I-7 (amino acid residues 136-154) and 3E3 (residues 1-54, both from Spectral Diagnostics Inc., Toronto, Canada, used at a concentration of 0.5 ⁇ g/ml), pAb P1 (residues 1-26, Bios Pacific, Emeryville, Calif., 0.5 ⁇ g/ml); and mAb 10F2 (residues 188-199, Sanofi Diagnostics Pasteur, Marnes-la-Coquette, France, 0.25 ⁇ g/ml); anti-cTnT antibodies such as P1-P3 (BiosPacific, Emeryville, Calif.); JTL-12 (Sigma Chemical Co., St.
  • HRP horseradish peroxidase
  • rabbit anti-goat IgG both from Jackson Laboratories, West Grove, Pa.
  • Serum cTnI levels were determined using WB-DSA in 10 patients in the emergency department of a teaching hospital complaining of chest pain. In 6 out of 10 cases, cTnI was detected, using Western Blot-Direct Serum Analysis, in the serum of patients presenting with non-diagnostic ECG, where routine clinical testing showed non-significant elevations of biochemical cardiac markers.
  • Patient 2 was a 73-year-old male, with a family history of cardiovascular disease, who had experienced a myocardial infarction within the last six months. He presented to emergency with chest pain of 1.5 hours duration, but with no other associated cardiac symptoms. The results of his ECG showed inferior-lateral T wave inversion with inferior Q waves. CK, CK-MB, and cTnI values determined by commercially available kits were not significantly elevated. Analysis of serum samples by WB-DSA from patient 2 showed cTnI to be present at admission and to remain detectable throughout, until the last sample was taken 21 hours later. This patient was discharged from the hospital with the diagnosis of unstable angina.
  • results from these patients demonstrate the superior analytical sensitivity, compared to routine clinical tests, of WB-DSA in the detection of myofilament proteins such as cTnI (and its modification products) in the serum of patients presenting with symptoms of ACS.
  • this method allowed for earlier detection of a myofilament protein, in this example cTnI, in serum of patients with symptoms of ACS, compared to routine clinical tests.
  • WB-DSA was capable of detecting this myofilament protein in serum.
  • ACS represents a spectrum of cardiac pathophysiology
  • unique patterns of myofilament protein modifications may also be detectable in serum using WB-DSA at various points along this spectrum.
  • the status of cTnI and/or cTnT and their patterns of modifications in serum reflect the state of the myocardium, since cTnI and/or cTnT can be modified in the myocardium prior to release into the circulation.
  • the method for monitoring the state of the myocardium of a patient by monitoring degradation of myofilament proteins such as cTnI and/or cTnT in their serum by WB-DSA is also provided by the present invention.
  • Polyacrylamide gel electrophoresis was performed under denaturing and reducing conditions using a sample buffer containing 0.33% SDS, 0.33% CHAPS, 0.33% NP-40, 0.1 M DTT, 4 M urea, and 50 mM Tris-HCl, pH 6.8 in 50% glycerol.
  • Serum was diluted 12.5-times in sample buffer to prevent precipitation of serum proteins during boiling. Diluted samples were then boiled for 10 minutes, to assure separation of the troponins from serum proteins and to break-up binary and ternary complexes.
  • Western blot analysis was then carried out with the following anti-cTnI antibodies (with epitopes to): mAb 8I-7 (amino acid residues 136-154) or 3E3 (residues 1-54, both from Spectral Diagnostics Inc., Toronto, Canada, used at a concentration of 0.5 ⁇ g/ml); pAb P1 (residues 1-26, Bios Pacific, Emeryville, Calif., 0.5 ⁇ g/ml); mAb 10F2 (residues 188-199, Sanofi Diagnostics Pasteur, Marnes-la-Coquette, France, 0.25 ⁇ g/ml).
  • cTnT was probed with pAb anti-cTnT (residues 3-15, Bios Pacific, Emeryville, Calif., 0.5 ⁇ g/ml), detecting all isoforms of cTnT.
  • Primary antibodies were detected with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG or rabbit anti-goat IgG (both from Jackson Laboratories, West Grove, Pa.) and signals visualized using chemiluminescence substrate (Boehringer Mannheim) and X-OmatTM Scientific Imaging Film (Eastman Kodak Company, Rochester, N.Y.). All antibodies were diluted in 1% blocking reagent and incubated for 1 hour at room temperature.
  • HRP horseradish peroxidase
  • dephosphorylation of serum has been performed as follows: one hundred units of calf intestinal alkaline phosphatase (AP, New England Biolabs, Beverly, Mass.) and 1.6 ⁇ l of 10 ⁇ dephosphorylation buffer (50 mM Tris, 100 mM NaCl, 10 mM MgCl 2 , 1 mM DTT pH 7.9) were added to 4 ⁇ l of serum (approx. 100 mg/ml serum proteins) and incubated for 30 minutes at 30° C. (1 unit of AP hydrolyses 1 nmole of p-nitrophenylphosphate/minute at 30° C. and pH 8.5). Reactions were terminated by addition of 4 ⁇ l of 5 ⁇ sample buffer and boiling for 5 minutes. The activity of AP in serum was confirmed by its ability to dephosphorylate 32 P-labeled myelin basic protein when added to normal serum.
  • 10 ⁇ dephosphorylation buffer 50 mM Tris, 100 mM NaCl, 10 mM MgCl 2 ,
  • Serum samples were obtained from a prospective case series of patients presenting, within four hours onset of symptoms of ACS, to a hospital emergency department. Serum samples of ten representative cases were selected from the first 45 cases of ACS enrolled, who also had non-diagnostic ECG and non-significant elevations in the biochemical cardiac markers CK, CK-MB, and cTnI using commercially available kits. Patients underwent a history and clinical examination, a 12 lead ECG was recorded and serial blood was drawn at presentation, and subsequently at 1, 2, 4, 6 and 16-24 hours for routine clinical testing of biochemical cardiac markers and for analysis by WB-DSA. Serum samples were stored at ⁇ 80° C. until analyzed. Final discharge diagnosis from the emergency department was based on standard criteria of history, physical examination, ECG changes, and biochemical cardiac markers.
  • CK-MB fraction A 20% elevation in CK values was considered to be a significant increase. Testing for the CK-MB fraction was considered negative if the concentration was ⁇ 8 ⁇ g/L and positive if the concentration was >8 ⁇ g/L, with a relative index (CK-MB ⁇ 100/CK) greater than 3%. For cTnI, the minimum detectable concentration reported for Immuno 1 is 0.1 ⁇ g/L.
  • serum from a healthy individual was spiked with human recombinant intact cTnI1-200 and cTnI1-209 (the primary cTnI degradation product observed in stunned myocardium from isolated hearts) (McDonough et al. Circ. Res. 1999 84:9-20; Gao et al. Circ. Res. 1997 80:393-399; Van Eyk et al. Circ. Res. 1998 82:261-271) and resolved alongside each patient's samples. Serum from each patient was also resolved and probed with only secondary antibody to control for cross reactivity with the patients' IgG.

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US20060135875A1 (en) * 2003-05-19 2006-06-22 Ischemia Technologies, Inc. Apparatus and methods for risk stratification of patients with chest pain of suspected cardiac origin
US7618782B1 (en) 1999-10-18 2009-11-17 Queen's University At Kingston Methods of diagnosing muscle damage
US20100184210A1 (en) * 2006-08-10 2010-07-22 Peter Rossmanith Method for isolating cells
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JP5179011B2 (ja) 2002-10-09 2013-04-10 ディーエムアイ バイオサイエンシズ インコーポレイテッド 胎盤虚血および多臓器不全のモニタリング
US9814422B2 (en) 2007-08-06 2017-11-14 The Regents Of The University Of California Compositions for solubilizing cells and/or tissue
EP2396654B1 (fr) * 2009-02-13 2016-02-10 The Regents of The University of California Composition et procédé diagnostic à partir de tissu

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