WO1998026293A1 - Procedes de diagnostic d'encephalopathies spongiformes transmissibles par detection d'isoformes proteiniques 14-3-3 - Google Patents
Procedes de diagnostic d'encephalopathies spongiformes transmissibles par detection d'isoformes proteiniques 14-3-3 Download PDFInfo
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- WO1998026293A1 WO1998026293A1 PCT/US1997/022820 US9722820W WO9826293A1 WO 1998026293 A1 WO1998026293 A1 WO 1998026293A1 US 9722820 W US9722820 W US 9722820W WO 9826293 A1 WO9826293 A1 WO 9826293A1
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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
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
- G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
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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
- G01N2800/28—Neurological disorders
- G01N2800/2814—Dementia; Cognitive disorders
- G01N2800/2828—Prion diseases
Definitions
- This invention relates to methods of detecting the presence of transmissible spongiform encephalopathies (TSEs). More specifically it relates to detecting TSEs by determining the presence or amount of 14-3-3 isoforms in the cerebrospinal fluid of a subject.
- TSEs transmissible spongiform encephalopathies
- TSEs transmissible spongiform encephalopathies
- TSE Gerstmann-Staussler-Scheinker syndrome
- Fatal Familial Insomnia and Kuru.
- the TSEs include sheep scrapie, bovine spongiform encephalopathy, transmissible mink encephalopathy, and chronic wasting disease of captive mule deer and elk (Gajdusek
- TSEs can currently be performed by brain biopsy or upon postmortem examination. Neither of these option is favorable.
- Postmortem diagnosis cannot reduce the risk of transmission from living patients and does not address the concerns regarding patient management.
- Premortem brain biopsy is highly invasive and causes health risks to animals, patients, and health care personnel. Moreover, brain biopsy may often miss the site of pathology.
- CSF cerebrospinal fluid
- the 14-3-3 proteins are a highly conserved group of proteins found in a broad range of species, including plants, yeast, Drosophila and mammals. They exist at low levels in many tissues, but are most highly concentrated in the brain, (see, e.g., Aitken et al. (1992) Trends Biochem. Sci. 17:498:501). There is increasing evidence that 14-3-3 proteins are critical to cell transformation and mitotic signaling, (see, e.g. Ford et al. (1994) Science 265:533-535; Freed et al. (1994) Science 265:1713-1716; Irie et al. (1994) Science 265:1716-1719 and Fu et al. (1994) Science 266:126-129).
- isoforms of 14-3-3 including, for example, ⁇ , ⁇ , ⁇ , ⁇ , ⁇ and ⁇ . (Morrison (1994) Science 266:56-57). It is, however, the surprising discovery of this invention that these various different isoforms of the 14-3-3 family may be specifically diagnostic of TSEs. Polyclonal antibodies against 14-3-3 (all isoforms), 14-3-3 ⁇ , 14-3-3 ⁇ , and 14-3-3 ⁇ are immunoreactive with a 30 kDa marker band from CJD CSF. However, 14-3-3 ⁇ and 14-3- 3 ⁇ antibodies are not present in TSE-positive samples. This invention also describes for the first time how antibodies to 14-3-3 isoforms is a pre-mortem diagnostic for BSE in cattle.
- the present invention provides methods of diagnosing transmissible spongiform encephalopathies by detecting the 14-3-3 isoforms in the cerebrospinal fluid of an animal.
- Figure 1 is a half-tone reproduction of a silver-stained 2DE gel of cerebrospinal fluid from a patient with Creutzfeldt- Jakob disease. Spot 130 and 131 are denoted with arrows and correlate specifically with spongiform pathology in patients affect by
- FIG. 2 panels A through F, are half-tone reproductions of an immunostained one-dimensional SDS-PAGE blots with polyclonal antibodies to 14-3-3 isoforms.
- lane 1 contains cerebrospinal fluid from a patient with Creutzfeldt- Jakob disease and lane 2 contains the cerebrospinal fluid from a normal individual.
- Panel A shows staining with anti-14-3-3 (all isoforms);
- Panel B shows staining with anti-14-3-3 ⁇ ;
- Panel C shows anti-14-3-3 ⁇ ;
- Panel D shows anti-14-3-3 ⁇ ;
- Panel E shows anti-14-3-3 ⁇ ;
- Panel F shows anti-14-3-3 ⁇ .
- the diagnostic band migrates at 30 kDa as shown by the arrows.
- Figure 3 is a half-tone reproduction of 2DE gel of cerebrospinal fluid from a bovine affected by bovine spongiform encephalopathy.
- the region marked in the box represents the region that should contain spots 130/131. No spots comigrating with human cerebrospinal fluid 130/131 are seen.
- Figure 4 is a half-tone reproduction of ID gels on two bovine cerebrospinal fluid samples blotted onto PVDF or nitrocellulose.
- the diagnostic band migrates at approximately 30 kDa by SDS-PAGE.
- Figure 5 is a half-tone reproduction of a ID gel of bovine samples blotted onto PVDF membrane and immunostained with 14-3-3 ⁇ antibody.
- Lane 1 is a sample from normal cow brain extract
- lanes 2-5 are premortem samples from cerebrospinal fluid from BSE-affected cattle
- lanes 6-9 are spinal fluid samples from control cattle (non-BSE- affected).
- the 14-3-3 band migrates at a position corresponding to 30 kDa and its appearance correlates with BSE pathology.
- 14-3-3 protein is used herein to refer to mean any member of isoform of the 14-3-3 class of proteins as it is commonly known to those of skill in the art (see, e.g., Ichimura et al. (1988) Proc. Nat'l, Acad. Sci, USA 85:7084-7088; Zupan et al. (1992) JBiol. Chem., 267, 8707-87 10; Aitken et al (1992) Trends. Biochem. Sci., 17: 498-501 Burbtlo et al. (1995) Current Biology, 5:95-96; Robinson et al (1994) Biochem. J 299:
- Assays that detect 14-3-3 are intended to detect the level of endogenous (native) 14-3-3 present in subject biological sample (e.g., CSF). However, exogenous 14-3-3 (14-3-3 protein from a source extrinsic to the biological sample) may be added to various assays to provide a label or to compete with the native
- 14-3-3 in binding to an anti-14-3-3 antibody.
- 14-3-3 mimetic may be used in place of exogenous 14-3-3 in this context.
- An "14-3-3 mimetic", as used herein, refers to a molecule that bears one or more 14-3-3 epitopes such that it is specifically bound by an antibody that specifically binds native 14-3-3.
- glycosylation is intended to mean the covalent addition of sugar or sugar-related molecules to any protein or polynucleotide.
- a glycosylated protein has one or more covalently bound sugar-related molecules and one protein can have multiple glycosylated forms.
- glycoform and isoforms are used interchangeably and are intended to mean to the different glycosylated variants of one protein.
- the glycoform may be identified herein by its isoelectric focusing point (pi) and its molecular weight (MW) in kilodaltons (kD).
- pi and molecular weight values are intended to include a range which includes any variations which may occur due to experimental conditions as would be known by one of skill in the art.
- pis will generally range up or down 0.5 from the value given. At higher pHs, the range may be + 1.
- Molecular weight ranges will usually be within 5 kilodaltons of the value given, although glycoproteins may migrate anomalously in SDS PAGE gels and give show MW values which are higher than the actual MW.
- cerebrospinal fluid or "CSF” is intended to include whole cerebrospinal fluid or derivatives or fractions thereof well known to those of skill in the art.
- a cerebrospinal fluid sample can include various fractionated forms of cerebrospinal fluid or can include various diluents added to facilitate storage or processing in a particular assay.
- Such diluents are well known to those of skill in the art and include various buffers, preservatives and the like.
- 2-dimensional gel electrophoresis or “2-D gel electrophoresis” or “2DE” is intended to refer to the two dimensional migration of proteins in solution of suspension in the presence of an electrical field. Methods of 2DE are well known to those of skill in the art. See generally, R. Scopes (1982) Protein Purification,
- 2DE relies on isoelectric focusing (IEF) in either carrier ampholyte gradient gels or immobilized pH gradients for one dimension and SDS-polyacrylamide gels for the second dimension.
- IEF isoelectric focusing
- SDS-polyacrylamide gels for the second dimension.
- the amount of polyacrylamide to be used in making the gels can be readily determined by a skilled artisan.
- synthetic peptide refers to any amino acid or polypeptide which results from chemical synthesis or assembly.
- Methods of synthesizing peptides are known in the art and include for example, solution or solid phase peptide synthesis of either the t-Boc or Fmoc types. Methods of synthesizing peptides may be found, for example, in Current Protocols in Molecular Biology, Ausubel et al. eds. (1996)
- an "immunoassay” is an assay that utilizes an antibody to specifically bind to the analyte.
- the immunoassay is characterized by the use of specific binding to a particular antibody as opposed to other physical or chemical properties to isolate, target, and quantify the analyte.
- an “antibody” refers to a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes or fragments of immunoglobulin genes.
- the recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as the myriad immunoglobulin variable region genes.
- Light chains are classified as either kappa or lambda.
- Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.
- the basic immunoglobulin (antibody) structural unit is known to comprise a tetramer.
- Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light” (about 25 kD) and one "heavy” chain (about 50-70 kD).
- the N- terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition.
- the terms variable light chain (V,) and variable heavy chain (V H ) refer to these variable regions of the light and heavy chains respectively.
- Antibodies may exist as intact immunoglobulins or as a number of well characterized fragments produced by digestion with various peptidases.
- pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab)' 2 , a dimer of Fab which itself is a light chain joined to V H -C H 1 by a disulfide bond.
- the F(ab)' 2 may be reduced under mild conditions to break the disulfide linkage in the hinge region thereby converting the F(ab)' 2 dimer into an Fab' monomer.
- the Fab' monomer is essentially an Fab with part of the hinge region (See, Fundamental
- antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such Fab' fragments may be synthesized de novo either chemically or by utilizing recombinant DNA methodology.
- antibody as used herein also includes antibody fragments either produced by the modification of whole antibodies or synthesized de novo using recombinant DNA methodologies.
- the specified antibodies bind to a particular protein and do not bind in a significant amount to other proteins present in the sample.
- Specific binding to a protein under such conditions may require an antibody that is selected for its specificity for a particular protein.
- antibodies can be raised to the human 14-3-3 protein that bind 14-3-3 and not to any other proteins present in a biological sample (e.g., CSF).
- immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein.
- solid- phase ELISA immunoassays are routinely used to select monoclonal antibodies specifically immunoreactive with a protein. See Harlow and Lane (1988) Antibodies. A Laboratory Manual, Cold Spring Harbor Publications, New York, for a description of immunoassay formats and conditions that can be used to determine specific immunoreacti vity .
- the term "subject” refers a mammal and includes, but is not limited to, humans, bovine, mink, sheep, elk and deer.
- This invention provides methods for the detection of transmissible spongiform encephalopathies (TSEs) by detecting the presence or absence or quantification of a 14-3-3 protein in a sample of cerebrospinal fluid (CSF). While the 14-3-3 proteins were previously known, it is the surprising discovery of this invention that different 14-3-3 isoforms can be diagnostic of TSE. The detection methods of this invention thus provide new uses for this previously known class of proteins. Using the methods of the present invention, TSEs can be diagnosed by premortem testing. Table 1 shows the sensitivity and specificity of immunoassays for 14-3-3 ⁇ isoform antibodies in immunostained one-dimensional gels (ID). Table 2 shows the sensitivity and specificity of immunoassays for 14-3-3 ⁇ isoform antibodies in immunostained two- dimensional gels (2DE).
- ID immunostained one-dimensional gels
- 2 shows the sensitivity and specificity of immunoassays for 14-3-3 ⁇ isoform antibodies in immunostained two- dimensional gels (2DE).
- Tables 1 and 2 show that 14-3-3 ⁇ antibodies are both a sensitive and specific method of detecting TSEs.
- the invention provides methods for TSEs by determining whether specific 14-3-3 isoforms are present or absent in CSF. For example, antibodies to 14-3-3 ⁇ and ⁇ , do not detect the 30 kDa band diagnostic of TSEs. In contrast, 14-3-3 ⁇ , ⁇ and ⁇ are diagnostic of TSEs. As detailed below in the Experimental section, these different isoforms can therefore be used to diagnose TSEs.
- This invention also provides for methods of diagnosing animal TSEs by detecting 14-3-3 isoforms. As shown in Figure 5, and detailed below, the CSF from all ten animals later confirmed positive for BSE, had a 14-3-3 immunoreactive band. The 14-3-3 isoform antibody was not reactive with CSF samples obtained from non-BSE or with an extract of normal bovine brain.
- nervous system diseases can be detected by the presence and amount of 14-3-3 isoforms in cerebrospinal fluid from animals and humans.
- the 14-3-3 proteins can be detected and quantified by any of a number of means well known to those of skill in the art.
- analytic biochemical methods such as electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography, mass spectrometry and the like, or various immunological methods such immunodiffusion (single or double), immunoelectrophoresis, radioimmunoassay(RIA), enzyme-linked immunosorbent assays (ELISAs), immunofluorescent assays, western blotting, and the like.
- analytic biochemical methods such as electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography, mass spectrometry and the like
- immunological methods such immunodiffusion (single or double), immunoelectrophoresis, radioimmunoassay(RIA), enzyme-linked immunosorbent assays (ELISAs), immunofluorescent assays, western blotting, and the like.
- the 14-3-3 proteins are detected in an electrophoretic protein separation, more preferably in a two-dimensional electrophoresis, while in a most preferred embodiment, the 14-3-3 proteins are detected using an immunoassay.
- an electrophoretic protein separation more preferably in a two-dimensional electrophoresis
- the 14-3-3 proteins are detected using an immunoassay. The collection of biological samples and subsequent testing for 14-3-3 proteins is discussed in more detail below.
- the 14-3-3 proteins are preferably quantified in cerebrospinal fluid derived from a mammal, including human patients and animals.
- Obtaining and storing CSF are well known to those of skill in the art.
- CSF is obtained by lumbar puncture.
- the CSF may be diluted by the addition of buffers or other reagents well known to those of skill in the art and may be stored for up to 24 hours at 2-8°C, or at -20°C or lower for longer periods, prior to measurement of 14-3-3. Any of a number of standard aqueous buffer solutions, employing one of a variety of buffers, such as phosphate, Tris, or the like, at physiological pH can be used.
- the CSF is stored at -70°C without preservative indefinitely.
- the presence or absence of 14-3-3 isoforms in cerebrospinal fluid can be determined using electrophoretic methods.
- Means of detecting proteins using electrophoretic techniques are well known to those of skill in the art (see generally, R.
- the 14-3-3 proteins are detected using two-dimensional electrophoresis.
- a particularly preferred separation relies on isoelectric focusing (IEF) in carrier ampholyte gradient gels or immobilized pH gradients for one dimension and 14% polyacrylamide gels for the second dimension.
- IEF isoelectric focusing
- Proteins separated on 2DE gels can be visualized by any method known in the art. For example, Coomassie blue staining, gold staining, silver staining and the like may be used to visualize proteins directly on the gels, (see, generally, Current Protocols in
- the proteins on the 2DE gel can be transferred or blotted onto a membrane and probed with an antibody. Methods of blotting and immunostaining are known to those of skill in the art. (see, e.g., Ausubel (1996), supra, ⁇ 10.8).
- the present invention detects the presence and amount of 14-3-3 first by silver staining and, subsequently, by immunostaining.
- the 14-3-3 proteins are detected or quantified in CSF using any of a number of well recognized immunological binding assays (see, e.g., U.S. Patents).
- Immunological binding assays typically utilize a "capture agent" to specifically bind to and often immobilize the analyte (in this case 14-3-3).
- the / ⁇ capture agent is a moiety that specifically binds to the analyte.
- the capture agent is an antibody that specifically binds 14-3-3 protein(s).
- the antibody (anti-14-3-3) may be produced by any of a number of means well known to those of skill in the art (see, e.g. Methods in Cell Biology Volume 37 Antibodies in Cell Biology, Asai, ed. Academic Press, Inc. New York (1993); and Basic and Clinical
- the antibody may be a whole antibody or an antibody fragment. It may be polyclonal or monoclonal, and it may be produced by challenging an organism (e.g. mouse, rat, rabbit, etc.) with a 14-3-3 protein or an epitope derived therefrom. Alternatively, the antibody may be produced de novo using recombinant DNA methodology.
- the antibody can also be selected from a phage display library screened against 14-3-3 (see, e.g. Vaughan et al. (1996) Nature Biotechnology, 14:309-314 and references therein). Commercially available 14-3-3 isoform antibodies are also available from, for example, Santa Cruz Biotechnology.
- Immunoassays also often utilize a labeling agent to specifically bind to and label the binding complex formed by the capture agent and the analyte
- the labeling agent may itself be one of the moieties comprising the antibody/analyte complex.
- the labeling agent may be a labeled 14-3-3 protein or a labeled anti-14-3-3 antibody, Alternatively, the labeling agent may be a third moiety, such as another antibody, that specifically binds to the antibody/ 14-3 -3 complex.
- the labeling agent is a second human 14-3-3 antibody bearing a label.
- the second 14-3-3 antibody may lack a label, but it may, in turn, be bound by a labeled third antibody specific to antibodies of the species from which the second antibody is derived.
- the second can be modified with a detectable moiety, such as biotin, to which a third labeled molecule can specifically bind, such as enzyme-labeled streptavidin.
- proteins capable of specifically binding immunoglobulin constant regions such as protein A or protein G may also be used as the label agent. These proteins are normal constituents of the cell walls of streptococcal bacteria. They exhibit a strong non- immunogenic reactivity with immunoglobulin constant regions from a variety of species. See, generally Kronval, et al. J. Immunol; 111:1401—1406 (1973), and Akerstrom, et al, J.
- incubation and/or washing steps may be required after each combination of reagents. Incubation steps can vary from about 5 seconds to several hours, preferably from about 5 minutes to about 24 hours. However, the incubation time will depend upon the assay format, analyte, volume of solution, concentrations, and the like. Usually, the assays will be carried out at ambient temperature, although they can be conducted over a range of temperatures, such as 10°C to 40°C.
- Immunoassays for detecting CSF 14-3-3 protein may be either competitive or noncompetitive.
- Noncompetitive immunoassays are assays in which the amount of captured analyte (in this case 14-3-3) is directly measured.
- the capture agent anti-14-3-3 antibodies
- the capture agent can be bound directly to a solid substrate where they are immobilized. These immobilized antibodies then capture 14-3-3 present in the test sample.
- the 14-3-3 thus immobilized is then bound by a labeling agent, such as a second human 14-3-3 antibody bearing a label.
- a labeling agent such as a second human 14-3-3 antibody bearing a label.
- the second labeling agent such as a second human 14-3-3 antibody bearing a label.
- 14-3-3 antibody may lack a label, but it may, in turn, be bound by a labeled third antibody specific to antibodies of the species from which the second antibody is derived.
- the second can be modified with a detectable moiety, such as biotin, to which a third labeled, molecule can specifically bind, such as enzyme-labeled strepavidin.
- the amount of analyte (14-3-3) present in the sample is measured indirectly by measuring the amount of an added (exogenous) analyte (14-3-3) displaced (or competed away) from a capture agent (anti 14-3-3 antibody) by the analyte present in the sample.
- a capture agent in this case an antibody that specifically binds 14-3-3.
- the amount of 14-3-3 bound to the antibody is inversely proportional to the concentration of 14-3-3 present in the sample before the exogenous 14-3-3 is added to the reaction mix.
- the antibody is immobilized on a solid substrate.
- the amount of 14-3-3 bound to the antibody may be determined either by measuring the amount of 14-3-3 present in an 14-3-3/antibody complex, or alternatively by measuring the amount of remaining uncompeted 14-3-3.
- the amount of 14-3-3 may be detected by providing a labeled 14-3-3 molecule.
- a hapten inhibition assay is another preferred competitive assay.
- a known analyte in this case 14-3-3 is immobilized on a solid substrate.
- a known amount of anti-14-3-3 antibody is added to the sample, and the sample is then contacted with the immobilized 14-3-3.
- the amount of anti-14-3-3 antibody bound to the immobilized 14-3-3 is inversely proportional to the amount of 14-3-3 present in the sample.
- the amount of immobilized antibody may be detected by detecting either the immobilized fraction of antibody or the fraction of the antibody that remains in solution. Detection may be direct where the antibody is labeled or indirect by the subsequent addition of a labeled moiety that specifically binds to the antibody as described above.
- Western blot (immunoblot) analysis is used to detect and quantify the presence of 14-3-3 in the sample.
- the technique generally comprises separating sample proteins by gel electrophoresis on the basis of molecular weight, transferring the separated proteins to a suitable solid support, (such as a nitrocellulose filter, a nylon filter, or derivatized nylon filter), and incubating the sample with the antibodies that specifically bind 14-3-3.
- a suitable solid support such as a nitrocellulose filter, a nylon filter, or derivatized nylon filter
- 14-3-3 on the solid support may be directly labeled or alternatively may be subsequently detected using labeled antibodies (e.g., labeled sheep anti-mouse antibodies) that specifically bind to the anti-14-3-3.
- labeled antibodies e.g., labeled sheep anti-mouse antibodies
- a particularly preferred protocol for Western Blot detection of human or animal 14-3-3 protein in CSF is provided in Example 1.
- LISA liposome immunoassays
- the particular label or detectable group used in the assay is not a critical aspect of the invention, so long as it does not significantly interfere with the specific binding of the antibody used in the assay.
- the detectable group can be any material having a detectable physical or chemical property.
- Such detectable labels have been well developed in the field of immunoassays and, in general, most any label useful in such methods can be applied to the present invention.
- a label is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means.
- Useful labels in the present invention include magnetic beads (e.g.
- DynabeadsTM DynabeadsTM
- fluorescent dyes e.g., fluorescein isothiocyanate, texas red, rhodamine, and the like
- radiolabels e.g., H, 125 1, 35 S, 14 C, or 32 P
- enzymes e.g., horse radish peroxidase, alkaline phosphatase and others commonly used in an ELISA
- colorimetric labels such as colloidal gold or colored glass or plastic (e.g. polystyrene, polypropylene, latex, etc.) beads.
- the label may be coupled directly or indirectly to the desired component of the assay according to methods well known in the art.
- Non-radioactive labels are often attached by indirect means.
- a ligand molecule e.g., biotin
- the ligand then binds to an anti-ligand (e.g., streptavidin) molecule which is either inherently detectable or covalently bound to a signal system, such as a detectable enzyme, a fluorescent compound, or a chemiluminescent compound.
- an anti-ligand e.g., streptavidin
- a ligand has a natural anti-ligand, for example, biotin, thyroxine, and cortisol, it can be used in conjunction with the labeled, naturally occurring anti-ligands.
- a haptenic or antigenic compound can be used in combination with an antibody.
- the molecules can also be conjugated directly to signal generating compounds, e.g., by conjugation with an enzyme or fluorophore.
- Enzymes of interest as labels will primarily be hydrolases, particularly phosphatases, esterases and glycosidases, or oxidoreductases, particularly peroxidases.
- Fluorescent compounds include fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, etc.
- Chemiluminescent compounds include luciferin, and 2,3-dihydrophtlialazinediones, e.g., luminol. (For a review of various labeling or signal producing systems which may be used, see, U.S. Patent No. 4,391,904).
- Means of detecting labels are well known to those of skill in the art.
- means for detection include a scintillation counter or photographic film as in autoradiography.
- the label is a fluorescent label, it may be detected by exciting the fluorophore with the appropriate wavelength of light and detecting the resulting fluorescence. The fluorescence may be detected visually, by means of photographic film, by the use of electronic detectors such as charge coupled devices (CCDs) or photomultipliers and the like.
- CCDs charge coupled devices
- enzymatic labels may be detected by providing the appropriate substrates for the enzyme and detecting the resulting reaction product.
- simple colorimetric labels may be detected simply by observing the color associated with the label.
- conjugated gold often appears pink, while various conjugated beads appear the color of the bead.
- Some assay formats do not require the use of labeled components.
- agglutination assays can be used to detect the presence of the target antibodies.
- antigen-coated particles are agglutinated by samples comprising the target antibodies.
- none of the components need be labeled and the presence of the target antibody is detected by simple visual inspection.
- the solid surface may be a membrane (e.g. , nitrocellulose), a microtiter dish
- the desired component may be covalently bound or noncovalently attached through nonspecific bonding.
- organic and inorganic polymers both natural and synthetic may be employed as the material for the solid surface.
- Illustrative polymers include polyethylene, polypropylene, poly(4-methylbutene), polystyrene, polymethacrylate,
- materials which may be employed include paper, glasses, ceramics, metals, metalloids, semiconductive materials, cements or the like.
- substances that form gels such as proteins (e.g., gelatins), lipopolysaccharides, silicates, agarose and polyacrylamides can be used.
- Polymers which form several aqueous phases such as dextrans, polyalkylene glycols or surfactants, such as phospholipids, long chain (12-24 carbon atoms) alkyl ammonium salts and the like are also suitable. Where the solid surface is porous, various pore sizes may be employed depending upon the nature of the system.
- a plurality of different materials may be employed, particularly as laminates, to obtain various properties.
- protein coatings such as gelatin can be used to avoid non-specific binding, simplify covalent conjugation, enhance signal detection or the like.
- the surface will usually be poly functional or be capable of being polyfunctionalized.
- Functional groups which may be present on the surface and used for linking can include carboxylic acids, aldehydes, amino groups, cyano groups, ethylenic groups, hydroxyl groups, mercapto groups and the like.
- the manner of linking a wide variety of compounds to various surfaces is well known and is amply illustrated in the literature. See, for example,
- Noncovalent binding is typically nonspecific absorption of a compound to the surface.
- the surface is blocked with a second compound to prevent nonspecific binding of labeled assay components.
- the surface is designed such that it nonspecifically binds one component but does not significantly bind another.
- a surface bearing a lectin such as Concanavalin A will bind a carbohydrate containing compound but not a labeled protein that lacks glycosylation.
- Various solid surfaces for use in noncovalent attachment of assay components are reviewed in U.S. Patent Nos. 4,447,576 and 4,254,082. The following examples are provided to illustrate but not limit the present invention.
- Cerebrospinal fluid samples were kindly provided by various neurologists and veterinarians. The diagnoses were made by the referring physicians according to standard clinical criteria in addition to pathological studies as appropriate. The CSF was collected and the samples were immediately frozen at -70°C before shipment to our laboratory. Upon receipt, samples were thawed and aliquoted for use. Samples for 2-DE were prepared by the addition of 9M urea, 2% 2-mercaptoethanol, 2% NP-40, 0.8% BioLyte pH 3-10 carrier ampholytes, and 0.002% Bromophenol blue. For SDS-PAGE, 50 ⁇ L CSF was added to 20 ⁇ L Laemmli sample buffer, heated for 5 minutes at 95°C, and loaded.
- the 14-3-3 SDS-PAGE immunoassay was performed according to a method adapted from Brown et al. (1986) N. Engl. J. Med. 314:547-551. Fifty microliters of cerebrospinal fluid was mixed with 10 microliters of sample buffer (5% glycerol, 1% 2- mercaptoethanol, 1% sodium dodecyl sulfate, and a trace of bromophenol blue in the final solution) and boiled for 5 minutes. Samples were separated by sodium dodecyl sulfate- polyacrylamide-gel electrophoresis (4% stacking gel with 12 percent resolving gel at 75 V for three hours). Proteins were blotted to either PVDF (Millipore) or nitrocellulose
- the polyclonal 14-3-3 antibodies used were purchased from ⁇ Santa Cruz Biotechnology (catalog numbers: sc-629, sc-628, sc-731, sc-732, sc-1020, and sc-1019) and were diluted 1:500.
- the anti-rabbit-horse radish peroxidase-conjugated antibody (Promega) was diluted 1:5000. With the exception of final washes, all steps during the immunodetection were performed in the presence of 5% milk in TBST. Chemiluminescent detection (Pierce) on Kodak XAR film was used.
- Figure 1 shows results of the silver stained 2-DE gel pattern of CSF taken from a patient diagnosed with CJD. Spots 130 and 131 are denoted by the arrows and these spots selectively appear in the CSF of patients with CJD. In an attempt to develop an immunoassay for CJD, these spots were characterized. Amino acid sequencing of comigrating spots from normal human brain has revealed that these spots are proteins which belong to the 14-3-3 family of proteins. The amino acid sequence data suggests that 130/131 are derived from 14-3-3 ⁇ . However, we describe herein for the first time antibodies tested against various isoforms of the 14-3-3 family of proteins for use with an SDS-PAGE immunoassay because of the sequence similarity among these isoforms.
- 14-3-3 isoform antibodies recognize a 30 kDa band on ID protein gels
- Figure 2 depicts the results obtained with antibodies specific to different 14-3-3 isoforms in CJD screening.
- lane 1 is CSF obtained from a patient diagnosed with CJD and lane 2 is CSF from a control patient.
- the CJD marker band of interest migrates at 30 kDa by SDS-PAGE.
- Panel A depicts the results with an antibody used to detect all isoforms of 14-3-3 and it is mildly reactive at 30 kDa.
- Panels B, C, and D utilize antibodies against the ⁇ , ⁇ , and ⁇ isoforms of 14-3-3, respectively. All of these antibodies are reactive with the 30 kDa band.
- Panels E and F show the use of antibodies against 14-3- 3 ⁇ and ⁇ , respectively and, interestingly, both of these antibodies do not detect any 30 kDa band. Taken together, these results suggest that specific isoforms may be found in CJD
- the 14-3-3 family of proteins forms both homodimers and heterodimers among its isoforms.
- the positive results obtained with the and ⁇ , ⁇ , and ⁇ antibodies may reflect the release of specific multimers of the 14-3-3 family from neuronal tissue.
- the ⁇ isoform currently demonstrates the highest correlation to a diagnosis of CJD and clinical pathology.
- FIG. 3 depicts a typical silver stained 2D gel from BSE CSF. A region is marked in Figure 3 to denote the region in which
- the specificity of human 14-3-3 isoform antibodies was also tested on bovine samples.
- the human and bovine sequences for several of the 14-3-3 isoforms are similar.
- the 14-3-3 ⁇ isoform is nearly identical for human and bovines.
- the human sequence has an additional asparagine residue at the C-terminus (A. Aitken, personal communication).
- the polygonal antibody obtained (Santa Cruz Biotechnology) is antigenic against the C-terminal sequence of human 14-3-3 ⁇ .
- Figure 4 depicts our results with a 14-3-3 ⁇ immunostain against 2 samples of bovine CSF. When these proteins are blotted to PVDF (Millipore), the antibody is able to recognize the 30 kDa band of interest.
- Example 2 Samples were analyzed essentially as described in Example 1. CSF samples were stored at -70°C to minimize protein degradation. 50 microliters of CSF was mixed with 20 microliters Laemmli SDS buffer and heated for 5 minutes. A crude extract of bovine brain was prepared by methods known in the art. 10 micrograms of protein in Laemmli buffer was loaded into each lane of a 12%T polyacrylamide gel, transferred to PVDF membrane and immunostained. The 14-3-3 ⁇ antibody (Santa Cruz Biotechnology), which is specific for the ⁇ isoform of 14-3-3 was used for detection. Moreover, as described in Example 2, the electrophoresed proteins were blotted onto PVDF (Millipore) material. Enhanced chemiluminescence (Pierce) was used for detection.
- PVDF Micropore
- a 30 kDa, 14-3-3 immunoreactive band from an extract of normal bovine brain (lane 1) has the same mobility as a band from BSE samples (lanes 2 through 5) that is not seen in control CSF (lanes 6 through 9). All ten animals with BSE had this band while it was absent in five of six controls. The one control sample which had a small amount of 14-3-3 was believed to be the result of technical error.
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Abstract
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AU56012/98A AU5601298A (en) | 1996-12-12 | 1997-12-11 | Methods of detecting transmissible spongiform encephalopathies by detecting 14-3-3 protein isoforms |
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Cited By (8)
Publication number | Priority date | Publication date | Assignee | Title |
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FR2821431A1 (fr) * | 2001-02-28 | 2002-08-30 | Louis Lery | Reactif destine au depistage ante-morten d'atnc |
WO2002074986A2 (fr) * | 2001-03-21 | 2002-09-26 | Exonhit Therapeutics S.A. | Test sanguin de diagnostic precoce pre-symptomatique pour les encephalopathies |
WO2003023406A2 (fr) * | 2001-09-05 | 2003-03-20 | Proteome Sciences Plc. | Methode de diagnostic pour encephalopathies spongiformes transmissibles |
WO2004040316A2 (fr) * | 2002-10-30 | 2004-05-13 | Proteome Sciences Plc. | Methodes destinees a diagnostiquer une encephalopathie spongiforme transmissible |
EP2021792A1 (fr) * | 2006-05-09 | 2009-02-11 | The University of British Columbia | Protéine dissoutes marqueurs de l'arthrite |
US9079947B2 (en) | 2007-11-27 | 2015-07-14 | The University Of British Columbia | 14-3-3 eta antibodies and uses thereof for the diagnosis and treatment of arthritis |
US10132806B2 (en) | 2011-10-21 | 2018-11-20 | Augurex Life Sciences Corp. | Antigens derived from citrullinated 14-3-3 and uses thereof in the diagnosis of rheumatoid arthritis |
US11054419B2 (en) | 2009-03-11 | 2021-07-06 | Anthony Marotta | Compositions and methods for characterizing arthritic conditions |
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US4892814A (en) * | 1987-06-22 | 1990-01-09 | The United States Of America As Represented By The Department Of Health And Human Services | Method for distinguishing Creutzfeldt-Jakob disease from other dementias |
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WO2002068961A1 (fr) * | 2001-02-28 | 2002-09-06 | Louis Lery | Reactif destine au despitage ante-mortem d"atnc |
FR2821431A1 (fr) * | 2001-02-28 | 2002-08-30 | Louis Lery | Reactif destine au depistage ante-morten d'atnc |
WO2002074986A2 (fr) * | 2001-03-21 | 2002-09-26 | Exonhit Therapeutics S.A. | Test sanguin de diagnostic precoce pre-symptomatique pour les encephalopathies |
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JP2009536318A (ja) * | 2006-05-09 | 2009-10-08 | ザ ユニバーシティ オブ ブリティッシュ コロンビア | 溶解したタンパク質関節炎マーカー |
US9791458B2 (en) | 2006-05-09 | 2017-10-17 | University Of British Columbia | Dissolved protein arthritis markers |
EP2021792A1 (fr) * | 2006-05-09 | 2009-02-11 | The University of British Columbia | Protéine dissoutes marqueurs de l'arthrite |
US9079947B2 (en) | 2007-11-27 | 2015-07-14 | The University Of British Columbia | 14-3-3 eta antibodies and uses thereof for the diagnosis and treatment of arthritis |
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US10132806B2 (en) | 2011-10-21 | 2018-11-20 | Augurex Life Sciences Corp. | Antigens derived from citrullinated 14-3-3 and uses thereof in the diagnosis of rheumatoid arthritis |
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