Title: Method of detecting axonally-derived protein tau in patients with traumatic CNS injury
United States Patent: 6,589,746
Issued: July 8, 2003
Inventors: Zemlan; Frank P. (Cincinnati, OH)
Assignee: University of Cincinnati (Cincinnati, OH)
Appl. No.: 694627
Filed: October 23, 2000
Patients having several neurological diseases have been shown to have elevated levels of axonally-derived proteins (i.e. tau and neurofilament proteins) in cerebrospinal fluid (CSF) and in brain tissue. Three monoclonal antibodies (MAbs) recognizing CSF tau proteins were developed. The MAbs were found to label a ladder of 30 kD to 50 kD tau proteins in CSF from patients with disease states producing axonal damage such as head trauma or CNS tumor but not in CSF from controls. High levels of tau protein in CSF were shown to be diagnostic of axonal degeneration in head trauma. An ELISA assay was developed with these MAbs to aid in the diagnosis of patients with axonal damage.
DETAILED DESCRIPTION OF INVENTION
The proteins that are useful in the present invention are a novel class of axonally-derived proteins occurring in human CSF and blood including cleaved tau proteins and cleaved neurofilament proteins. As used herein, axonally-derived proteins refer to a group of proteins that occur in axons of CNS neurons and are released into the extracellular space during degeneration of said axons. As used herein, the term full length tau refers to any of the six non- cleaved isoforms of this protein that demonstrate an apparent molecular weight of 48 to 68 kDa and whose genomic and amino acid sequences are described in Goedert et al. (1989). The class of novel cleaved tau proteins that are useful in the invention were discovered in human cerebrospinal fluid and have been purified. These novel cleaved tau proteins may be distinguished from previously described full length tau proteins, in that they: 1) have reduced apparent molecular weights in comparison to full length tau, 2) are comprised of the interior portion of the tau sequence that includes ser199 to ser396 of tau, and 3) lack the N-terminal and C-terminal amino acids of tau. As used herein, the term neurofilament-L refers to a protein with an apparent molecular weight of 68 kDa and whose genomic and amino acid sequences are described in Julien et al. (1987). As used herein, the term neurofilament-M refers to a protein with an apparent molecular weight of 160 kDa and whose genomic and amino acid sequences are described in Myers et al. (1987). As used herein, the term neurofilament-H refers to a protein with an apparent molecular weight of 200 kDa and whose genomic and amino acid sequences are described in Lees et al. (1988). As used herein, the term neurofilament66 refers to a protein with an apparent molecular weight of 66 kDa and whose genomic and amino acid sequences are described in Chan and Chiu (1995).
Antibodies specifically reactive with the novel cleaved tau proteins are included within the scope of the invention. A "specifically reactive" antibody is one that is capable of binding with a particular molecule to thereby couple said molecule to the antibody. The term "epitope" refers to that portion of a hapten that can be recognized and bound by an antibody. The present disclosure indicates that the antigen employed for monoclonal antibody production, a cleaved form of tau found in human cerebrospinal fluid, possesses more than one epitope. An antigen is a molecule capable of inducing an animal to produce an antibody capable of binding to an epitope of that antigen. The specific reaction referred to above is meant to indicate that an antibody will bind with a significantly higher affinity to its corresponding antigen as opposed to the multitude of other antigens occurring in the human body. For example, there is provided by this invention several MAbs (i.e. cTau-7, cTau-8 and cTau-12) raised against the cleaved form of tau found in CSF. These cTau antibodies demonstrated sufficiently higher affinity for cleaved tau than for full length tau such that selective immunolabeling of cleaved tau occurred with equivalent protein loads on Western blots.
In particular, the invention includes a method for detecting and quantitating an axonally- derived protein in a human subject, comprising:
(a) contacting a blood sample from a human subject that is suspected of containing detectable levels of an axonally-derived protein with a molecule capable of binding to the axonally-derived protein; and
(b) detecting the molecule bound to the axonally-derived protein.
The invention additionally includes the method as above, wherein the binding molecule is selected from the group consisting of:
(a) an antibody substantially free of natural impurities;
(b) a monoclonal antibody; and
(c) a fragment of (a) or (b).
The invention additionally includes the method as above, wherein the detecting molecule is detectably labeled and where a combination of such binding molecules is used.
The invention additionally includes a method for determining the presence of a condition in a human subject, said condition including, but not limited to, the group consisting of Alzheimer's Disease, the presence of neuroectodermal tumors, the presence of malignant astrocytomas, and the presence of gliomas.
The invention additionally includes a method of diagnosing the presence of neurological disease in a human subject suspected of having neurological disease, which comprises:
(a) incubating a biological sample from said subject suspected of containing an axonally-derived protein with a molecule capable of identifying an axonally-derived protein; and
(b) detecting the molecule, which is bound in the sample, wherein the detection indicates that the subject has neurological disease.
The invention additionally includes a method of diagnosing the presence of neuroectodermal tumors in a human subject suspected of having neuroectodermal tumors which comprises:
(a) incubating a biological sample from said subject suspected of containing an axonally-derived protein with a molecule capable of identifying an axonally-derived protein; and
(b) detecting the molecule, which is bound in the sample, wherein the detection indicates that the subject has neuroectodermal tumors.
Antibodies directed against an axonally-derived protein can be used, as taught by the present invention, to detect and diagnose neurological disease. Various histological staining methods, including immunohistochemical staining methods, may also be used effectively according to the teaching of the invention. Silver stain is but one method of visualizing axonally-derived protein. Other staining methods useful in the present invention will be obvious to the artisan, the determination of which would not involve undue experimentation.
One screening method for determining whether a given compound is an axonally-derived protein functional derivative comprises, for example, immunoassays employing radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA) methodologies, based on the production of specific antibodies (monoclonal or polyclonal) to an axonally-derived protein. Venipuncture (blood), spinal tap (cerebral spinal fluid (CSF)), urine and other body secretions, such as sweat and tears, are used as biological samples. For example, in one form of RIA, the substance under test is mixed with diluted antiserum in the presence of radiolabeled antigen. In this method, the concentration of the test substance will be inversely proportional to the amount of labeled antigen bound to the specific antibody and directly related to the amount of free labeled antigen. Other suitable screening methods will be readily apparent to those of skill in the art.
The present invention also relates to methods of detecting an axonally-derived protein or functional derivatives in a sample or subject. For example, antibodies specific for an axonally- derived protein, or a functional derivative, may be detectably labeled with any appropriate marker, for example, a radioisotope, an enzyme, a fluorescent label, a paramagnetic label, or a free radical.
Alternatively, antibodies specific for an axonally-derived protein, or a functional derivative, may be detectably labeled with DNA by the technique of immunopolymerase chain reaction. The polymerase chain reaction (PCR) procedure amplifies specific nucleic acid sequences through a series of manipulations including denaturation, annealing of oligonucleotide primers, and extension of the primers with DNA polymerase (see, for example, U.S. Pat. No. 4,683,202).
Various amounts of the test material can be immobilized on the surface of microtiter wells. The wells are subsequently incubated with an axonally-derived protein monoclonal antibody, washed, and then incubated with biotinylated axonally-derived protein DNA molecules that have been conjugated to streptavidin-protein chimera (Id.). This chimera binds biotin (via the streptavidin moiety) and the Fc portion of an immunoglobulin G molecule. The wells are then washed to remove unbound conjugates. Any axonally-derived protein present in the test material will be bound by the axonally-derived protein monoclonal antibody, which in turn, is bound by the protein A moiety of the biotinylated axonally-derived protein DNA-streptavidin-protein A conjugate. Then, the axonally-derived protein DNA sequences are amplified using PCR. The PCR products are then analyzed by agarose gel electrophoresis after staining with ethidium bromide.
Methods of making and detecting such detectably labeled antibodies or their functional derivatives are well known to those of ordinary skill in the art. The term "antibody" refers both to monoclonal antibodies, which are a substantially homogeneous population and to polyclonal antibodies, which are heterogeneous populations. Polyclonal antibodies are derived from the sera of animals immunized with an antigen. Monoclonal antibodies (MAbs) to specific antigens may be obtained by methods known to those skilled in the art. See, for example, U.S. Pat. No. 4,376,110. Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD and any subclass thereof. The term "antibody" is also meant to include both intact molecules as well as fragments thereof, such as, for example, Fab and F(ab')2, which are capable of binding antigen. Fab and F(ab')2 fragments lack the Fc fragment of intact antibody, clear more rapidly from the circulation, and may have less non-specific tissue binding than an intact antibody.
It will be appreciated that Fab and F(ab')2 and other fragments of the antibodies useful in the present invention may be used for the detection and quantitation of an axonally-derived protein according to the methods disclosed herein in order to detect and diagnose neurological disease in the same manner as an intact antibody. Such fragments are typically produced by proteolytic cleavage, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab')2 fragments).
An antibody is said to be "capable of binding" a molecule if it is capable of specifically reacting with the molecule to thereby bind the molecule to the antibody. The term "epitope" is meant to refer to that portion of any molecule capable of being bound by an antibody that can also be recognized by that antibody. Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and have specific three dimensional structural characteristics as well as specific charge characteristics.
An "antigen" is a molecule capable of being bound by an antibody that is additionally capable of inducing an animal to produce antibody capable of binding to an epitope of that antigen. An antigen may have one, or more than one epitope. The specific reaction referred to above is meant to indicate that the antigen will react, in a highly selective manner, with its corresponding antibody and not with the multitude of other antibodies that may be evoked by other antigens.
The antibodies, or fragments of antibodies, useful in the present invention may be used to quantitatively or qualitatively detect the presence of cells that contain the axonally-derived protein antigens. Thus, the antibodies (or fragments thereof) useful in the present invention may be employed histologically to detect or visualize the presence of an axonally-derived protein.
Such an assay for an axonally-derived protein typically comprises incubating a biological sample from said subject suspected of having such a condition in the presence of a detectably labeled binding molecule (e.g., antibody) capable of identifying an axonally-derived protein, and detecting said binding molecule which is bound in a sample.
Thus, in this aspect of the invention, a biological sample may be treated with nitrocellulose, or other solid support that is capable of immobilizing cells, cell particles or soluble proteins. The support may then be washed with suitable buffers followed by treatment with the detectably labeled axonally-derived protein-specific antibody. The solid phase support may then be washed with the buffer a second time to remove unbound antibody. The amount of bound label on said solid support may then be detected by conventional means.
By "solid phase support" is intended any support capable of binding antigen or antibodies. Well-known supports, or carriers, include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, agaroses, and magnetite. The nature of the carrier can be either soluble to some extent or insoluble for the purposes of the present invention. The support material may have virtually any possible structural configuration so long as the coupled molecule is capable of binding to an antigen or antibody. Thus, the support configuration may be spherical, as in a bead, or cylindrical, as in the inside surface of a test tube, or the external surface of a rod. Alternatively, the surface may be flat such as a sheet, test strip, etc. Preferred supports include polystyrene beads. Those skilled in the art will note many other suitable carriers for binding monoclonal antibody or antigen, or will be able to ascertain the same by use of routine experimentation.
One embodiment for carrying out the diagnostic assay of the present invention on a biological sample containing an axonally-derived protein, comprises:
(a) contacting a detectably labeled axonally-derived protein-specific antibody with a solid support to effect immobilization of said axonally-derived protein-specific antibody or a fragment thereof;
(b) contacting a sample suspected of containing an axonally-derived protein with said solid support;
(c) incubating said detectably labeled axonally-derived protein-specific antibody with said support for a time sufficient to allow the immobilized axonally-derived protein-specific antibody to bind to the axonally-derived protein;
(d) separating the solid phase support from the incubation mixture obtained in step (c); and
(e) detecting the bound label and thereby detecting and quantifying axonally-derived protein.
Alternatively, labeled axonally-derived protein-specific antibody/axonally-derived protein complexes in a sample may be separated from a reaction mixture by contacting the complex with an immobilized antibody or protein which is specific for an immunoglobulin, e.g., Staphylococcus protein A, Staphylococcus protein G, anti-IgM or anti-IgG antibodies. Such anti-immunoglobulin antibodies may be polyclonal, but are preferably monoclonal. The solid support may then be washed with a suitable buffer to give an immobilized axonally-derived protein/labeled axonally-derived protein-specific antibody complex. The label may then be detected to give a measure of an axonally-derived protein.
This aspect of the invention relates to a method for detecting an axonally-derived protein or a fragment thereof in a sample comprising:
(a) contacting a sample suspected of containing an axonally-derived protein with an axonally-derived protein-specific antibody or fragment thereof which binds to axonally-derived protein; and
(b) detecting whether a complex is formed.
The invention also relates to a method of detecting an axonally-derived protein in a sample, further comprising:
(c) contacting the mixture obtained in step (a) with an Fc binding molecule, such as an antibody, Staphylococcus protein A, or Staphylococcus protein G, which is immobilized on a solid phase support and is specific for the axonally-derived protein-specific antibody to give an axonally-derived protein/axonally-derived protein-specific antibody immobilized antibody complex;
(d) washing the solid phase support obtained in step (c) to remove unbound axonally-derived protein/axonally-derived protein-specific antibody complex;
(e) and detecting the label bound to said solid support.
Of course, the specific concentrations of detectably labeled antibody and axonally-derived protein, the temperature and time of incubation, as well as other assay conditions may be varied, depending on various factors including the concentration of an axonally-derived protein in the sample, the nature of the sample, and the like. The binding activity of a given lot of anti-axonally-derived protein antibody may be determined according to well-known methods. Those skilled in the art will be able to determine operative and optimal assay conditions for each determination by employing routine experimentation.
Other such steps as washing, stirring, shaking, filtering and the like may be added to the assays as is customary or necessary for the particular situation.
One of the ways in which the axonally-derived protein-specific antibody can be detectably labeled is by linking the same to an enzyme. This enzyme, in turn, when later exposed to its substrate, will react with the substrate in such a manner as to produce a chemical moiety that can be detected, for example, by spectrophotometric, fluorometric or by visual means. Enzymes which can be used to detectably label the axonally-derived protein-specific antibody include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-V-steroid isomerase, yeast alcohol dehydrogenase, alpha -glycerophosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta -galactosidase, ribonuclease, urease, catalase, glucose-VI-phosphate dehydrogenase, glucoamylase and acetylcholinesterase.
Detection may be accomplished using any of a variety of immunoassays. For example, by radioactively labeling the axonally-derived protein-specific antibodies or antibody fragments, it is possible to detect axonally-derived protein through the use of radioimmunoassays.
The radioactive isotope can be detected by such means as the use of a gamma counter or a scintillation counter or by autoradiography. Isotopes that are particularly useful for the purpose of the present invention are: 3H, 125I, 131I, 35S, 14C, and preferably 125I.
It is also possible to label the axonally-derived protein-specific antibody with a fluorescent compound. When the fluorescently labeled antibody is exposed to light of the proper wavelength, its presence can then be detected due to fluorescence. Among the most commonly used fluorescent labelling compounds are fluorescein isothiocyanate, rhodamine, phycoerytherin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.
The axonally-derived protein-specific antibody can also be detectably labeled using fluorescence emitting metals such as 152Eu, or others of the lanthanide series. These metals can be attached to the axonally-derived protein-specific antibody using such metal chelating groups as diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).
The axonally-derived protein-specific antibody also can be detectably labeled by coupling it to a chemiluminescent compound. The presence of the chemiluminescent-tagged axonally-derived protein-specific antibody is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction. Examples of particularly useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.
The axonally-derived protein-specific antibody may also be labeled with biotin and then reacted with avidin. A biotin-labeled DNA fragment will be linked to the axonally-derived protein-biotinylated monoclonal antibody by an avidin bridge. axonally-derived protein molecules are then detected by polymerase chain reaction (PCR) amplification of the DNA fragment with specific primers.
Likewise, a bioluminescent compound may be used to label the axonally-derived protein-specific antibody of the present invention. Bioluminescence is a type of chemiluminescence found in biological systems in which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent protein is determined by detecting the presence of luminescence. Important bioluminescent compounds for purposes of labeling are luciferin, luciferase and aequorin.
Detection of the axonally-derived protein-specific antibody may be accomplished by a scintillation counter, for example, if the detectable label is a radioactive gamma emitter, or by a fluorometer, for example, if the label is a fluorescent material. In the case of an enzyme label, the detection can be accomplished by calorimetric methods that employ a substrate for the enzyme. Detection may also be accomplished by visual comparison of the extent of enzymatic reaction of a substrate in comparison with similarly prepared standards.
The detection of foci of such detectably labeled antibodies is indicative of a disease or dysfunctional state as previously described. For the purposes of the present invention, the axonally-derived protein that is detected by this assay may be present in a biological sample. Any sample containing an axonally-derived protein can be used. However, one of the benefits of the present diagnostic invention is that invasive tissue removal may be avoided. Therefore, preferably, the sample is a biological solution such as, for example, cerebrospinal fluid, amniotic fluid, blood, serum, urine and the like. However, the invention is not limited to assays using only these samples, it being possible for one of ordinary skill in the art to determine suitable conditions that allow the use of other samples.
For example, the three-site monoclonal antibody-based immunoradiometric assays (M-IRMA) may be used to measure axonally-derived protein levels in a biological fluid, such as CSF, blood, plasma or serum. While it is possible to obtain, by spinal tap, on a routine basis, CSF from individuals suspected of having neurological disease, it is an intrusive method. Thus, the diagnosis of neurological disease can be established by a simple, non-invasive blood immunoassay that reveals axonally-derived protein levels greatly increased over normal levels.
There are many different in vivo labels and methods of labeling known to those of ordinary skill in the art. Examples of the types of labels that can be used in the present invention include radioactive isotopes and paramagnetic isotopes. Those of ordinary skill in the art will know of other suitable labels for binding to the antibodies used in the invention, or will be able to ascertain such, using routine experimentation. Furthermore, the binding of these labels to the antibodies can be done using standard techniques common to those of ordinary skill in the art.
An important factor in selecting a radionuclide for in vivo diagnosis is that the half-life of a radionuclide be long enough so that it is still detectable at the time of maximum uptake by the target, but short enough so that deleterious radiation upon the host is minimized. Ideally, a radionuclide used for in vivo imaging will lack a particulate emission, but produce a large number of photons in the 140-200 keV range, which maybe readily detected by conventional gamma cameras.
For in vivo diagnosis radionuclides may be bound to antibody either directly or indirectly by using an intermnediary functional group. Intermediary functional groups that are often used in binding radioisotopes that exist as metallic ions to immunoglobulins are DTPA and EDTA. Typical examples of ions that can be bound to immunoglobulins are 99mTc, 123I, 111In, 131I, 97Ru, 67Cu, 67Ga, 125I, 68Ga, 72As, 89Zr, and 201T1.
For diagnostic in vivo imaging, the type of detection instrument available is a major factor in selecting a given radionuclide. The radionuclide chosen must have a type of decay that is detectable for a given type of instrument. In general, any conventional method for visualizing diagnostic imaging can be utilized in accordance with this invention. For example, PET, gamma, beta, and MRI detectors can be used to visualize diagnostic imagining.
The antibodies useful in the invention can also be labeled with paramagnetic isotopes for purposes of in vivo diagnosis. Elements that are particularly useful, as in Magnetic Resonance Imaging (MRI), include 157Gd, 55Mn, 162Dy, and 56Fe.
The antibodies (or fragments thereof) useful in the present invention are also particularly suited for use in in vitro immunoassays to detect the presence of an axonally-derived protein in body tissue, fluids (such as CSF, blood, plasma or serum), or cellular extracts. In such immunoassays, the antibodies (or antibody fragments) may be utilized in liquid phase or, preferably, bound to a solid-phase carrier, as described above.
Those of ordinary skill in the art will know of other suitable labels that may be employed in accordance with the present invention. The binding of these labels to antibodies or fragments thereof can be accomplished using standard techniques commonly known to those of ordinary skill in the art. Coupling techniques mentioned in the latter are the glutaraldehyde method, the periodate method, the dimaleimide method, the m-maleimidobenzyl-N-hydroxy-succinimide ester method, all of which methods are incorporated by reference herein.
Removing a histological specimen from a patient, and providing the combination of labeled antibodies of the present invention to such a specimen may accomplish in situ detection. The antibody (or fragment) is preferably provided by applying or by overlaying the labeled antibody (or fragment) to a biological sample. Through the use of such a procedure, it is possible to determine not only the presence of an axonally-derived protein, but also the distribution of an axonally-derived protein on the examined tissue. Using the present invention, those of ordinary skill will readily perceive that any of a wide variety of histological methods (such as staining procedures) can be modified in order to achieve such in situ detection.
The binding molecules of the present invention may be adapted for utilization in an immunometric assay, also known as a "two-site" or "sandwich" assay. In a typical immunometric assay, a quantity of unlabeled antibody (or fragment of antibody) is bound to a solid support that is insoluble in the fluid being tested (i.e., CSF, blood, plasma or serum) and a quantity of detectably labeled soluble antibody is added to permit detection and/or quantitation of the ternary complex formed between solid-phase antibody, antigen, and labeled antibody.
Typical, and preferred, immunometric assays include "forward" assays in which the antibody bound to the solid phase is first contacted with the sample being tested to extract the antigen from the sample by formation of a binary solid phase antibody-antigen complex. After a suitable incubation period, the solid support is washed to remove the residue of the fluid sample, including unreacted antigen, if any, and then contacted with the solution containing an unknown quantity of labeled antibody (which functions as a "reporter molecule"). After a second incubation period to permit the labeled antibody to complex with the antigen bound to the solid support through the unlabeled antibody, the solid support is washed a second time to remove the unreacted labeled antibody. This type of forward sandwich assay may be a simple "yes/no" assay to determine whether antigen is present or may be made quantitative by comparing the measure of labeled antibody with that obtained for a standard sample containing known quantities of antigen.
In another type of "sandwich" assay, which may also be useful with the antigens of the present invention, the so-called "simultaneous" and "reverse" assays are used. A simultaneous assay involves a single incubation step as the antibody bound to the solid support and labeled antibody are both added to the sample being tested at the same time. After the incubation is completed, the solid support is washed to remove the residue of fluid sample and uncomplexed labeled antibody. The presence of labeled antibody associated with the solid support is then determined as it would be in a conventional "forward" sandwich assay.
In the "reverse" assay, stepwise addition first of a solution of labeled antibody to the fluid sample followed by the addition of unlabeled antibody bound to a solid support after a suitable incubation period is utilized. After a second incubation, the solid phase is washed in conventional fashion to free it of the residue of the sample being tested and the solution of unreacted labeled antibody. The determination of labeled antibody associated with a solid support is then determined as in the "simultaneous" and "forward" assays.
The above-described in vitro or in vivo detection methods may be used in the detection and diagnosis of neurological disease without the necessity of removing tissue. Such detection methods may be used to assist in the determination of the stage of neurological deterioration in neurological disease by evaluating and comparing the concentration of an axonally-derived protein in the biological sample.
As used herein, an effective amount of a diagnostic reagent (such as an antibody or antibody fragment) is one capable of achieving the desired diagnostic discrimination and will vary depending on such factors as age, condition, sex, extent of disease of the subject, counter-indications, if any, and other variables to be adjusted by the physician. The amounts of such materials that are typically used in a diagnostic test are generally between 0.1 to 5 mg, and preferably between 0.1 to 0.5 mg.
The assay of the present invention is also ideally suited for the preparation of a kit. Such a kit may comprise a carrier means being compartmentalized to receive in close confinement therewith one or more container means such as vials, tubes and the like, each of said container means comprising the separate elements of the immunoassay.
For example, there may be a container means containing a first antibody immobilized on a solid phase support, and a further container means containing a second detectably labeled antibody in solution. Further container means may contain standard solutions comprising serial dilutions of the axonally-derived protein to be detected. The standard solutions of an axonally-derived protein may be used to prepare a standard curve with the concentration of axonally-derived protein plotted on the abscissa and the detection signal on the ordinate. The results obtained from a sample containing an axonally-derived protein may be interpolated from such a plot to give the concentration of the axonally-derived protein.
Claim 1 of 16 Claims
1. A method for detecting and quantitating axonally-derived tau protein in a human subject, comprising:
(a) contacting a blood sample from a human subject with an antibody capable of binding to the tau protein;
(b) detecting the bound tau protein; and
(c) determining the amount of bound tau protein based upon detection of the bound protein.