JP7173815B2 - Non-invasive method to detect target molecules - Google Patents

Description

Embodiments of the present invention relate to non-invasive methods and compositions for collecting, detecting, measuring and identifying target molecules. In some embodiments, the methods and compositions relate to target molecules in gastrointestinal washing fluid (GLF) or feces.

REFERENCE TO SEQUENCE LISTING This application is being filed in electronic form along with a Sequence Listing. The Sequence Listing is located in USA — 007 WO. It is provided as a file entitled TXT. The information in electronic form of the Sequence Listing is hereby incorporated by reference in its entirety.

Disorders associated with the gastrointestinal (GI) tract and hepatobiliary tract and organs/tissues associated with the GI tract include cancers such as gastric, esophageal, liver and pancreatic cancers. Pancreatic cancer (eg, pancreatic adenocarcinoma) is a malignant growth of the pancreas that primarily arises in cells of the pancreatic duct. The disease is the ninth most common form of cancer, but is the fourth and fifth cause of cancer death in men and women, respectively. Cancer of the pancreas is most often fatal, with a 5-year survival rate of less than 3%.

The most common symptoms of pancreatic cancer include jaundice, abdominal pain and weight loss, which along with other presenting factors are often non-specific in nature. Therefore, diagnosing pancreatic cancer at an early stage of tumor growth is often difficult, requires extensive work-up, and is often discovered incidentally during exploratory laparotomy. Endoscopic ultrasound is one exemplary non-surgical technique available for diagnosing pancreatic cancer. However, reliable detection of pancreatic cancer differentiation from small tumors as well as focal pancreatitis is difficult. Most patients with pancreatic cancer are now diagnosed at a late stage when the tumor has already spread beyond the pancreas, invaded surrounding organs, and/or has extensively metastasized. Non-Patent Document 1, which is incorporated herein by reference in its entirety. Late detection of disease is common in the majority of patients diagnosed with advanced disease that often leads to death, and only a minority of patients are detected with early disease.

Invasive techniques for diagnosing disorders and diseases associated with the GI tract are inconvenient and pose significant risks to the subject. Therefore, there is a need for non-invasive methods and compositions for detecting and identifying target molecules from the GI tract or related organs/tissues. In some embodiments, determining whether a biomarker is useful as a biomarker associated with a particular characteristic such as disease, disease predisposition, positive response to a treatment regimen or no response or negative response to a treatment regimen A target molecule can be evaluated to do so. Additionally, biomarkers derived from the GI tract or related organs/tissues can be used to determine whether an individual has any of the specific characteristics listed above.

Gold et al., Crit. Rev. Oncology/Hematology (2001) 39:147-54

Embodiments of the present invention relate to non-invasive methods and compositions for collecting, detecting, measuring and identifying target molecules. In some embodiments, the methods and compositions relate to target molecules in gastrointestinal washing fluid (GLF) or feces.

Some embodiments are a method for assessing the physiological status of a subject comprising obtaining gastrointestinal lavage from the subject and detecting target molecules from outside the gastrointestinal system in the gastrointestinal lavage. and a method comprising:

Some embodiments are a method for assessing the physiological status of a subject comprising obtaining a stool sample from the subject and detecting a target molecule from outside the gastrointestinal system in the stool sample. and a method comprising:

In some embodiments, the gastrointestinal wash is obtained from the subject by partially cleansing the subject's gastrointestinal system.

In some embodiments, the gastrointestinal wash includes fecal material. In some embodiments, the fecal sample comprises a gastrointestinal wash.

In some embodiments, target molecules include polypeptides, antibodies, bile acids, metabolites or glycans. In some embodiments, target molecules include biomarkers. In some embodiments, the biomarker is associated with disease, positive response to therapy, partial response to therapy, negative response to therapy, or no response to therapy. In some embodiments, the target molecule is associated with the presence of cancer or a predisposition to cancer. In some embodiments, the cancer is pancreatic, colorectal, liver or stomach cancer. In some embodiments, the target molecule is derived from an accessory digestive gland. In some embodiments, the accessory digestive gland is a salivary gland, pancreas, gallbladder or liver.

Some embodiments include administering a wash solution to the subject. In some embodiments, the wash solution is administered orally. In some embodiments, the cleaning solution comprises a component selected from the group consisting of polyethylene glycol, magnesium sulfate, sodium sulfate, potassium sulfate, magnesium citrate, ascorbic acid, sodium picosulfate and bisacodyl. In some embodiments, the cleaning solution is selected from the group consisting of GOLYTELY, HALFLYTELY, NULYTELY, SUPREP, FLEET's PHOSPHO-SODA, magnesium citrate and common equivalents thereof.

Some embodiments include performing a colonoscopy on the subject.

In some embodiments, the subject is a mammal. In some embodiments, the subject is human.

Some embodiments are a method for identifying a biomarker comprising a test gastrointestinal wash from a plurality of test subjects with a condition or physiological state of interest and without said condition or said physiological state obtaining a control gastrointestinal wash from a plurality of control subjects; determining levels of at least five target molecules in the test gastrointestinal wash and the control gastrointestinal wash; identifying target molecules that are present at significantly different levels in , thereby identifying biomarkers.

In some embodiments, the gastrointestinal wash includes fecal material.

In some embodiments, the target molecule is selected from the group consisting of polypeptides, bile acids, antibodies, metabolites, glycans and combinations thereof.

Some embodiments comprise determining the levels of at least ten target molecules in test gastrointestinal lavage and control gastrointestinal lavage. Some embodiments comprise determining the levels of at least 20 target molecules in test and control gastrointestinal lavage fluids. Some embodiments comprise determining levels of at least 30 target molecules in test and control gastrointestinal lavage fluids. Some embodiments comprise determining levels of at least 50 target molecules in test and control gastrointestinal lavage fluids. Some embodiments comprise determining levels of at least 100 target molecules in test and control gastrointestinal lavage fluids.

In some embodiments, the biomarker is associated with disease, positive response to therapy, partial response to therapy, negative response to therapy, or no response to therapy.

In some embodiments, the biomarker is associated with the presence of cancer or a predisposition to cancer. In some embodiments, the cancer is pancreatic cancer, liver cancer or stomach cancer.

In some embodiments, at least one target molecule is derived from an accessory digestive gland. In some embodiments, the accessory digestive gland is a salivary gland, pancreas, gallbladder or liver.

Some embodiments include administering a wash solution to test and control subjects. In some embodiments, the wash solution is administered orally. In some embodiments, the cleaning solution comprises a component selected from the group consisting of polyethylene glycol, magnesium sulfate, sodium sulfate, potassium sulfate, magnesium citrate, ascorbic acid, sodium picosulfate and bisacodyl. In some embodiments, the cleaning solution is selected from the group consisting of GOLYTELY, HALFLYTELY, NULYTELY, SUPREP and FLEET's PHOSPHO-SODA, magnesium citrate and common equivalents thereof.

Some embodiments include performing colonoscopies on test and control subjects.

In some embodiments, test and control subjects are mammals. In some embodiments, test and control subjects are humans.

Some embodiments are a method for identifying a biomarker comprising obtaining a test stool sample from a plurality of test subjects having a condition of interest and a control stool sample from a plurality of control subjects; determining the levels of at least five target molecules in the sample and the control stool sample; and identifying target molecules that are present at significantly different levels in the test stool sample relative to the levels in the control stool sample, thereby identifying a biomarker.

In some embodiments, the fecal sample comprises a gastrointestinal wash.

In some embodiments, the target molecule is selected from the group consisting of polypeptides, bile acids, antibodies, metabolites, glycans and combinations thereof.

Some embodiments comprise determining the levels of at least ten target molecules in test stool samples and control stool samples. Some embodiments comprise determining the levels of at least 20 target molecules in stool samples and control stool samples. Some embodiments comprise determining the levels of at least 30 target molecules in stool samples and control stool samples. Some embodiments comprise determining the levels of at least 50 target molecules in stool samples and control stool samples. Some embodiments comprise determining levels of at least 100 target molecules in stool samples and control stool samples.

In some embodiments, the biomarker is associated with disease, positive response to treatment or negative response to treatment.

In some embodiments, the biomarker is associated with the presence of cancer or a predisposition to cancer. In some embodiments, the cancer is pancreatic, colorectal, liver or stomach cancer.

In some embodiments, at least one target molecule is derived from an accessory digestive gland. In some embodiments, the accessory digestive gland is a salivary gland, pancreas, gallbladder or liver.

In some embodiments, test and control subjects are mammals. In some embodiments, test and control subjects are humans.

Some embodiments are kits for detecting a target molecule in a gastrointestinal lavage, comprising a lavage for oral administration to a subject, a container for collecting the gastrointestinal lavage from the subject, and a gastrointestinal lavage. and agents for detecting exogenously derived target molecules.

Some embodiments are kits for detecting a target molecule in a fecal sample, comprising a lavage fluid for oral administration to a subject, a container for collecting the fecal sample from the subject, and a gastrointestinal tract. and agents for detecting exogenously derived target molecules.

Some embodiments include protease inhibitors.

In some embodiments, target molecules include polypeptides, antibodies, bile acids, metabolites or glycans. In some embodiments, target molecules include biomarkers. In some embodiments, the biomarker is associated with disease, positive response to treatment or negative response to treatment.

In some embodiments, the target molecule is associated with the presence of cancer or a predisposition to cancer. In some embodiments, the cancer is pancreatic cancer, liver cancer, colorectal cancer, or gastric cancer.

In some embodiments, the target molecule is derived from an accessory digestive gland. In some embodiments, the accessory digestive gland is a salivary gland, pancreas, gallbladder or liver.

In some embodiments, the cleaning solution comprises a component selected from the group consisting of polyethylene glycol, magnesium sulfate, sodium sulfate, potassium sulfate, magnesium citrate, ascorbic acid, sodium picosulfate and bisacodyl. In some embodiments, the cleaning solution is selected from the group consisting of GOLYTELY, HALFLYTELY, NULYTELY, SUPREP, FLEET's PHOSPHO-SODA, magnesium citrate and common equivalents thereof.
For example, the present invention provides the following items:
(Item 1)
A method for assessing the physiological status of a subject, comprising:
obtaining a gastrointestinal wash from the subject;
detecting target molecules from outside the gastrointestinal system in said gastrointestinal lavage fluid.
(Item 2)
A method for assessing the physiological status of a subject, comprising:
obtaining a stool sample from the subject;
detecting target molecules from outside the gastrointestinal system in said fecal sample.
(Item 3)
2. The method of item 1, wherein the gastrointestinal lavage fluid is obtained from the subject by partially cleansing the subject's gastrointestinal system.
(Item 4)
2. The method of item 1, wherein the gastrointestinal lavage comprises fecal material.
(Item 5)
3. The method of item 2, wherein the fecal sample comprises gastrointestinal washings.
(Item 6)
6. The method of any one of items 1-5, wherein said target molecule comprises a polypeptide, antibody, bile acid, metabolite or glycan.
(Item 7)
7. Method according to any one of items 1 to 6, wherein said target molecule comprises a biomarker.
(Item 8)
8. The method of item 7, wherein said biomarker is associated with disease, positive response to therapy, partial response to therapy, negative response to therapy or no response to therapy.
(Item 9)
9. The method of any one of items 1-8, wherein said target molecule is associated with the presence of cancer or a predisposition to cancer.
(Item 10)
9. The method of item 8, wherein the cancer is pancreatic cancer, colorectal cancer, liver cancer or gastric cancer.
(Item 11)
11. The method of any one of items 1-10, wherein said target molecule is derived from an accessory digestive gland.
(Item 12)
12. The method of item 11, wherein the accessory digestive gland is salivary gland, pancreas, gallbladder or liver.
(Item 13)
13. The method of any one of items 1-12, further comprising administering a lavage fluid to the subject.
(Item 14)
14. The method of item 13, wherein the wash solution is administered orally.
(Item 15)
14. The method of item 13, wherein the cleaning solution comprises a component selected from the group consisting of polyethylene glycol, magnesium sulfate, sodium sulfate, potassium sulfate, magnesium citrate, ascorbic acid, sodium picosulfate and bisacodyl.
(Item 16)
14. The method of item 13, wherein the cleaning solution is selected from the group consisting of GOLYTELY, HALFLYTELY, NULYTELY, SUPREP, FLEET's PHOSPHO-SODA, magnesium citrate and common equivalents thereof.
(Item 17)
17. The method of any one of items 1-16, further comprising performing a colonoscopy on the subject.
(Item 18)
18. The method of any one of items 1-17, wherein the subject is a mammal.
(Item 19)
19. The method of any one of items 1-18, wherein the subject is a human.
(Item 20)
A method for identifying a biomarker comprising:
obtaining a test gastrointestinal wash from a plurality of test subjects having the condition or physiological state of interest and a control gastrointestinal wash from a plurality of control subjects who do not have the condition or physiological state;
determining levels of at least five target molecules in the test gastrointestinal wash and the control gastrointestinal wash;
identifying target molecules that are present at significantly different levels in said test gastrointestinal lavage relative to levels in said control gastrointestinal lavage, thereby identifying a biomarker.
(Item 21)
21. The method of item 20, wherein the gastrointestinal lavage comprises fecal matter.
(Item 22)
22. The method of any one of items 20-21, wherein said target molecule is selected from the group consisting of polypeptides, bile acids, antibodies, metabolites, glycans and combinations thereof.
(Item 23)
23. The method of any one of items 20-22, comprising determining the levels of at least 10 target molecules in the test gastrointestinal wash and the control gastrointestinal wash.
(Item 24)
23. The method of any one of items 20-22, comprising determining levels of at least 20 target molecules in said test gastrointestinal wash and said control gastrointestinal wash.
(Item 25)
23. The method of any one of items 20-22, comprising determining levels of at least 30 target molecules in the test gastrointestinal wash and the control gastrointestinal wash.
(Item 26)
23. The method of any one of items 20-22, comprising determining levels of at least 50 target molecules in the test gastrointestinal wash and the control gastrointestinal wash.
(Item 27)
23. The method of any one of items 20-22, comprising determining levels of at least 100 target molecules in said test gastrointestinal wash and said control gastrointestinal wash.
(Item 28)
28. The method of any one of items 20-27, wherein said biomarker is associated with disease, positive response to treatment, partial response to treatment, negative response to treatment or no response to treatment. .
(Item 29)
28. The method of any one of items 20-27, wherein said biomarker is associated with the presence of cancer or a predisposition to cancer.
(Item 30)
29. The method of item 28, wherein said cancer is pancreatic cancer, liver cancer or gastric cancer.
(Item 31)
31. The method of any one of items 20-30, wherein at least one target molecule is derived from an accessory digestive gland.
(Item 32)
32. The method of item 31, wherein the accessory digestive gland is salivary gland, pancreas, gallbladder or liver.
(Item 33)
33. The method of any one of items 20-32, further comprising administering a wash solution to said test subject and said control subject.
(Item 34)
34. The method of item 33, wherein the wash solution is administered orally.
(Item 35)
34. The method of item 33, wherein the cleaning solution comprises a component selected from the group consisting of polyethylene glycol, magnesium sulfate, sodium sulfate, potassium sulfate, magnesium citrate, ascorbic acid, sodium picosulfate and bisacodyl.
(Item 36)
34. The method of item 33, wherein the washing solution is selected from the group consisting of GOLYTELY, HALFLYTELY, NULYTELY, SUPREP, FLEET's PHOSPHO-SODA, magnesium citrate and common equivalents thereof.
(Item 37)
37. The method of any one of items 20-36, further comprising performing a colonoscopy on the test subject and the control subject.
(Item 38)
38. The method of any one of items 20-37, wherein said test subject and said control subject are mammals.
(Item 39)
39. The method of any one of items 20-38, wherein said test subject and said control subject are human.
(Item 40)
A method for identifying a biomarker comprising:
obtaining a test stool sample from a plurality of test subjects with a condition of interest and a control stool sample from a plurality of control subjects;
determining levels of at least five target molecules in said test stool sample and said control stool sample; and target molecules present at significantly different levels in said test stool sample relative to levels in said control stool sample. and thereby identifying a biomarker.
(Item 41)
41. The method of item 40, wherein the fecal sample comprises gastrointestinal washings.
(Item 42)
42. The method of any one of items 40-41, wherein said target molecule is selected from the group consisting of polypeptides, nucleic acids, bile acids, antibodies, metabolites, glycans and combinations thereof.
(Item 43)
43. The method of any one of items 40-42, comprising determining levels of at least 10 target molecules in said test stool sample and said control stool sample.
(Item 44)
43. The method of any one of items 40-42, comprising determining levels of at least 20 target molecules in said stool sample and said control stool sample.
(Item 45)
43. The method of any one of items 40-42, comprising determining levels of at least 30 target molecules in said stool sample and said control stool sample.
(Item 46)
43. The method of any one of items 40-42, comprising determining levels of at least 50 target molecules in said stool sample and said control stool sample.
(Item 47)
43. The method of any one of items 40-42, comprising determining levels of at least 100 target molecules in said stool sample and said control stool sample.
(Item 48)
48. The method of any one of items 40-47, wherein said biomarker is associated with disease, positive response to therapy or negative response to therapy.
(Item 49)
49. The method of any one of items 40-48, wherein said biomarker is associated with the presence of cancer or a predisposition to cancer.
(Item 50)
50. The method of item 49, wherein said cancer is pancreatic cancer, colorectal cancer, liver cancer or gastric cancer.
(Item 51)
51. The method of any one of items 40-50, wherein at least one target molecule is derived from an accessory digestive gland.
(Item 52)
52. The method of item 51, wherein the accessory digestive gland is salivary gland, pancreas, gallbladder or liver.
(Item 53)
53. The method of any one of items 40-52, wherein said test subject and said control subject are mammals.
(Item 54)
54. The method of any one of items 40-53, wherein said test subject and said control subject are human.
(Item 55)
A kit for detecting a target molecule in gastrointestinal lavage, comprising:
a wash solution for oral administration to a subject;
a container for collecting the gastrointestinal lavage fluid from the subject;
and agents for detecting target molecules originating outside the gastrointestinal system.
(Item 56)
A kit for detecting a target molecule in a stool sample, comprising:
a wash solution for oral administration to a subject;
a container for collecting the fecal sample from the subject;
and agents for detecting target molecules originating outside the gastrointestinal system.
(Item 57)
57. The kit of any one of items 55-56, further comprising a protease inhibitor.
(Item 58)
58. The kit of any one of items 55-57, wherein said target molecule comprises a polypeptide, antibody, bile acid, metabolite or glycan.
(Item 59)
59. Kit according to any one of items 55-58, wherein said target molecule comprises a biomarker.
(Item 60)
60. The kit of item 59, wherein said biomarkers are associated with disease, positive response to therapy or negative response to therapy.
(Item 61)
61. The kit of any one of items 55-60, wherein said target molecule is associated with the presence of cancer or a predisposition to cancer.
(Item 62)
62. The kit of item 61, wherein said cancer is pancreatic cancer, liver cancer, colorectal cancer or gastric cancer.
(Item 63)
63. The kit of any one of items 55-62, wherein said target molecule is derived from an accessory digestive gland.
(Item 64)
64. The kit of item 63, wherein said accessory digestive gland is salivary gland, pancreas, gallbladder or liver.
(Item 65)
65. Any one of items 55 to 64, wherein the cleaning solution comprises a component selected from the group consisting of polyethylene glycol, magnesium sulfate, sodium sulfate, potassium sulfate, magnesium citrate, ascorbic acid, sodium picosulfate and bisacodyl. Kit as described.
(Item 66)
65. The kit of any one of items 55-64, wherein said wash solution is selected from the group consisting of GOLYTELY, HALFLYTELY, NULYTELY, SUPREP, FLEET's PHOSPHO-SODA, magnesium citrate and common equivalents thereof. .

FIG. 1 is a graphical representation of the relative abundance of glycan structures derived from various glycoproteins present in fractions of gastrointestinal lavage fluid. Derivatized glycans were eluted from the C18 reverse-phase column on a Q-TOF MS with approximately 20-25% acetonitrile in 0.2% formic acid. The mass spectrometer scanned from m/z 150-2000 every second in MS-only mode to acquire derivatized glycan profile data. FIG. 2 is a graphical representation of the relative abundance of compounds, including metabolites such as cholic acid, present in fractions of gastrointestinal lavage fluid. Data were acquired on a Waters Q-TOF mass spectrometer using MassLynx software using input from the LC system. The MS scanned over the mass range from m/z 100 to m/z 2000 every second.

Embodiments of the present invention relate to non-invasive methods and compositions for collecting, detecting, measuring and identifying target molecules. In some embodiments, the methods and compositions relate to target molecules in gastrointestinal washing fluid (GLF) or feces.

Gastrointestinal lavage is widely used as a preparation of the lower gastrointestinal (GI) tract for colonoscopy or colorectal surgery (e.g., DiPalma JA. et al., which is incorporated herein by reference in its entirety). 1984) Gastroenterology 86:856-60). The specific pathophysiology of diseases of the intestinal tract has been investigated by measuring proteins in the GLF (Evgenikos N et al. (2000) Br J Surg vol. 87, each of which is hereby incorporated by reference in its entirety). : 808-13, Brydon WG, Ferguson A. (1992) Lancet 340:1381-2, Choudari CP et al. (1993) Gastroenterology 104:1064-71, Ferguson A et al. : 120-4, Handy LM et al. (1996) Scand J Gastroenterol 31:700-5, and Stanley AJ et al. (1996) Gastroenterology 111:1679-82).

Fecal protein measurements can be useful for investigating various pathophysiology such as protein-losing enteropathy and mucosal inflammation. However, in some embodiments described herein, faeces may be used, although GLF contains less substances that interfere with the assay, and GLF rapidly crosses the GI tract. GLF is preferred over stool as a sample for detecting and identifying biomarkers because it causes less protein breakdown by digestive enzymes and bacterial proteases than stool samples. Furthermore, since the rate of fluid passage along the intestine can be estimated, it is possible to estimate the rate of protein release from the mucosa.

In some embodiments, GLF may be produced by orally administering a lavage fluid to a subject and passing a large volume of fluid through the intestinal tract, where the lavage fluid is a mixture of salts and other compounds such as polyethylene glycol and bisacodyl. It can contain substances. The irrigation fluid causes an influx of liquid into the colon, which flushes out solids. Irrigation solutions are commonly used to cause cleansing of the GI tract, as are commonly used in preparation for colonoscopies and other methods used to examine the GI tract. be. These fluids, which have flowed out of or remain in the largely cleared GI tract, are useful in assessing a variety of diseases because of the mouth-to-anus continuity along the GI tract. be. As a result, any and all organs, including the GI tract, that allow fluid to enter the GI tract are candidates for the methods and compositions provided herein.

GI Tract and Associated Organs/Tissues Some of the methods and compositions provided herein relate to organs/tissues associated with the GI tract, including the GI tract and accessory digestive glands. As is well known in the art, the GI tract includes the upper GI tract and the lower GI tract. The upper GI tract includes the oral or buccal oral cavity, esophagus, stomach and duodenum. The lower GI tract includes the jejunum, ileum and large intestine and the anus. The large intestine includes the appendix, cecum, colon and rectum.

Organs and tissues associated with the GI tract include structures outside the GI tract. Examples of such structures include the salivary glands, such as the parotid, submandibular and sublingual salivary glands, the pancreas, such as the exocrine pancreas, the gallbladder, bile ducts, and gastrointestinal appendages such as the liver. Additional examples of structures associated with the GI tract and outside the GI tract include the pancreatic duct, the biliary tree and bile ducts.

Gastrointestinal Laundry Fluid (GLF)
Generally, the lavage fluid can be administered orally to the subject, the oral lavage fluid is passed through the subject's GI tract, and the resulting GLF is collected from the subject. As used herein, the term "subject" can include animals such as mammals, eg, humans. As noted above, GLF provides a cleaner sampling of the GI tract than examination of fecal/stool samples. GLF appears to reduce the variability associated with food intake, type and digestive status.

Some embodiments described herein are useful for detecting target molecules or for screening, triage, disease detection, diagnosis, prognosis, treatment of diseases and conditions of the gastrointestinal tract or related organs/tissues. Includes analysis of GLF for response, treatment selection and personalized medicine. Some embodiments include analysis of GLF samples to rule out certain diseases and conditions from the possible diseases or conditions that a subject may be suffering from. Some embodiments include analysis of GLF to indicate the need for further testing for diagnosis. Further embodiments are for establishing new disease diagnoses, further classifying previous diagnoses, determining susceptibility to potential therapeutic regimens, and/or assessing response to previous or ongoing therapeutic regimens. Includes analysis of GLF.

Methods for Obtaining GLF Some embodiments of the methods and compositions provided herein involve obtaining GLF from a subject. Methods of obtaining GLF are well known in the art. For example, during medical and/or diagnostic procedures such as sigmoidoscopy, colonoscopy, radiography, preparation for patients undergoing intestinal surgery, the bowel and colon are thoroughly cleaned and It is important to be clean. In particular, it is imperative that as much fecal material as possible be removed from the colon to allow adequate visualization of the intestinal mucosa. This is important, for example, before diagnostic procedures such as flexible sigmoidoscopy or colonoscopy, diagnostic tests commonly performed to screen patients for diseases of the colon. Furthermore, it is important that the intestinal tract is thoroughly cleansed in order to obtain satisfactory radiographs of the colon. The same situation also applies when the colon is pre-operatively prepared for surgery, where removal of fecal waste is critical to patient safety. To prepare the colon for endoscopic examination, current cleansing procedures include upright ambulatory colonic irrigation. An upright ambulatory wash may comprise orally administering to a subject a wash composition comprising 4 L of a polyethylene glycol/electrolyte solution (U.S. Patent Application Publication No. 20070298008, which is incorporated by reference in its entirety). Some embodiments include antegrade and retrograde lavage.

Oral lavage compositions generally include solutions of electrolytes such as sodium, potassium and magnesium salts of sulfuric acid, bicarbonate, chloride, phosphate or citrate. Some such compositions may also contain polyethylene glycol, which may act as a non-absorbable osmotic agent. Typical compositions include polyethylene glycol with an electrolyte solution, and optionally compositions including bisacodyl or ascorbic acid and sulfate salts such as sodium, magnesium or potassium sulfate. In some embodiments, oral lavage solutions may include magnesium citrate. In some embodiments, oral lavage solutions may include sodium picosulfate. One exemplary composition of an oral lavage solution containing polyethylene glycol along with an electrolyte solution is GOLYTELY (Braintree Labs. Inc.). GOLYTELY is formulated according to the following: 59 g polyethylene glycol, 5.68 g sodium sulfate, 1.69 g sodium bicarbonate, 1.46 g sodium chloride, 0.745 g potassium chloride and water to make up 1 liter (see that Davis et al. (1980) Gastroenterology 78:991-995, which is incorporated in its entirety. Ingestion of GOLYTELY results in voluminous liquid stools with minimal changes in the subject's water and electrolyte balance. Another example of an oral lavage composition containing polyethylene glycol with an electrolyte solution is NULYTELY (Braintree Labs. Inc.). One exemplary oral lavage composition containing polyethylene glycol with an electrolyte solution and bisacodyl is HALFLYTELY (Braintree Labs. Inc.). One exemplary oral lavage composition containing a sulfate salt such as sodium sulfate, magnesium sulfate, or potassium sulfate is SUPREP (Braintree Labs. Inc.). One exemplary composition of an oral irrigation solution containing polyethylene glycol with an electrolyte solution and ascorbic acid is MOVIPREP (Salix Pharmaceuticals, Inc.).

Polyethylene glycol is effective as an oral lavage composition when large amounts of polyethylene glycol are administered in large volumes of dilute salt solutions. Typically, about 250-400 g of polyethylene glycol is administered to a subject in about 4 L of aqueous electrolyte solution. Oral administration of polyethylene glycol can be used to produce bowel movements over a period of time, eg, overnight. About 10-100 grams of polyethylene glycol in 8 ounces of water may be effective, although the dosage required will vary. A dose of about 68-85 g of polyethylene glycol can be effective to produce an overnight bowel movement without severe diarrhea. The volume of solution of polyethylene glycol in the isotonic fluid can be an effective amount of an osmotic laxative. A volume of about 0.5 L to about 4 L may be effective. Preferably, the effective volume is between about 1.5L and about 2.5L. Oral administration of 2 L of isotonic solution is effective.

Further examples of oral lavage compositions include non-phosphate hypertonic solutions in combination with osmotic laxatives such as polyethylene glycol (US Patent Application Publication No. 20090258090, which is incorporated by reference in its entirety). Mixtures of sulfates excluding phosphates, such as effective amounts of one or more of the following sulfates Na ₂ SO ₄ , MgSO ₄ and K ₂ SO ₄ may be effective (eg, SUPREP). Some embodiments contain from about 0.1 g to about 20.0 g of Na ₂ SO ₄ , with about 1.0 g to 10.0 g of Na ₂ SO ₄ being useful. _Dosages of about 0.01 g to about 40.0 g MgSO4 can be effective. Doses of Na ₂ SO ₄ from about 0.1 g to about 20.0 g, as well as doses from 1.0 to 10.0 g can also be used to advantage. Dosages of K ₂ SO ₄ from about 0.01 g to about 20.0 g can be effective to effect cleansing, with K 2 SO 4 from about 0.1 g to about 10.0 g and K A dose of ₂ SO ₄ may also be useful. Addition of an osmotic laxative such as polyethylene glycol (PEG) may improve the efficacy of the salt mixtures. Doses of PEG from about 1.0 g to about 100 g are effective. Doses of PEG from about 10.0 g to about 50 g are also effective, as are doses of about 34 g. For ease of administration, the salt mixture may be dissolved in a convenient volume of water. A volume of less than 1 liter of water is well tolerated by most subjects. The mixture may be dissolved in any small volume of water, with volumes between 100 and 500 ml being useful. The effective dose may be divided and administered to the patient in two or more doses over an appropriate period of time. In general, administration of two doses of equal parts of the effective dose, separated by 6-24 hours, provides satisfactory clearance. Some embodiments include interruption of normal oral intake for a defined period of time before and during administration of the oral lavage composition.

Some cleaning compositions contain laxatives such as bisacodyl. In some embodiments, a laxative may be co-administered to the subject with the cleansing composition. As will be appreciated, such co-administration includes, for example, administration of a laxative up to several hours prior to administration of the cleansing composition to the subject, combined with administration of the cleansing composition to the subject. This can include administering the laxative or administering the laxative up to several hours after administering the cleansing composition to the subject. Examples of laxatives and their effective doses include: Aloe, 250-1000 mg; Bisacodyl, about 5-80 mg; Cassanthranol, 30-360 mg; Cascara aromatic fluid extract, 2-24 ml; Cascada Sagrada Fluid Extract, 0.5-5.0 ml; Castor Oil, 15-240 ml; Danthrone, 75-300 mg; Dehydrocholic Acid, 250-2000 mg; phenolphthalein, 30-1000 mg; sennosides A and B, 12-200 mg; and picosulfate, 1-100 mg.

Further examples of cleaning compositions include aqueous solutions of concentrated phosphates. Aqueous phosphate concentrates have an osmotic effect on the intraluminal contents of the GI tract, and intestinal elimination occurs with a large influx of water and electrolytes from the body into the colon. One exemplary composition includes 480 g/L sodium dihydrogen phosphate and 180 g/L disodium hydrogen phosphate in a stabilized aqueous buffer solution (PHOSPHO-SODA from FLEET, C.S. Fleet Co., Inc.). Subjects will normally require ingestion of 2-3 ounce doses of this composition separated by 3-12 hour intervals for a total of 6 ounces (180 ml).

GLF can be collected from a subject before, during or after a medical or diagnostic procedure. In some embodiments, the subject may collect the GLF using a container such as, for example, a toilet insert that captures the fluid. Enzyme inhibitors and denaturants may be used to preserve the quality of GLF. In some embodiments, the pH of the sample may be adjusted to help stabilize the sample. In some embodiments, the GLF sample may be further processed to remove some or all solids and/or bacteria, such as by centrifugation. In some embodiments, the GI tract may not be completely cleansed by administering an oral lavage composition. For example, a subject may be administered a portion of the full dose of oral lavage composition required to completely cleanse the subject's GI tract. In some embodiments, GLF may include fecal material. In further embodiments, the fecal material may comprise GLF.

Target Molecules Some embodiments described herein relate to methods or compositions useful for detecting target molecules in samples obtained from the GI tract. As used herein, "target molecule" includes any molecule that can be detected or measured or identified in a sample obtained from the GI tract. Such samples include GLF obtained from a subject and fecal samples obtained from a subject. Examples of target molecules include peptides, polypeptides, proteins, mutant proteins, proteins resulting from alternative splicing, post-translationally modified proteins such as glycosylated proteins, phosphorylated proteins, antibodies. (e.g. autoantibodies, IgG, IgA and IgM), antibody fragments, sugars such as monosaccharides, disaccharides, oligosaccharides and glycans, lipids, small molecules such as metabolites, pharmaceutical compositions, metabolized pharmaceutical compositions and molecules such as prodrugs. Further examples of target molecules include bile salts and bile acids such as cholic acid. Further examples include chenodeoxycholic acid, glycocholic acid, taurocholic acid, deoxycholic acid and lithocholic acid. Target molecules can be from the GI tract and outside the GI tract, eg, from organs and/or tissues associated with the GI tract, such as the accessory digestive glands. In some embodiments, cell fragments and other biproducts such as cells, including red blood cells, white blood cells and endothelial cells, organisms such as bacteria, protozoa, and viruses and viral particles are produced from GLF or faeces. It can be detected in a sample. In some embodiments, the target molecule can be any of the proteins or portions thereof listed in Tables 1-10 herein or any portion thereof. In some embodiments, the portion of the proteins listed in any of Tables 1-10 is at least 10, at least 15, at least 20 of any of the proteins listed in Tables 1-10, It may contain at least 25, at least 50 or more than 50 contiguous amino acids. In some embodiments, the target molecule may comprise, consist essentially of, or consist of a polypeptide of one of SEQ ID NOs:01-804. A polypeptide consisting essentially of one of SEQ ID NOs:01-804 has additional or substituted amino acids relative to those in SEQ ID NOs:01-804, such additional or substituted amino acids are detected by the polypeptide. May be included if not precluded from being possible.

Target molecules also include biomarkers. As used herein, the term "biomarker" is associated with a disease, a predisposition to a disease, a positive response to a particular treatment regimen, no response to a particular treatment regimen, or a negative response to a particular treatment regimen. Including any target molecule present in a GLF or stool sample. In some embodiments, biomarkers can be correlated with identification, measurement and/or diagnosis or prognosis of disease.

In some embodiments, the target molecule is a constituent of a subject's fluid selected from the group consisting of blood, saliva, gastric juice, liver secretions, bile, duodenal juice, and pancreatic juice. In some embodiments, the target molecule is expressed in the subject's upper gastrointestinal tract or the subject's lower gastrointestinal tract. In some embodiments, the target molecule is in a subject selected from the group consisting of buccal cavity, esophagus, stomach, biliary tree, gallbladder, duodenum, jejunum, ileum, appendix, cecum, colon, rectum and anal canal. is expressed at the position of

In some embodiments, the target molecule is a protein or other compound found in GLF, such as lactoferrin, eosinophil-derived neurotoxin, eosinophil cationic protein, bilirubin (Bil), alkaline phosphatase (ALP) , aspartate aminotransferase, hemoglobin or eosinophil peroxidase. In some embodiments, the target molecule is a protein found in feces, such as heptaglobulin, hemopexin, alpha-2-macroglobulin, cadherin-17, calprotectin, carcinoembryogenic antigen. , metalloproteinase-1 (TIMP-1), S100A12, K-ras or p53. In some embodiments, the target molecule is a protein found in pancreatic juice, such as inferior gradient-2 (AGR2), insulin-like growth factor binding protein-2, CEACAM6, MUC1, CA19-9, serine proteinase-2 (PRSS2) preproprotein, pancreatic lipase-related protein-1 (PLRP1), chymotrypsinogen B (CTRB), elastase 3B (ELA3B), tumor rejection antigen (pg96), azurocidin, hepatocellular carcinoma-intestine-pancreas/pancreatitis It does not contain associated protein I (HIP/PAP-I), matrix metalloproteinase-9 (MMP-9), oncogene DJ1 (DJ-1) or α-1B-glycoprotein precursor (A1BG).

Methods of Characterizing Target Molecules Some embodiments of the methods and compositions provided herein involve characterizing target molecules in GLF or fecal samples. Characterizing the target molecule can include, for example, identifying the target molecule, detecting the target molecule and/or quantifying the target molecule. Methods for identifying, detecting and quantifying target molecules are well known in the art.

Some embodiments involve identifying, determining the presence or absence of, and/or quantifying target molecules, including peptides, polypeptides and/or proteins. Such target molecules can be characterized by a variety of methods such as immunoassays, including radioimmunoassays, enzyme-linked immunoassays and two-antibody sandwich assays, as described herein. A variety of immunoassay formats are also useful, including competitive and non-competitive immunoassay formats, antigen capture assays and two-antibody sandwich assays (Self and Cook, (1996) Curr. Opin. Biotechnol. 7, which is incorporated by reference in its entirety). Volume: pp. 60-65). Some embodiments include one or more antigen capture assays. In an antigen capture assay, antibodies are bound to a solid phase and a sample is added such that the antigen, eg, GLF or a target molecule in a stool sample, is bound by the antibody. After unbound protein has been removed by washing, the amount of bound antigen can optionally be quantified using, for example, a radioassay (Harlow et al., incorporated by reference in its entirety). and Lane, (1988) Antibodies A Laboratory Manual Cold Spring Harbor Laboratory: New York). Immunoassays can be performed under conditions such as antibody excess or antigen competition to quantify the amount of antigen and thus determine the levels of target molecules in GLF or fecal samples.

Enzyme-linked immunosorbent assay (ELISA) may be useful in certain embodiments provided herein. Enzymes such as horseradish peroxidase (HRP), alkaline phosphatase (AP), β-galactosidase or urease can be linked, for example, to anti-HMGB1 antibodies or to secondary antibodies for use in the methods of the invention. A horseradish-peroxidase detection system, for example, can be used with the chromogenic substrate tetramethylbenzidine (TMB), which in the presence of hydrogen peroxide yields a soluble product that is detectable at 450 nm. Other convenient enzyme-linked systems include, for example, alkaline phosphatase detection systems that can be used with the chromogenic substrate p-nitrophenyl phosphate, which yields a soluble product readily detectable at 405 nm. Similarly, a β-galactosidase detection system can be used with the chromogenic substrate o-nitrophenyl-β-D-galactopyranoside (ONPG), which yields a soluble product detectable at 410 nm, or a urease detection system can be used with urea - can be used with substrates such as bromocresol purple (Sigma Immunochemicals). Useful enzyme-linked primary and secondary antibodies can be obtained from several commercial sources, such as Jackson Immuno-Research (West Grove, Pa.), as further described herein.

In certain embodiments, target molecules in GLF or fecal samples can be detected and/or measured using chemiluminescent detection. For example, in certain embodiments, specific antibodies against particular target molecules are used to capture target molecules present in a biological sample, e.g., GLF or a fecal sample, and detect target molecules present in the sample. To do so, antibodies that are specific for target molecule-specific antibodies and labeled with chemiluminescent labels are used. Any chemiluminescent label and detection system can be used in the method. Chemiluminescent secondary antibodies are commercially available from a variety of sources including Amersham. Methods for detecting chemiluminescent secondary antibodies are known in the art.

Fluorescence detection can also be useful for detecting target molecules in certain methods provided herein. Useful fluorochromes include DAPI, fluorescein, Hoechst 33258, R-phycocyanin, B-phycoerythrin, R-phycoerythrin, rhodamine, Texas Red and Lissamine. Fluorescein- or rhodamine-labeled antibodies or fluorescein- or rhodamine-labeled secondary antibodies may be useful in the present invention.

Radioimmunoassays (RIAs) can also be useful in certain methods provided herein. Such assays are well known in the art. Radioimmunoassays can be performed, for example, using ¹²⁵ I-labeled primary or secondary antibodies (Harlow and Lane, supra, 1988).

The signal from the detectable reagent is, for example, a spectrophotometer to detect color from a chromogenic substrate; a radiation counter to detect radiation, such as a gamma counter for detection of ¹²⁵ I; It can be analyzed using a fluorometer to detect fluorescence in the presence of light of that wavelength. If an enzyme-linked assay is used, quantitative analysis of the amount of target molecule is performed using a spectrophotometer such as the EMAX Microplate Reader (Molecular Devices; Menlo Park, Calif.) according to the manufacturer's instructions. can be implemented The assays of the invention can be automated or robotically controlled, as desired, and can detect signals from multiple samples simultaneously.

In some embodiments, a capillary electrophoresis-based immunoassay (CEIA), which can optionally be automated, can be used to detect and/or measure target molecules. Immunoassays are also described, for example, in Schmalzing and Nashabeh, Electrophoresis 18:2184-93 (1997) and Bao, J. Am. Chromatogr. B. Biomed. Sci. 699:463-80 (1997) with laser-induced fluorescence. Liposomal immunoassays, such as flow injection liposomal immunoassays and liposomal immunosensors, can also be used to detect target molecules or to determine levels of target molecules, according to certain methods provided herein ( Rongen et al. (1997) J. Immunol. Methods 204:105-133, which is incorporated by reference in its entirety.

Sandwich enzyme immunoassays may also be useful in certain embodiments. In a two-antibody sandwich assay, the primary antibody is bound to a solid support and the antigen is allowed to bind to the primary antibody. By measuring the amount of secondary antibody that binds to it, the amount of target molecule is quantified.

Quantitative Western blotting can also be used in the methods provided herein to detect target molecules or to determine levels of target molecules. Western blots can be quantified by well-known methods such as scanning densitometry. As an example, protein samples are electrophoresed on a 10% SDS-PAGE Laemmli gel. For example, a primary murine monoclonal antibody against the target molecule is reacted with the blot and preliminary slot blot experiments are used to confirm that antibody binding is linear. A goat anti-mouse horseradish peroxidase conjugated antibody (BioRad) was used as the secondary antibody and signal detection was by chemiluminescence, e.g., the Renaissance chemiluminescence kit (New England Nuclear; Boston, Mass.) according to the manufacturer's instructions. is used according to the Blot autoradiographs are analyzed using a scanning densitometer (Molecular Dynamics; Sunnyvale, Calif.) and normalized to the positive control. Values are reported, for example, as the ratio between the observed value and the positive control (densitometry index). Such methods are described, for example, in Parra et al., J. Am. Vasc. Surg. 28:669-675 (1998).

As described herein, in some embodiments for detecting target molecules or determining levels of target molecules include, for example, enzyme-linked immunosorbent assays, radioimmunoassays, and quantitative western analysis. Other immunoassays may be useful. Such assays typically rely on one or more antibodies. As will be appreciated by those of skill in the art, the methods described herein can be used to readily distinguish between proteins having alternative forms of post-translational modification, such as phosphorylated and glycosylated proteins.

Target molecules, such as protein target molecules, can be characterized by a variety of methods. Proteins, polypeptides and peptides may be subjected to techniques such as protein precipitation, chromatography (e.g. reverse phase chromatography, size exclusion chromatography, ion exchange chromatography, liquid chromatography), affinity capture and differential extractions. It can be isolated by various methods well known in the art.

An isolated protein may undergo enzymatic digestion or chemical cleavage to produce polypeptide fragments and peptides. Such fragments can be identified and quantified. A particularly useful method for analysis of polypeptide/peptide fragments and other target molecules is mass spectrometry (US Patent Application No. 20100279382, which is incorporated by reference in its entirety). Several mass spectrometry-based quantitative proteomics methods have been developed to identify the proteins contained in each sample and to determine the relative abundance of each identified protein across samples (each of which is incorporated by reference). 20:S23-29 (2002), Aebersold, J. Am.Soc.Mass Spectrom.14:685-695 (2003), Aebersold, J. Am. 187 Supplement 2: S315-320 (2003), Patterson and Aebersold, Nat. Genet. (2003), Aebersold, R. and Cravatt, Trends Biotechnol.20: S1-2 (2002), Aebersold and Goodlett, Chem. Aebersold, Curr. Opin. Biotechnol. 14:110-118 (2003)). Generally, proteins in each sample are labeled to acquire isotopic signatures that identify their sample of origin and provide the basis for accurate mass spectrometric quantification. Samples with different isotopic signatures are then combined and analyzed, usually by multidimensional chromatographic tandem mass spectrometry. The resulting collision-induced dissociation (CID) spectra were then assigned to the peptide sequences and relative signal intensities for each detected protein in each sample were based on the relative signal intensities of differently isotopically labeled peptides of the same sequence. abundance is calculated.

An additional technique for identifying and quantifying target molecules, label-free quantitative proteomics methods. Such methods include (i) sample preparation including protein extraction, reduction, alkylation and digestion, (ii) sample separation by liquid chromatography (LC or LC/LC) and analysis by MS/MS, (iii) Includes data analysis including peptide/protein identification, quantification and statistical analysis. Each sample can be prepared separately and then subjected to separate LC-MS/MS or LC/LC-MS/MS runs (Zhu W. et al., J. of Biomedicine and Biotech.(2010) Article ID 840518, page 6). As one exemplary technique, the mass of a peptide is combined with its corresponding chromatographic elution time as a peptide characteristic that uniquely defines the peptide sequence, the LC-MS, accurate mass and time (AMT) tag approach, and There is a method called AMT tags were compared to previously acquired LC-MS/ Peptide sequences can be identified by matching with the MS sequence information. By exploiting the linear correlation observed between measured peptide peak areas and their abundance, these peptides can be relatively quantified by the signal intensity ratios of their corresponding peaks compared between MS runs. (Tang, K. et al. (2004) J. Am. Soc. Mass Spectrom. 15:1416-1423, and Chelius, D. and Bondarenko, P.V. (2002) J. Proteome Res. 1:317-323). Data from multiple LC-MS runs of each sample can be analyzed using statistical tools such as Student's t-test (Wiener, M. C. et al., (2004), which is incorporated by reference in its entirety). ) Anal. Chem. 76:6085-6096). To detect peptides with statistically significant differences in abundance between samples, the amplitude of the signal intensity obtained from multiple LC-MS runs at each point of acquisition time and m/z was duplicated. of samples can be compared.

As will be appreciated, various mass spectrometry systems can be used in methods for identifying and/or quantifying polypeptides/peptide fragments. As a mass spectrometer with high mass accuracy, high sensitivity and high resolution, ion trap, triple quadrupole and time-of-flight mass spectrometer, quadrupole time-of-flight mass spectrometer and Fourier transform ion cyclotron mass spectrometer (FT-ICR-) MS). Mass spectrometers are usually equipped with matrix-assisted laser desorption (MALDI) or electrospray ionization (ESI) ion sources, although other methods of peptide ionization can also be used. In ion trap MS, analytes are ionized by ESI or MALDI and then placed in an ion trap. The trapped ions can then be separately analyzed by MS upon selective ejection from the ion trap. Fragments can also be generated and analyzed in an ion trap. Sample molecules such as released polypeptides/peptide fragments can be analyzed by single-stage mass spectrometry, eg, with MALDI-TOF or ESI-TOF systems. Analytical methods of mass spectrometry are well known to those of skill in the art (e.g., Yates, J. (1998) Mass Spect. 33:1-19, Kinter and Sherman, each of which is incorporated by reference in its entirety). (2000) Protein Sequencing and Identification Using Tandem Mass. Spectrometry, John Wiley & Sons, New York, and Aebersold and Goodlett, (2001) Chem. For high-resolution polypeptide fragment separation, liquid chromatography ESI-MS/MS or automated LC-MS/MS utilizing capillary reversed-phase chromatography as the separation method can be used (incorporated by reference in its entirety, Yates et al., Methods Mol. Biol. 112:553-569 (1999)). Data-dependent collision-induced dissociation (CID) with dynamic exclusion can also be used as a mass spectrometry method (Goodlett et al., Anal. Chem. 72:1112-1118 (2000), which is incorporated by reference in its entirety). ).

Once the peptides are analyzed by MS/MS, the resulting CID spectra can be compared against databases for determination of the identity of the isolated peptides. Methods for protein identification using single peptides have been previously described (Aebersold and Goodlett, Chem. Rev. 101:269-295 (2001), each of which is incorporated by reference in its entirety. ), Yates, J. Mass Spec., 33:1-19 (1998), David N. et al., Electrophoresis, 20:3551-67 (1999)). It is possible that one or a few peptide fragments can be used to identify the parent polypeptide from which the fragment was derived, especially if the peptide provides a unique identifying property of the parent polypeptide. Additionally, identification of a single peptide, alone or in combination with knowledge of the site of glycosylation, can be used to identify the parent glycopolypeptide from which the glycopeptide fragment was derived. As will be appreciated, methods involving MS can be used to characterize proteins, fragments thereof, as well as other types of target molecules described herein.

Some embodiments may include enriching the protein and/or the protein fraction of GLF. Exemplary methods can include protein precipitation, chromatography such as reverse phase chromatography, size exclusion chromatography, ion exchange chromatography, liquid chromatography as well as affinity capture, fractional extraction and centrifugation. Proteins and/or protein fractions can be further examined using crude protein methods such as top-down proteomics or gel chromatography such as SDS-PAGE.

Some embodiments involve identifying, determining the presence or absence of, and/or quantifying target molecules comprising glycosylated proteins and/or glycans. Glycosylated proteins and glycans can be analyzed by various methods well known in the art. Changes in glycosylation can be indicative of disease or disease states. A particular target molecule may therefore comprise a particular glycosylated protein and/or glycan. As will be appreciated, glycans can be constituents of glycoproteins, proteoglycans or other glycan-containing compounds.

Some embodiments involve identifying, determining the presence or absence of, and/or quantifying target molecules that include metabolites. Metabolites can be analyzed in GLF or fecal samples using a variety of methods. For example, GLF or fecal samples can be analyzed for metabolites using methods such as chromatography. Several components of the metabolome include bile acids and other small organic compounds. Metabolites may include peptides present in GLF or fecal samples.

Methods for Identifying Biomarkers In some embodiments, target molecules detected in GLF or fecal samples are evaluated to associate them with a particular condition, such as a disease or physiological condition. It can be determined whether it is a biomarker. Such biomarkers can indicate a particular disease, disease predisposition, prognosis, positive response to a particular treatment regimen or negative response to a particular treatment regimen. In some embodiments, the presence, absence, or level of a biomarker may be associated with a particular condition, such as a disease or physiological condition. In some embodiments, the presence or absence or level of a biomarker can be statistically correlated with a particular condition, such as a disease or physiological condition. In some embodiments, a physiological condition can include disease. In some embodiments, comparing the level of biomarker expression in a subject with a condition, such as a disease or physiological condition, to the level of biomarker expression in a subject without the condition or physiological condition allows correlation of biomarkers with a particular condition, such as a disease or physiological condition.

In some embodiments, differential expression of a biomarker in a subject with the condition compared to expression of the biomarker in a subject without the condition is indicative of the condition or physiological state. As used herein, "differential expression" refers to the difference in expression levels of a biomarker in a subject with a condition, such as a disease or physiological condition, and a subject without a disease or physiological condition, etc. Point. For example, the term "differential expression" can refer to the presence or absence of a biomarker in a subject with a condition, such as a disease or physiological condition, compared to a subject without the condition or physiological condition. In some embodiments, differential expression is expressed in a subject with a disease or physiological condition, such as a condition, compared to the level of expression of a biomarker in a subject without the disease or physiological condition, etc. can refer to differences in the expression levels of biomarkers in

Differences in biomarker levels can be determined by measuring the amount or level of biomarker expression using the methods provided herein. In some embodiments, differential expression can be determined as a ratio of levels of one or more biomarker products between reference subjects/populations with or without the condition or physiological condition. , where the ratio is statistically significant. Differential expression between populations can be determined to be statistically significant as a function of p-value. When using p-values to determine statistical significance, biomarkers, p-values are preferably less than 0.2. In another embodiment, a biomarker is differentially expressed if the p-value is less than 0.15, 0.1, 0.05, 0.01, 0.005, 0.0001, etc. identified as When determining differential expression based on a ratio, the biomarker product has a ratio of levels of expression in the first sample compared to the second sample that is greater than 1.0; Differentially expressed if less than 0. For example, ratios greater than 1.0 include, for example, ratios greater than 1.1, 1.2, 1.5, 1.7, 2, 3, 4, 10, 20, and the like. Ratios less than 1.0 include, for example, ratios less than 0.9, 0.8, 0.6, 0.4, 0.2, 0.1, 0.05, and the like. In another embodiment, the biomarker has a ratio of the mean level of expression of the first population compared to the mean level of expression of the second population greater than or less than 1.0 can be differentially expressed in some cases. For example, ratios greater than 1.0 include ratios greater than 1.1, 1.2, 1.5, 1.7, 2, 3, 4, 10, 20, etc., and ratios less than 1.0 include , for example, ratios less than 0.9, 0.8, 0.6, 0.4, 0.2, 0.1, 0.05, and the like. In another embodiment, the biomarker is differentially expressed if the ratio of the level of expression in the first sample compared to the mean of the second population is greater than or less than 1.0. expressed at a ratio greater than, for example, 1.1, 1.2, 1.5, 1.7, 2, 3, 4, 10, 20 or less than 1, such as 0.9, 0.8, 0 ratios of 0.6, 0.4, 0.2, 0.1, 0.05.

In some embodiments, the biomarker is a test GLF or test stool sample obtained from at least one test subject with the condition or physiological condition and at least one control without the condition or physiological condition. measuring the level of at least one target molecule in a control GLF or control stool sample obtained from the subject; by comparing the levels of at least one target molecule in a stool sample, wherein a significant difference in the level of at least one target identifies a biomarker. Some embodiments include test GLF or test stool samples obtained from multiple test subjects with the condition or physiological condition and control GLF obtained from multiple control subjects without the condition or physiological condition. or measuring and comparing multiple target molecules in control stool samples. In some embodiments, at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 targets Molecules can be measured and compared. In some embodiments, the GLF or fecal sample is at least May be obtained from 95 or 100 test subjects. In some embodiments, the GLF or fecal sample is at least May be obtained from 95 or 100 control subjects. In some embodiments, a significant difference in levels of a target molecule in a test GLF or test stool sample compared to a control GLF or control stool sample can be statistically significant.

Kits Some embodiments of the methods and compositions provided herein are used to detect target molecules in GLF or stool samples, to determine the presence or absence of target molecules in GLF or stool samples, to Kits for quantifying target molecules in stool samples or for identifying target molecules in GLF or stool samples. Some such kits may contain a cleansing composition for oral administration to a subject. In some embodiments, the cleaning solution may include ingredients such as polyethylene glycol, magnesium sulfate, sodium sulfate, potassium sulfate, magnesium citrate and bisacodyl. In some embodiments, the cleaning solution may include polyethylene glycol (eg, GOLYTELY, HALFLYTELY, NULYTELY, MOVIPREP), optionally with an electrolyte solution that also includes bisacodyl or ascorbic acid. In some embodiments, the wash solution may include a phosphate (eg, FLEET's PHOSPHO-SODA). In some embodiments, the cleaning solution may include sulfates such as sodium sulfate, magnesium sulfate, or potassium sulfate (eg, SUPREP). In some embodiments, the wash solution may contain magnesium citrate. In some embodiments, the wash solution may include sodium picosulfate.

In some embodiments, the kit can also include a container for collecting GLF and/or fecal samples from the subject. A container for collecting GLF may include a toilet insert that captures GLF or a fecal sample or the like. In some embodiments, the container may contain materials for stabilizing and/or preserving the target molecule, such as one or more isolated protease inhibitors. In some embodiments, the container may contain an agent for detecting the target molecule, determining the presence or absence of the target molecule, quantifying the target molecule, or identifying the target molecule.

Diseases Some embodiments of the methods and compositions provided herein relate to diagnosis, prognosis of particular diseases. Some embodiments include diseases and disorders associated with the GI tract and associated organs. Exemplary diseases include cancers of the GI tract and associated organs, such as stomach cancer, liver cancer, pancreatic cancer. Further examples of diseases include pancreatitis, pancreatic adenocarcinoma, gastrointestinal neuroendocrine tumors, gastric adenocarcinoma, colon adenocarcinoma, hepatocellular carcinoma, cholangiocarcinoma, adenoccarcinoma, ulcerative colitis and Crohn's disease. Several diseases are associated with inflammatory bowel disease (IBD). As used herein, the term "inflammatory bowel disease" can refer to a broad class of diseases characterized by inflammation of at least part of the gastrointestinal tract. IBD symptoms include inflammation of the intestinal tract and can result in abdominal muscle cramps and persistent diarrhea. Inflammatory bowel disease, including ulcerative colitis (UC), Crohn's disease (CD), indeterminate colitis, chronic colitis, discrete or patchy disease, ileal inflammation, extracolonic inflammation, granulation depending on ruptured crypts Tumor inflammation, aphthous ulcer, transmural inflammation, microscopic colitis, diverticulitis and empty colitis. Further examples of diseases include celiac sprue, malabsorption disorders and other conditions of the digestive tract, liver, pancreas and biliary tree.

Some embodiments of the methods and compositions provided herein benefit from the selection of treatment (often referred to as personalized medicine), positive subject response to treatment, negative response to treatment or Relating to determining no response. Some such embodiments include determining a patient's partial response to a treatment regimen. For example, at an initial time point, the presence of biomarkers, absence of biomarkers or levels of biomarkers can be determined in a GLF or stool sample obtained from a subject. At a second time point after treatment has begun and/or treatment has been completed, the presence of biomarkers, absence of biomarkers or levels of biomarkers can be determined in a GLF or stool sample obtained from the subject. Presence of biomarkers in GLF or stool samples at a second time point compared to presence of biomarkers, absence of biomarkers, or levels of biomarkers in GLF or stool samples obtained from the first time point; Absence or differences in biomarker levels can indicate a subject's positive response to treatment, negative response to treatment, partial response to treatment, or no response to treatment. Alternatively, subjects can be given a treatment regimen and classified as having a positive response, a negative response, a partial response, or no response. The presence, absence or level of target molecules in each subject group can be determined, and target molecules with a statistically significant association with each category of response can be identified. Some embodiments also consider the determination of the subject's positive response, negative response, partial response or no response to a previous or current treatment regimen, and the future It also includes deciding on a treatment plan. Thus, previous or current treatment regimens may be modified based on decisions made by the methods provided herein.

Further embodiments include methods for determining the physiological status of a subject by evaluating multiple biomarkers. Some such methods involve determining the presence, absence and/or levels of multiple biomarkers. The presence, absence and/or levels of multiple biomarkers can be used to assess a subject's physiological characteristics, such as the subject's likelihood of developing a disease and/or the subject's likely response to a therapeutic regimen for treating a particular disease. It can be correlated with the likelihood of the situation. In some such methods, by correlating the presence, absence and/or levels of a plurality of biomarkers to determine the likelihood that the subject has or will develop the disease, the A "clinical risk score" can be determined (e.g., Soonmyung P. et al. (2004) New Eng. J. of Medicine 351:2817-2826, which is incorporated herein by reference in its entirety), and Cho CS et al., (2008) J. Am. Coll. Surg. 206:281-291).

Although the present invention has been described in some detail for purposes of clarity and understanding, various changes in form and detail can be made by those skilled in the art without departing from the true scope of the invention. you will understand.

(Example 1)
Proteomic Analysis of Sulfate-Based GLF This analysis evaluated the ability of sulfate-based GLF to support proteomic analysis. To identify target molecules in GLF obtained using a sulfate-based cleaning composition, SUPREP was administered to three human subjects and proteins in the resulting GLF were analyzed by mass spectrometry. . In this example, GLF was collected from a subject as part of a colonoscopy procedure.

At the time of collection, complete protease inhibitor tablets (ROCHE) were added and the samples were spun at 1000 rpm for 30 minutes at 4°C. The supernatant was spun again at 14,000 xg for 30 minutes to pellet bacteria and debris. 1.8 ml of supernatant was precipitated with 6 volumes of acetone, followed by extraction with an equal volume of chloroform, followed by separation on a C-2 reverse-phase SPE column (Sep-Pak, Waters). did. The column was washed with 3 column volumes each of 0.1% trifluoroacetic acid (TFA), 10%, 20% and 30% acetonitrile (ACN) in 0.1% TFA and , was eluted with 3 column volumes of 60% ACN. Samples were dried by centrifugal lyophilization, resuspended in 100 μl of 50 mM ammonium bicarbonate/10 mM Tris(2-carboxyethyl)phosphine, and digested with 2 μl of 10 mM sequencing grade trypsin (Promega, Madison, WI). .

Data were acquired on an LTQ-Orbitrap mass spectrometer using input from the LC system. The A solvent contained 3% B and 0.2% formic acid in water. B solvent contained 0.2% formic acid in 3% A and acetonitrile. Solvents were HPLC grade from Fisher. For a 120 minute run, the starting solvent is 5% B and left for 7 minutes. The gradient was changed to 10% by 13 minutes, 40% by 83 minutes, 90% by 103 minutes, then decreased from 90% to 5% at 111 minutes. It was then re-equilibrated for the next injection. Three injections were performed for each sample for reproducibility determinations.

The MS scanned over the mass range from 400 m/z to 2000 m/z every second (Orbitrap) and the LTQ (Trap) acquired up to 5 MSMS (peptide sequence) spectra in parallel. Data were acquired using standard Thermo Xcalibur software. MS data (Orbitrap) were stable down to 2-3 ppm and background ions were used for mass drift estimation. MSMS data (LTQ) measured down to approximately 0.6 Da, while parental masses were obtained from low ppm Orbitrap data. Peptides were eluted from a C18 LC column using triplicate injections to ensure data reliability and reproducibility. Search files were generated from triplicate injections from each wash preparation (patient sample) and converted to MGF (Mascot Generic Format) files using a combination of Xcalibur and Mascot software packages.

Database searches were performed using the taxonomy identified as homo sapiens, a mass accuracy of (MS) 10 ppm for parent ions and (MS/MS) 0.6 Da for fragment ions, using the RefSeq database (http://www. .ncbi.nlm.nih.gov/RefSeq/) using the Mascot search engine (Matrix Science, UK) and "not an enzyme" was selected. Searches without enzyme specificity were performed due to the presence of digestive enzymes in the samples that could modify or truncate the peptides being tested. The RefSeq database was supplemented by the addition of antibody sequences contained in the SwissProt protein database because these antibody sequences are not part of the standard RefSeq list.

Higher Mascot scores indicate better protein hits and can be correlated with relative protein levels. A score threshold of ">40" indicates a p-value significance of <0.05 as determined by the Mascot scoring system based on searches of this database without enzyme specificity; Consistent with <0.01. Standard Mascot scoring was used in which only the highest score for each peptide detected was added, even if sampled during MS/MS multiple rounds. For all data included, scores were all >40 in at least one sample per protein lineage. For added confidence, the number of significant peptides was also reported and a minimum criterion of at least 2 peptides was chosen. Very few had less than 3 peptides. All significant peptides counted corresponded to different sequences (individual peptides) from their respective proteins. Scores and number of significant peptides are reported in the format x/y, where x is the score and y is the number of significant peptides. If no protein was detected in a particular sample, it is listed as "ND". Proteins were reported as protein names and provided with "gi" numbers as defined by NCBI's protein database. Sequences contained within each of the "gi" numbers in the NCBI databases listed throughout this application are incorporated herein by reference. Where a protein is named as a pre-protein or other immature form, the mature form of the protein is equally implied, including alterations such as removal of signal sequences and addition of post-translational modifications. In all cases, proteins were named by their gene-derived sequence to provide consistency.

Table 1 lists examples of the most abundant proteins identified in GLF from three separate patients, defined as patients 3, 4 and 6, shown in the format above. As can be seen from Table 1, a large number of proteins can be identified from GLF, many of which may be associated with the pancreas. Other proteins include DMBT1 (gi number 148539840) which may be associated with colon and other cancers. Antibodies and putative glycosylated proteins were also identified.

In another experiment, subjects were administered SUPREP according to the manufacturer's guidelines, and the resulting GLF was self-collected by the subject into a collection container placed in the toilet immediately prior to colonoscopy. was done. The GLF proteome was analyzed by MS as described above. Results are summarized in Table 2 showing the Mascot scores of the most abundant species present. Results showed some urine contamination. A similar proteomic profile was observed for samples subsequently collected during colonoscopy. Table 2 shows that a number of different proteins were identified in the GLF collected by the subjects. Identified proteins, consistent with the data in Table 1, included DMBT1, pancreatic proteins and antibodies.

The foregoing analysis demonstrates that a large number of target molecules can be detected in samples obtained using sulfate-based GLF.

(Example 2)
Proteomic analysis of polyethylene glycol-based GLF This analysis evaluated the ability of polyethylene glycol-based GLF to support proteomic analysis. To identify target molecules in GLF obtained using a polyethylene-based cleaning composition, a polyethylene glycol-based cleaning composition was administered to two human subjects, and proteins in the resulting GLF were It was analyzed by mass spectrometry as described in Example 1. Removal of polyethylene glycol was mostly accomplished by chloroform extraction of the washings. A number of different proteins were identified in the GLF obtained from those subjects administered the polyethylene glycol-based cleansing composition. Examples of the most abundant identified proteins identified, consistent with those observed in previous tables, are shown in Table 3.

In another experiment, subjects were administered a PEG-based cleansing composition and subjects self-collected the resulting GLF into a collection container placed in the toilet just prior to colonoscopy. The GLF proteome was analyzed by MS as described herein. A number of different proteins were identified in self-collected GLF samples. Examples of the most abundant identified proteins and the corresponding Mascot scores and number of significant peptides for each protein are listed in Table 4. A larger protein list showed evidence of urine contamination. A similar proteomic profile was observed for samples subsequently collected during colonoscopy.

The proteome of GLF resulting from administration of a sulfate-based cleansing composition, collected at that time as part of a colonoscopy procedure or self-collected by a subject, was compared. The Mascot scores of the most abundant proteins and the number of significant peptides are summarized in Table 5. Although there was a close correlation between the two proteomes observed, different isoforms were identified for at least two proteins. The selection of different isoforms may be the result of sequence data collection and search engines during MS/MS. Less protein was detected in self-collected samples that were more dilute than those collected during colonoscopy.

The foregoing analyzes demonstrate that a large number of target molecules can be detected in samples obtained using polyethylene glycol-based GLF.

(Example 3)
Proteomic Analysis of Magnesium Citrate-Based GLF This analysis evaluated the ability of magnesium citrate-based GLF to support proteomic analysis. To identify target molecules in GLF obtained from a human subject administered a magnesium citrate-based cleansing composition, the magnesium citrate-based cleansing composition is administered to the subject and the GLF is examined by colonoscopy. Collected from subjects as part of the microscopy procedure. The GLF proteome was analyzed by mass spectrometry as described in Example 1. A number of different proteins have been identified in GLF. Examples of the most abundant identified proteins are listed in Table 6. Many of the identified proteins were detected using different colonoscopy preparations, suggesting that the proteome is independent of the intestinal preparation used.

The foregoing analysis demonstrates that a large number of target molecules can be detected in samples obtained using magnesium citrate-based GLF.

(Example 4)
Detection of IgA-1 and IgA-2 Antibodies in Samples Obtained Using GLF in Combination with SSL-7 Enrichment The ability to detect IgA-1 and IgA-2 antibodies was assessed. To identify target molecules in the GLF using affinity to Staphylococcus aureus superantigen-like protein 7 (SSL-7), human subjects were administered a sulfate-based cleansing composition (SUPREP), SSL-7 Proteins were enriched in each GLF using affinity beads. GLF was collected from subjects as part of a colonoscopy procedure.

IgA-1 and IgA-2 were specifically isolated using SSL-7 affinity beads. To 1 ml of sample was added 20 μl of SSL-7 agarose (Invitrogen, San Diego, Calif.) and incubated overnight at 4° C. on a roller. The tube was spun at 1,000 xg for 2 minutes to pellet the beads and the supernatant was discarded. Beads were washed four times with 1× phosphate-buffered saline and eluted with 20 μl of 100 mM glycine, pH 2.7 in a shaker at 37° C., 600 rpm for 1 hour. Eluted antibody was diluted with 60 μl of 100 mM ammonium bicarbonate/10 mM Tris(2-carboxyethyl)phosphine and digested with 2 μl of sequencing grade trypsin (Promega, Madison, Wis.). Mass spectrometry and database searches were performed as described above. The most abundant identified proteins present in GLF with affinity for SSL-7 and their corresponding Mascot scores are summarized in Table 7. As observed in previous examples, antibodies are present in the GLF and their enrichment and analysis are possible using affinity reagents, thus allowing specific analysis of this subproteome in the GLF. enable The most abundant antibody was IgA. IgA has been consistently reported to be present in the intestinal tract.

The foregoing analysis demonstrates that IgA antibodies can be detected in samples obtained using GLF in combination with SSL-7 enrichment.

(Example 5)
Detection of IgA and IgM in Samples Obtained Using GLF in Combination with Protein L Enrichment This assay detects IgA and IgM antibodies in samples obtained using GLF in combination with Protein L enrichment. evaluated ability. To identify target molecules in GLFs using affinity for Protein L, human subjects were administered a sulfate-based wash composition (SUPREP) and Protein L affinity beads were used to target proteins in each GLF. Concentrated. GLF was collected from subjects as part of a colonoscopy procedure.

Protein L affinity beads were used to isolate antibodies containing kappa light chains. 20 μl of protein L agarose (Santa Cruz Biotechnology, Santa Cruz, Calif.) was added to 1 ml of sample and incubated overnight at 4° C. on a roller. The tube was spun at 1,000 xg for 2 minutes to pellet the beads and the supernatant was discarded. Beads were washed four times with 1× phosphate-buffered saline and eluted with 20 μl of 100 mM glycine, pH 2.7 in a shaker at 37° C., 600 rpm for 1 hour. Eluted antibody was diluted with 60 μl of 100 mM ammonium bicarbonate/10 mM Tris(2-carboxyethyl)phosphine and digested with 2 μl of sequencing grade trypsin (Promega, Madison, Wis.). The most abundant identified proteins present in GLF with affinity for Protein L and their corresponding Mascot scores and number of significant peptides are summarized in Table 8. As expected, antibody-derived IgA and related chains were again detected. IgM antibodies (gi number 193806374) were also detected as protein L is not completely specific for IgA antibodies.

The foregoing analyzes demonstrate that IgA and IgM antibodies can be detected in samples obtained using GLF in combination with protein L enrichment.

(Example 6)
Detection of proteins of bacterial origin in samples obtained using GLF This analysis evaluated the ability of GLF to facilitate the detection of proteins of bacterial origin. To identify target molecules associated with bacteria in the GLF, two human subjects were administered a sulfate-based cleansing composition (SUPREP) and the resulting GLF was used for colonoscopy procedures. Collected from subjects as part. 100 μl from each GLF was used to inoculate Super Optimal Broth (SOB) medium and incubated overnight at 37° C. with 220 rpm shaking. The pellet was lysed in 8 M urea in a bead beater and the lysate was diluted with 50 mM ammonium bicarbonate/10 mM tris(2-carboxyethyl)phosphine to 2 M urea and sequenced grade trypsin (Promega, Madison, Wis.). was digested using Data were acquired using a 120 minute run on the Orbitrap MS system as previously described. An MGF search file was generated and analyzed in the RefSeq database (http://www. .ncbi.nlm.nih.gov/RefSeq/) using the Mascot search engine (Matrix Science, UK). The most abundant identified proteins present in GLF associated with bacteria and their corresponding Mascot scores and number of significant peptides are summarized in Table 9. In the samples shown, the cultured bacterium was Escherichia coli. Other samples show different bacteria indicating that the lavage fluid still retains some of the intestinal bacteria.

The foregoing analyzes demonstrate that proteins of bacterial origin can be detected in samples obtained using GLF.

(Example 7)
Proteomic Analysis of Combined Samples Obtained from Several Subjects To further facilitate identification of the large number of target proteins detectable in samples obtained using GLF, 12 subjects Search files generated from separately acquired data were concatenated into a single search file and searched using the parameters previously specified for the Orbitrap data. A large number of proteins were analyzed in different GLF samples and proteins with (mainly) at least 3 unique significant peptides detected at a threshold of p<0.05 (Mascot score approximately 41) were selected. Only three of the proteins listed (5031863, 6466801 and 115430223 in gi) had fewer than three significant peptides, but these were approximately 400 proteins, well above the 95% confidence level for protein identification. had a Mascot score of Proteins identified in this combined analysis are listed in Table 10, along with the reported origin of the specific proteins and reported associated cancers. Table 10 also lists the SEQ ID NOs of identified peptides that had a Mascot score of 40 or greater for each unique identified protein. A number of identified proteins have been reported to be present in pancreatic juice. The references listed in Table 10 are provided in this application.

The foregoing analyzes demonstrate that numerous proteins can be detected in samples obtained using GLF.

(Example 8)
Proteomic analysis of fecal samples This analysis evaluated the ability of fecal samples to support proteomic analysis. To identify target molecules in fecal samples, human subjects were not administered the cleansing composition. Fecal samples were collected from subjects during normal bowel movements using a collection container placed in the toilet. A small fecal (stool) sample was homogenized in 0.1% TFA and then centrifuged at 13000×g. Proteins were precipitated with 6 volumes of acetone, resuspended in 0.1% TFA, extracted with an equal volume of chloroform, and then processed in an SPE column as described in Example 1. did. The most abundant identified proteins and their corresponding Mascot scores and number of significant peptides are summarized in Table 11. Proteins of presumably pancreatic origin were mostly detected in the samples, indicating that stool is the source for detection of these biomarker proteins after their discovery in GLF. However, the samples also contained several other non-human substances that made the analysis much more limited, especially for biomarker discovery.

The foregoing analyzes demonstrate that a large number of target molecules can be detected in fecal samples.

(Example 9)
Detection of Glycans in Samples Obtained Using GLF This analysis assessed the ability of glycans to be detected in samples obtained using GLF. To identify and analyze target molecules, including glycans, in GLF, GLF was collected from human subjects. 1.8 mL of GLF was added to 12 mL of ice-cold acetone and incubated for 1 hour to pellet the protein. Samples were centrifuged at 12,000 xg for 15 minutes to remove acetone. After washing with ice-cold acetone, the pellet was resuspended in 0.1% TFA and passed through a 5 mL syringe-style SepPak C2 column. Proteins were eluted with 60% acetonitrile/40% 0.1% TFA. After removing the solvent under vacuum, the protein fraction was redissolved in 100 μL of 50 mM ammonium bicarbonate and deglycosylated with 2 μL of PNGase F at 37° C. overnight in a shaker. After quenching with 1 mL of 0.1% TFA, glycans were collected as a flow-through fraction from a 1 mL syringe-style SepPak C18 column using a vacuum manifold. Dried glycans were treated with 4-ABEE (ethyl 4-aminobenzoate) and 25 μL of derivatization solution (90:10 MeOH:HAc containing 35 mM ABEE and 100 mM 2-PB) was added to the dried glycans and 65 Labeled by reductive amination by incubating at °C for 2 hours. Excess ABEE was removed by adding 1 mL of ethyl ether, vortexing and discarding the ether. After the second ether extraction, the sample was briefly placed in a SpeedVac to remove any remaining ether. The labeled glycans were then run on HPLC and eluted with between 20-25% acetonitrile from an Agilent C8 reverse phase column. This fraction was vacuum dried, redissolved in 50 μL of 0.1% TFA and run on a Waters Q-TOF LC-ESI-MS for glycan analysis. Derivatized glycans were eluted at approximately 20-25% acetonitrile in 0.2% formic acid from a C18 reverse phase column on a Q-TOF MS. The mass spectrometer was scanned at m/z 150-2000 per second in MS-only mode to acquire derivatized glycan profile data. Figure 1 summarizes these results and presents a graph of the relative abundance of glycan structures derived from various glycoproteins present in fractions of gastrointestinal lavage fluid. As shown in FIG. 1, glycan structures from some glycoproteins contain specific modifications associated with chain truncation. These modifications may be due to bacterial activity present in the GLF sample, as bacteria are known to be able to digest and consume glycans from proteins. However, such modifications may also be associated with disease, particularly cancer where aberrant glycosylation is associated with disease.

The foregoing analysis demonstrates that a large number of glycans can be detected in samples obtained using GLF.

(Example 10)
Detection of Metabolites in Samples Obtained Using GLF This analysis assessed the ability of metabolites to be detected in samples obtained using GLF. GLF obtained for metabolites such as cholic acid and other bile salts by administering magnesium citrate-based cleansing compositions to human subjects to identify and analyze target molecules, including metabolites, in GLF. was analyzed. The resulting GLF was collected from subjects as part of a colonoscopy procedure.

3 ml of GLF was centrifuged at maximum speed for 20 minutes and the supernatant was acidified with 0.1% TFA. The supernatant was applied to a C18 SPE column (Waters Sep-Pak), washed with 3 volumes of 0.1% TFA and eluted with 50% ACN in 0.1% TFA. The eluate was dried by centrifugal lyophilization and redissolved in 500 μl 0.1% TFA.

Data were acquired on a Waters Q-TOF mass spectrometer using input from an LC system. The A solvent contained 3% B and 0.2% formic acid in water. B solvent contained 0.2% formic acid in 3% A and acetonitrile. Solvents were HPLC grade from Fisher. The starting solvent was 5% B, held for 5 minutes, then changed to 40% by 25 minutes, 90% by 30 minutes, then reset to 5% at 36 minutes. The MS scanned over the mass range from 100 m/z to 2000 m/z every second. Data were acquired using standard MassLynx software. The eluted compounds that marked the cholic acid peak are summarized in FIG. A similar peak profile was observed on the Orbitrap instrument where the cholic acid peak was identified using standard and MS/MS data. Metabolites were identified, including cholic acid.

The foregoing analyzes demonstrate that metabolites can be detected in samples obtained using GLF.

The term "comprising," as used herein, is synonymous with "including," "containing," or "characterized by." , are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

All numbers expressing quantities of ingredients, reaction conditions, etc. used herein are to be understood as being modified in all instances by the term "about." Accordingly, unless indicated to the contrary, the numerical parameters set forth herein are approximations that may vary depending on the desired properties sought to be obtained. At a minimum, without intending to limit the application of the doctrine of equivalence of any claim in any application claiming priority to this application, each numerical It must be interpreted in consideration of the rounding approach.

The above description discloses several methods and materials of the present invention. The present invention is susceptible to modifications in methods and materials and changes in manufacturing methods and equipment. Such modifications will become apparent to those skilled in the art from a consideration of this disclosure or practice of the invention disclosed herein. As a result, it is not intended that the invention be limited to the particular embodiments disclosed herein, but is intended to cover all modifications and variations that fall within the true scope and spirit of the invention. .

REFERENCES Each of the following references is hereby incorporated by reference in its entirety.

All references cited herein, including but not limited to published and unpublished applications, patents and literature references, are hereby incorporated by reference in their entireties, and hereby Forms part of this specification. To the extent any publications and patents or patent applications incorporated by reference conflict with the disclosure contained herein, the specification will supersede and/or supersede any such conflicting material. shall be

Claims (27)

A method wherein the presence of a target molecule in gastrointestinal lavage is indicative of pancreatic cancer or a predisposition to pancreatic cancer in a subject, comprising:
detecting the target molecule in a gastrointestinal wash obtained from the subject by orally administering a wash composition to the subject to effect cleansing of the subject's gastrointestinal system, wherein the target molecule is , associated with pancreatic cancer or a predisposition to pancreatic cancer, and is selected from the group consisting of a polypeptide, an antibody , and a glycan. 2. The method of claim 1, wherein the subject's gastrointestinal system is lavaged. 2. The method of claim 1, wherein said target molecule comprises a polypeptide. 4. The method of claim 3, wherein said polypeptide is IgG, IgA or IgM. The cleaning composition is
(i) contains a component selected from the group consisting of polyethylene glycol, magnesium sulfate, sodium sulfate, potassium sulfate, magnesium citrate, ascorbic acid, sodium picosulfate and bisacodyl;
(ii) a solution comprising sulfuric acid, bicarbonate, chloride, sodium, potassium or magnesium salts of phosphate or citric acid, or combinations thereof; or (iii) a composition comprising polyethylene glycol with an electrolyte solution. , a composition comprising polyethylene glycol with an electrolyte solution and bisacodyl, and a composition comprising sodium sulfate, magnesium sulfate and potassium sulfate;
The method of claim 1. 2. The method of claim 1, wherein the cleaning composition comprises at least one of an osmotic agent or a laxative. The cleaning composition contains aloe, bisacodyl, cassanthranol, cascara aromatic fluid extract, cascara sagrada bark, cascada sagrada extract, cascara sagrada fluid extract, castor oil, danthrone, dehydrocholic acid, 7. The method of claim 6, comprising a laxative selected from the group consisting of phenolphthalein, sennoside A, sennoside B, picosulfate and combinations thereof. 2. The method of claim 1, further comprising exposing the gastrointestinal lavage fluid to a material for stabilizing or preserving the target molecule. 2. The method of claim 1, further comprising adjusting the pH of said gastrointestinal lavage fluid to stabilize or preserve said target molecule. 9. The method of claim 8, wherein the material comprises at least one of enzyme inhibitors, protease inhibitors or denaturants. 2. The method of claim 1, wherein said gastrointestinal lavage fluid is obtained by non-invasive self-collection by said subject. 2. The method of claim 1, wherein the gastrointestinal lavage fluid is obtained as part of a colonoscopy procedure. 2. The method of claim 1, wherein said subject is a mammal. 2. The method of claim 1, wherein said subject is human. 2. The method of claim 1, comprising detecting at least 5, 10, 20, 30, 50 or 100 target molecules in said gastrointestinal lavage fluid. 2. The method of claim 1, wherein said target molecule is derived from the pancreas. A kit for detecting the presence of a target molecule in gastrointestinal lavage as an indicator of pancreatic cancer or a predisposition to pancreatic cancer in a subject, comprising:
a cleansing composition suitable for oral administration to said subject to effect cleansing of the subject's gastrointestinal system;
a container for collecting a sample comprising said gastrointestinal lavage fluid from said subject;
and an agent for detecting said target molecule in said gastrointestinal lavage fluid, said target molecule associated with pancreatic cancer or a predisposition to pancreatic cancer and selected from the group consisting of polypeptides, antibodies and glycans. kit. 18. The kit of Claim 17, wherein said target molecule comprises a polypeptide. 19. The kit of claim 18, wherein said polypeptide is IgG, IgA or IgM. 18. The kit of claim 17, further comprising materials for stabilizing or preserving said target molecule. 21. The kit of claim 20, further comprising materials for adjusting the pH of said sample to stabilize or preserve said target molecule. 21. The kit of Claim 20, wherein said material comprises at least one of an enzyme inhibitor, a protease inhibitor or a denaturant. The cleaning composition is
(i) contains a component selected from the group consisting of polyethylene glycol, magnesium sulfate, sodium sulfate, potassium sulfate, magnesium citrate, ascorbic acid, sodium picosulfate and bisacodyl;
(ii) a solution comprising sulfuric acid, bicarbonate, chloride, sodium, potassium or magnesium salts of phosphate or citric acid, or combinations thereof; or (iii) a composition comprising polyethylene glycol with an electrolyte solution. , a composition comprising polyethylene glycol with an electrolyte solution and bisacodyl, and a composition comprising sodium sulfate, magnesium sulfate and potassium sulfate;
18. A kit according to claim 17. 18. The kit of Claim 17, wherein the cleaning composition comprises at least one of an osmotic agent or a laxative. The cleaning composition contains aloe, bisacodyl, cassanthranol, cascara aromatic fluid extract, cascara sagrada bark, cascada sagrada extract, cascara sagrada fluid extract, castor oil, danthrone, dehydrocholic acid, 25. The kit of claim 24, comprising a laxative selected from the group consisting of phenolphthalein, sennoside A, sennoside B, picosulfate and combinations thereof. 18. The kit of claim 17, comprising additional agents for detecting at least 5, 10, 20, 30, 50 or 100 target molecules in said gastrointestinal lavage fluid. 18. The kit of claim 17, wherein said targeting molecule is derived from the pancreas.

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