Inventors: Kerkay; Julius (8040 Barbara Dr., Strongsville, OH 44136) Appl. No.: 736352 Filed: May 21, 1985

Primary Examiner: Rosen; Sam Assistant Examiner: Saunders; David A. Attorney, Agent or Firm: Woodling, Krost & Rust United States Patent 4,818,708 April 4, 1989

Parent Case Text
This application is a continuation of application Ser. No. 08/055,778, filed May 5, 1993, now abandoned, which is a continuation of application Ser. No. 07/451,975, filed Dec. 20, 1989, now abandoned, which is a continuation in part of application Ser. No. 07/287,207, filed Dec. 21, 1988, now abandoned.

Claims
What is claimed is:

1. A method for detecting the presence of nPTP in a human subject, said method comprising:

(a) contacting cerebrospinal fluid or brain tissue removed from said human subject with one or more antibodies that specifically bind PTP and nPTP; and

(b) detecting any of said antibodies which are bound to, or detecting any of said antibodies which are not bound to, nPTP.

2. A method for detecting the presence of nPTP in a human fetus or embryo, said method comprising:

(a) removing amniotic fluid from a female human pregnant with said fetus or embryo

(b) contacting the amniotic fluid of (a) with one or more antibodies that specifically bind PTP and nPTP; and

(c) detecting any of said antibodies which are bound to, or detecting any of said antibodies which are not bound to, nPTP.

3. The method of claim 2, wherein said method aids in the prenatal diagnosis of Down's Syndrome or a neural tube defect.

4. The method of claim 2, wherein said method aids in the prenatal diagnosis of Down's Syndrome in a human fetus or embryo, and wherein detection of at least approximately 20 ng/ml of nPTP indicates that said fetus or embryo has Down's Syndrome.

5. The method of claim 2, wherein said method aids in the prenatal diagnosis of a neural tube defect in a human fetus or embryo, and wherein detection of at least approximately 200 ng/ml of nPTP indicates that said fetus or embryo has a neural tube defect.

6. A method which aids in the diagnosis of Alzheimer's Disease in a human subject suspected of having Alzheimer's Disease which comprises:

(a) incubating a biological sample comprising cerebrospinal fluid or brain tissue from said subject in the presence of an antibody that specifically binds PTP and nPTP; and

(b) detecting any of said antibodies which are bound to, or detecting any of said antibodies which are not bound to, nPTP,

wherein detection of elevated levels of nPTP indicates that said subject has or should be further examined for other indications of Alzheimer's Disease.

7. The method of claim 6, wherein said incubating step further includes adding a known quantity of labeled PTP or nPTP whereby a competitive immunoassay is established.

8. The method of claim 7, wherein said label is capable of emitting radiation.

9. The method of claim 8, wherein said label is .sup.125 I.

10. A method which aids in the diagnosis of Down's Syndrome in a human subject suspected of having Down's Syndrome which comprises:

(a) incubating a biological sample comprising cerebrospinal fluid or brain tissue from said subject in the presence of one or more antibodies that specifically bind PTP and nPTP;

(b) detecting any of said antibodies which are bound to, or detecting any of said antibodies which are not bound to, nPTP in said sample; and

(c) comparing the results obtained in step (b) with results obtained with samples comprising known concentrations of purified PTP or nPTP, in order to determine the concentration of nPTP in said sample,

wherein detection of at least approximately 20 ng/ml of nPTP in said sample indicates that said subject has Down's Syndrome.

11. The diagnostic method of claim 10, wherein said detection is by an immunometric assay.

12. The method of claim 11, wherein said immunometric assay is a monoclonal antibody-based immunometric assay.

13. The method of claim 11, wherein said immunometric assay comprises two different antibodies bound to a solid phase support combined with a third different detectably labeled antibody in solution.

14. The method of claim 10, wherein said incubating step further includes adding a known quantity of labeled PTP or nPTP whereby a competitive immunoassay is established.

15. The method of claim 14, wherein said label is capable of emitting radiation.

16. The method of claim 15, wherein said label is .sup.125 I.

17. A method which aids in the diagnosis of a neural tube defect in a human subject suspected of having a neural tube defect which comprises:

(a) incubating a biological sample comprising cerebrospinal fluid or brain tissue from said subject which is suspected of containing elevated levels of nPTP, in the presence of one or more antibodies that specifically bind PTP and nPTP;

(b) detecting any of said antibodies which are bound to, or detecting any of said antibodies which are not bound to, nPTP in said sample; and

(c) comparing the results obtained in step (b) with results obtained with samples comprising known concentrations of purified PTP or nPTP, in order to determine the concentration of nPTP in said sample,

wherein detection of at least approximately 200 ng/ml of nPTP indicates that said subject has a neural tube defect.

18. The method of claim 17, wherein said detection is by an immunometric assay.

19. The method of claim 18, wherein said immunometric assay is a monoclonal antibody-based immunometric assay.

20. The method of claim 18, wherein said immunometric assay comprises two different antibodies bound to a solid phase support combined with a third different detectably labeled antibody in solution.

21. The method of claim 17, wherein said incubating step further includes adding a known quantity of labeled PTP nPTP whereby a competitive immunoassay is established.

22. The method of claim 21, wherein said label is capable of emitting radiation.

23. The method of claim 22, wherein said label is .sup.125 I.

24. A method which aids in the diagnosis of pancreatic disease or injury in a human subject suspected of having pancreatic disease or injury which comprises:

(a) incubating a sample of urine from said subject suspected of containing elevated levels of PTP in the presence of one or more antibodies that specifically bind PTP;

(b) detecting any of said antibodies which are bound to, or detecting any of said antibodies which are not bound to, PTP; and

(c) comparing the results obtained in step (b) with results obtained with samples comprising known concentrations of purified PTP or NPTP, in order to determine the concentration of PTP in said sample,

wherein detection of at least approximately 150 ng/ml of PTP in said sample indicates that said subject suffers from pancreatic disease or injury.

25. The method of claim 24, wherein said detection is by an immunometric assay.

26. The method of claim 25, wherein said immunometric assay is a monoclonal antibody-based immunometric assay.

27. The method of claim 25, wherein said immunometric assay comprises two different antibodies bound to a solid phase support combined with a third different detectably labeled antibody in solution.

28. The method of claim 24, wherein said incubating step further includes adding a known quantity of labeled PTP or nPTP whereby a competitive immunoassay is established.

29. The method of claim 28, wherein said label is capable of emitting radiation.

30. The method of claim 29, wherein said label is .sup.125 I.

31. A method which aids in the diagnosis of Down's Syndrome in a human subject suspected of having Down's Syndrome which comprises:

(a) incubating a biological sample comprising cerebrospinal fluid or brain tissue from said subject which is suspected of containing elevated levels of nPTP, in the presence of one or more antibodies that specifically bind PTP and nPTP; and

(b) detecting any of said antibodies which are bound to, or detecting any of said antibodies which are not bound to, nPTP,

wherein detection of elevated levels of nPTP indicates that said subject has or should be further examined for other indications of Down's Syndrome.

32. A method which aids in the diagnosis of a neural tube defect in a human subject suspected of having a neural tube defect which comprises:

(a) incubating a biological sample comprising cerebrospinal fluid or brain tissue from said subject which is suspected of containing elevated levels of nPTP, in the presence of one or more antibodies capable of binding PTP and nPTP; and

(b) detecting any of said antibodies which are bound to, or detecting any of said antibodies which are not bound to, NPTP,

wherein detection of elevated levels of nPTP indicates that said subject has or should be further examined for other indications of a neural tube defect.

33. A method for detecting the presence of nPTP in a human subject, said method comprising:

(a) preparing a soluble extract of cerebrospinal fluid, brain tissue, blood, urine, lymph or serum from said human subject;

(b) fractionating the soluble extract of (a) according to molecular size, and retaining fractions comprising material with an apparent molecular weight of from about 17 to about 20 kilodaltons;

(c) contacting one or more of the fractions retained in (b) with one or more antibodies that specifically bind nPTP; and

(d) detecting any of said bound antibodies which are bound to, or detecting any of said antibodies which are not bound to, nPTP.

34. A method which aids in the diagnosis of pancreatic or neural disease or injury in a human subject suspected of having pancreatic or neural disease or injury which comprises:

(a) contacting cerebrospinal fluid, blood or lymph of said subject with one or more labeled antibodies that specifically bind nPTP and PTP; and

(b) detecting by imaging any of said antibodies which are bound to PTP, nPTP or PTP and nPTP,

wherein the detection of an elevated level of PTP, nPTP or PTP and nPTP is performed by in situ imaging of said bound antibodies, and wherein detection of an elevated level of PTP, nPTP or PTP and nPTP indicates that said subject has or should be further examined for other indications of pancreatic or neural disease or injury.

35. A method which aids in the diagnosis of pancreatic or neural disease or injury in a human subject suspected of having pancreatic or neural disease or injury which comprises:

(a) contacting cerebrospinal fluid, blood or lymph of said subject with one or more labeled antibodies that specifically bind nPTP and PTP; and

(b) detecting by imaging any of said antibodies which are bound to PTP, nPTP or PTP and nPTP,

wherein the detection of an elevated level of PTP, nPTP or PTP and nPTP is performed by in vitro imaging of said bound antibodies, and wherein detection of an elevated level of PTP, nPTP or PTP and nPTP indicates that said subject has or should be further examined for other indications of pancreatic or neural disease or injury.

36. The method of claim 1, 2, 6, 7-23, 24-30, 33, 34 or 35, wherein said antibodies are 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) that specifically bind PTP and nPTP.

37. The method of claim 1, 2, 6, 31, 32, or 33, wherein said detection of PTP, nPTP or PTP and nPTP is performed by immunometric assay.

38. The method of claim 37, wherein said immunometric assay is a monoclonal antibody-based immunometric assay.

39. The method of claim 37, wherein said immunometric assay comprises two different antibodies bound to a solid phase support combined with a third different detectably labeled antibody in solution.

40. The method of claim 17, 3 or 5, wherein said neural tube defect is selected from the group consisting of:

(a) anencephaly;

(b) spina bifida;

(c) meningocele;

(d) meningomyelocele; and

(e) holoprosencephaly.

41. The method of any one of claims 1, 2, 6, 7-23, 24-30, 33, 34, 35, wherein said antibodies are produced by a hybridoma deposited at the American Type Culture Collection selected from the group consisting of HB 9934, HB 9935, and HB 9936.

Description
FIELD OF THE INVENTION

This invention relates to proteins associated with Alzheimer's Disease, Down's Syndrome, neural tube defects and pancreatic disease. The invention further relates to the genes encoding such proteins, immunodiagnostic and molecular diagnostic methods to diagnose these diseases.

Abbreviations

For brevity, the following abbreviations are used throughout this application: Pancreatic Thread Protein (PTP); Neural Pancreatic Thread Protein (nPTP); Immunoradiometric Assay (IRMA); Monoclonal Antibody (mAb); Alzheimer's Disease (AD); Down's Syndrome (DS); Neurofibrillary Tangles (NFTs); and Paired Helical Filaments (PHFs).

BACKGROUND OF THE INVENTION

Neurological Diseases

Alzheimer's Disease (AD) is the most frequent cause of dementia in the United States, affecting over two million individuals each year. It is a degenerative brain disorder characterized clinically by loss of memory, confusion, and gradual physical deterioration. It is the fourth most common cause of death. The etiology of the disease is virtually unknown but has been attributed to various viruses, toxins, heavy metals, as well as genetic defects. The disease is at present incurable.

Until quite recently, AD was thought to account for relatively few of the cases generally classified as senile dementia. Other factors can lead to such a condition, including repetitious mild strokes, thyroid disorders, alcoholism, and deficiencies of certain vitamins, many of which are potentially treatable. It can be appreciated, then, that a diagnostic test specific for AD would be very useful for the clinical diagnosis and proper clinical treatment of subjects presenting with symptoms common to all of these conditions.

The brains of individuals with AD exhibit characteristic pathological accumulations of congophilic fibrous material which occurs as neurofibrillary tangles (NFTs) within neuronal cell bodies, and neuritic (or senile) plaques. NFTs may also be found in the walls of certain cerebral blood vessels. The major organized structural components of NFTs are paired helical filaments (PHFs). Qualitatively indistinguishable amyloid deposits also occur in normal aged brains but in much smaller numbers with restricted topographical distribution.

There has been considerable recent investigative activity regarding the characterization of proteins found in neuritic plaques and NFTs of AD and other neurologic diseases. One of the amyloid proteins initially described by Glenner et al. has been cloned and sequenced (Glenner et al., Biochem. Biophys. Res. Commun. 120:1131-1135 (1984); U.S. Pat. No. 4,666,829). The A4 amyloid protein found in neuritic plaques and blood vessels has been determined to be a component of a 695 amino acid precursor; a protein postulated to function as a glycosylated cell surface receptor (Masters et al., Proc. Natl. Acad. Sci. USA 82:4245-4249 (1985), Kang et al., Nature 325:733-736 (1987)). The gene coding for A4 is located on chromosome 21 (Kang et al., ibid.; Goldgaber et al., Science 235:877-880 (1987); Tanzi et al., Science 235:880-885 (1987); St. George-Hyslop et al., Science 235:885-889 (1987)) but apparently is not linked to the familial form of the disease (Van Broekhoven et al., Nature 329:153-155 (1987)). There appears to be little, if any, protein sequence homology between amyloid A4 and .beta. protein, their higher molecular weight precursor, and nPTP described by the present invention (see discussion below) (Gross et al., J. Clin. Invest. 76:2115-2126 (1985)).

A number of other proteins thought to be associated with the disease have been described, including Ubiquitin, ALZ-50, microtubular-associated proteins .tau. and MAP2, and neurofilament protein (see, for example, Manetto et al., Proc. Natl. Acad. Sci. USA 85:4502-4505 (1988); Wolozin et al., Science 232:648-651 (1986); Selkoe, Neurobiol. Aging 7:425-432 (1986); Perry et al., in: Alterations of the Neuronal Cytoskeleton in Alzheimer's Disease, Plenum, N.Y., pp 137-149 (1987)). More recently, a serine protease inhibitor called .alpha..sub.1 -anti-chymotrypsin has been found in AD amyloid deposits (Abraham et al., Cell 52:487-501 (1988)).

Until this time, there has been no useful diagnostic test for AD. A definitive diagnosis is possible only postmortem, or during life through a brain biopsy, to reveal the presence of the characteristic plaques, tangles, PHFS, and other cerebrovascular deposits which characterize the disorder. Such an invasive surgical procedure is inherently dangerous and is therefore rarely utilized. As a result, the clinical misdiagnosis of AD is estimated to be approximately 20%-30%.

Down Syndrome (DS) results in mental retardation and is associated with a variable constellation of abnormalities caused by trisomy of at least a critical portion of chromosome 21 in some or all cells. No single physical sign is diagnostic and most stigmata are found in some normal persons. In rare patients, no chromosome abnormalities can be detected by routine cytogenetic analysis. Although DS can generally be detected pre- and post-natally by cytogenetic testing, an alternative diagnostic test which measured a parameter other than a gross karyotypic alteration would be useful in identifying and verifying the presence of DS in a subject, either pre- or post-natally.

Neural tube defects refer to defects which develop in the vertebrate embryo in a tube formed from differentiated middorsal ectoderm. In a developing fetus, the neural tube ultimately gives rise to the brain and spinal cord. Thus, defects in the neural tube often result in severe defects in these organs. For example, such defects could include anencephaly, the absence of the cerebral and cerebellar hemispheres of the brain, spina bifida (absence of vertebral arches of the spinal cord through which the spinal membranes (with or without spinal cord tissue) may protrude), meningocele (protrusion of the brain or spinal cord membranes through a defect in the skull or vertebral column), meningomyelocele (protrusion of the membranes and spinal cord through a defect in the vertebral column), or holoprosencephaly (failure of the forebrain to divide into hemispheres).

A simple prenatal diagnostic test, using amniotic fluid, for example, which could detect neural tube defects would be very useful in determining prenatal or early postnatal treatment such as, for example, immediate postnatal surgical intervention.

Pancreatic and other Diseases

Acute pancreatitis or acute pancreatic injury may be caused by multiple factors including alcohol, penetrating peptic ulcer, gallstones, drugs, trauma, uremia, etc. Diffuse abdominal pain, nausea and vomiting, fever, tachycardia, epigastric tenderness and rigidity are cardinal symptoms and physical findings. Often hemoconcentration and intravascular volume depletion are present. Total serum amylase activity of 3-5 times greater than normal has been the diagnostic anchor for such diseases, despite the lack of specificity of this test. Measurement of serum lipase has also been somewhat helpful in this regard. However, serum amylase and lipase may be elevated in this same range in a variety of serious and life-threatening illnesses, some of which are medical emergencies.

For example, it is well-known that serum lipase and total amylase activities may be elevated in perforated ulcer, intestinal obstruction, intestinal infarction, and renal insufficiency. In these conditions, where no pancreatic injury has occurred, the signs and symptoms may be quite similar to those of acute pancreatitis. The treatment of these extrapancreatic causes of elevated amylase and lipase activities, however, is quite different from that for pancreatitis. For example, surgery for acute pancreatitis is discouraged, whereas failure to perform surgery for intestinal infarction can have lethal consequences. Thus, the search for a more specific diagnostic test of acute and chronic pancreatic injury has great clinical significance.

It is therefore clear that a simple, standardized, and relatively inexpensive assay for diagnosing neural tube defects or pancreatic disease, as well as for specifically detecting DS and AD, would be an immensely useful diagnostic tool for the clinician and researcher alike.

Pancreatic Proteins

Pancreatic Thread Protein (PTP) is found in great abundance in the acinar cells of the pancreas and reaches concentrations of 1-2 mg/ml in normal pancreatic fluid as measured by a monoclonal antibody (mAb)-based immunoradiometric assay (M-IRMA) (Gross et al., J. Clin. Invest. 76:2115-2126 (1985)).

PTP in its monomeric form has an apparent molecular weight of approximately 14 kilodaltons (kD), consists of a single polypeptide chain and is rich in aromatic amino acids. The protein has unusual solubility characteristics: it undergoes a pH-dependent fibril formation at pH's between 5.4 and 9.2. The protein forms long "thread like" structures of 7-10 nm (by electron microscopy) when pancreatic fluid is allowed to stand for several hours at 4.degree. C. (Gross et al., J. Clin. Invest. 76:2115-2126 (1985)). Thus, PTP represents one of the major secretory products of the exocrine pancreas in man.

Another pancreatic protein called pancreatic stone protein (PSP) has been described by one research group (DeCaro et al., Biochem. Biophys. Res. Commun. 87:1176-1182 (1979)). Based on amino acid sequence, PSP appears identical to PTP (DeCaro et al., J. Biochem. 168:201-207 (1987)). A similar protein has been identified in bovine pancreas (Gross et al., Proc. Natl. Acad. Sci. USA 82:5627-5631 (1985)).

One group of investigators recently found that treatment of highly pure PHFs with pronase removed a 9.5 kD and 12 kD fragment which included the .tau. microtubular protein (Wischik et al., Proc. Natl. Acad. Sci. USA 85:4506-4510 (1988); Wischik et al., Proc. Natl. Acad. Sci. USA 85:4884-4888 (1988)). The insoluble core protein remaining following pronase digestion had repeating subunits to which a mAb was made. The mAb bound specifically to the core protein but did not bind the .tau. protein (Wischik et al., Proc. Natl. Acad. Sci. USA 85:4506-4510 (1988)). The solubility characteristics and physical appearance (under electron microscopy) of PTP (Gross et al., J. Clin. Invest. 76:2115-2126 (1985)) and the PHF core protein (Wischik et al., Proc. Natl. Acad. Sci. USA 85:4506-4510 (1988); Wischik et al., Proc. Natl. Acad. Sci. USA 85:4884-4888 (1988)) are similar.

SUMMARY OF THE INVENTION

A need exists for a definitive diagnostic test which can be performed on individuals suspected of having, or being at risk for, AD, DS, and other neurological diseases. The present invention satisfies such needs and provides further advantages.

The manner in which these and other objects are realized by the present invention will be apparent from the summary and detailed description set forth below.

Unless defined otherwise, various terms used herein have the same meaning as is well understood in the art to which the invention belongs. All cited publications are incorporated herein by reference.

Because of the insolubility of PTP at physiologic pH and the physical appearance of the fibrils by electron microscopy, the inventors saw a resemblance of PTP to some of the fibrils observed in neuritic plaques, NFTs, and particularly the PHFs, of AD, leading to their studies which resulted in the present invention.

The inventors have identified, by M-IRMA, high concentrations of a neural form of PTP, referred to as Neural PTP (nPTP) in AD and DS brain. nPTP has been found in all AD brains studied where characteristic neuropathologic changes of the disease exist. The saline-extractable soluble immunoreactivity shares at least three epitopes with the native pancreatic form of PTP and has a molecular weight of approximately 17 to 20 kD.

Quantitative measurements of nPTP immunoreactivity in various regions of AD brains revealed levels varying from 12 to 295 ng/gm tissue (Mean=116 ng/gm tissue) compared to 1-11 ng/gm tissue (Mean=5 ng/gm tissue) in comparable areas of control brains.

Immunocytochemistry performed with mAbs directed against the pancreatic form of PTP demonstrated that nPTP is localized within cells, within fine processes within the neuropil, or is extracellular in both AD and DS brains. Two types of cell contain nPTP: neurons and astrocytes. The affected neurons are the large pyramidal type which typically contain the NFTs well known in AD brain.

That nPTP accumulation within neurons is intrinsically important or integrally related to the evolution of AD lesions is corroborated by the presence of identical patterns of immunolabeling for nPTP in DS brains, but not in control brains. It is important to note that the same structural abnormalities of AD occur in brains of all middle-age individuals with Down's syndrome, whether or not they are demented. There is also a higher incidence of AD in family members of DS patients. Moreover, the regional differences in the densities of nPTP-containing neurons parallels the density distributions of NFTs in both AD and DS. This provides further evidence that nPTP is germane to the pathophysiology of AD. Whether nPTP accumulates within neuronal perikarya, as a result of aberrant cellular metabolism or transport is not yet known. Accordingly, one object of the present invention is to provide a relatively simple, sensitive, accurate, and painless diagnostic method for detecting AD, DS, or other neurological defects which involve incontinence of the bony coverings of central nervous system tissue, such as neural tube defects which would permit the escape of cerebrospinal fluid (CSF).

Another object of the present invention is to provide a highly specific assay for diagnosing and distinguishing AD and DS from other disorders. The assays described by the present invention are non-invasive, thus avoiding the painful and often hazardous removal of brain tissue samples. In view of the immense numbers of individuals potentially afflicted with AD, for example, the assays taught by the present invention will be relatively inexpensive to administer.

An additional object of the present invention is to provide a method for early diagnosis of neural tube defects. Prenatal diagnosis of these defects would allow for corrective actions to be taken prenatally or early postnatally.

Another object of the present invention is to provide a diagnostic method for detecting acute or chronic pancreatic disease, using a combination of antibodies as taught herein.

Furthermore, the assays of the present invention are capable of being reduced to a standardized format, easily and quickly performed.

The present invention additionally pertains to assays for detecting the presence of nPTP in the biological fluids of a human subject using histology, imaging, immunoassays, and the like as diagnostic methods for determining the presence of AD, DS, and neural tube defects, as well as detecting the presence of PTP as a diagnostic method for determining the presence of pancreatic disease.

In particular, the invention includes a method for detecting and quantitating nPTP in a human subject, comprising:

(a) contacting a biological sample from a human subject that is suspected of containing detectable levels of nPTP with a molecule capable of binding to the nPTP; and

(b) detecting the molecule bound to the nPTP.

The invention additionally includes the method as above, wherein the 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);

(d) a polynucleotide probe derived from the recombinant bovine PTP of this invention; and

(e) a polynucleotide probe derived from recombinant human PTP of this invention.

The invention additionally includes the method as above, wherein the detecting molecule is detectably labeled and where a combination of such 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, Down's Syndrome, anencephaly, spina bifida, meningocele, meningomyelocele, holoprosencephaly, and pancreatic disease.

The invention additionally includes the method as above, wherein the condition exists as a prenatal condition.

The invention additionally includes a method of diagnosing the presence of AD in a human subject suspected of having AD which comprises:

(a) incubating a biological sample from said subject suspected of containing nPTP with a molecule capable of identifying nPTP; and

(b) detecting the molecule which is bound in the sample, wherein the detection indicates that the subject has AD.

The invention additionally includes a method of diagnosing the presence of DS in a human subject suspected of having DS which comprises:

(a) incubating a biological sample from said subject suspected of containing nPTP with a molecule capable of identifying nPTP; and

(b) detecting the molecule which is bound in the sample, wherein the detection indicates that the subject has DS.

The invention additionally includes a method of diagnosing the presence of pancreatic disease in a human subject suspected of having pancreatic disease which comprises:

(a) incubating a biological sample from said subject, which is suspected of containing PTP, in the presence of a binding molecule capable of identifying PTP; and

(b) detecting the molecule which is bound in the sample, wherein the detection indicates that the subject has pancreatic disease.

The invention additionally includes the methods as above, wherein a biological sample is removed from a human subject prior to contacting the sample with the molecule.

The invention additionally includes the methods as above, wherein detecting any of the molecules bound to the protein is performed by in situ imaging.

The invention additionally includes the methods as above, wherein detecting of any of the molecule bound to the protein is performed by in vitro imaging.

The invention additionally includes the methods as above, wherein the biological sample is reacted with the molecule in a manner and under such conditions sufficient to determine the presence and the distribution of the protein.

The invention additionally includes the methods as above, wherein a detectably labeled binding molecule is administered to a human subject.

The invention additionally includes the methods as above, wherein the molecule is bound to the protein in vivo.

The invention additionally includes nPTP substantially free of any natural impurities with molecular weights of less than about 17 kD daltons, the nPTP having a molecular weight of about 17-20 kD. However, larger molecular weight forms may be detected and isolated with more vigorous extraction procedures.

The invention also includes a method for recovering nPTP substantially free of natural impurities which includes, but is not limited to, the following steps:

(a) recovering crude nPTP from a biological sample;

(b) subjecting the crude nPTP from step (a) to ion-exchange chromatography to obtain partially purified fractions of nPTP;

(c) subjecting the partially purified fractions of nPTP from step (b) to molecular sieve chromatography to obtain nPTP; and

(d) purifying nPTP to homogeneity by subjecting the nPTP from step (c) to gel chromatography to obtain nPTP substantially free of natural impurities with molecular weights of less than about 17 kD, said nPTP having a molecular weight of about 17-20 kD daltons.

The invention also includes the method as above, further comprising:

(e) subjecting the purified nPTP obtained in step (d) to affinity chromatography to obtain highly purified nPTP substantially free of natural impurities with molecular weights of less than about 17 kD, the nPTP having a molecular weight of about 17-20 kD.

The invention is particularly directed to a diagnostic method for determining the presence of AD in a human subject by detecting and measuring the concentration of nPTP by immunoassay, comprising:

(a) reacting a biological sample from a subject suspected of containing nPTP with an antibody or antibodies specific to nPTP;

(b) monitoring the reaction of step (a) to determine whether the antibodies have bound to nPTP, the concentration of nPTP indicating whether the subject has AD.

The invention is also directed to a diagnostic method for determining the presence of DS in a human subject by detecting and measuring the concentration of nPTP by immunoassay, comprising:

(a) reacting a biological sample from a subject suspected of containing nPTP with an antibody or antibodies specific to nPTP;

(b) monitoring the reaction of step (a) to determine whether the antibodies have bound to nPTP, the concentration of nPTP indicating whether the subject has DS.

Additionally, the invention is particularly directed to a diagnostic method for determining the presence of pancreatic disease in a human subject by detecting and measuring the concentration of PTP by immunoassay, comprising:

(a) reacting a biological sample from a subject suspected of containing PTP with an antibody or antibodies specific to PTP;

(b) monitoring the reaction of step (a) to determine whether the antibodies have bound to nPTP, the concentration of PTP indicating whether the subject has pancreatic disease.

The present invention also particularly relates to the diagnostic methods recited above, wherein the immunoassay comprises two different antibodies bound to a solid phase support combined with a third different detectably labeled antibody in solution.