JP2021185378A - Non-invasive methods of detecting target molecules - Google Patents

Abstract

To provide non-invasive methods of detecting target molecules.SOLUTION: 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 lavage fluid or feces. In one aspect, a method of assessing a physiological state of a subject is provided, comprising; obtaining a gastrointestinal lavage fluid from the subject; and detecting a target molecule originating from outside the gastrointestinal system in the gastrointestinal lavage fluid. In another aspect, a method of assessing a physiological state of a subject is provided comprising; obtaining a fecal sample from the subject; and detecting a target molecule originating from outside the gastrointestinal system in the fecal sample.SELECTED DRAWING: None

Description

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

References to Sequence Listings This application has been filed in electronic form with sequence listings. The sequence listing is approximately 231 KB in size, USA_007WO. It is provided as a file entitled TXT. The information in electronic form of the sequence listing is incorporated herein by reference in its entirety.

Disorders associated with the gastrointestinal (GI) and hepatobiliary tracts and organs / tissues associated with the GI canal include cancers such as gastric cancer, esophageal cancer, liver cancer and pancreatic cancer. Pancreatic cancer (eg, pancreatic adenocarcinoma) is a malignant growth of the pancreas that occurs primarily in the cells of the pancreatic duct. The disease, the ninth most common form of cancer, is the fourth and fifth cause of cancer death in men and females, respectively. Pancreatic cancer is almost always lethal and has 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 in fact non-specific. Therefore, diagnosing pancreatic cancer in the early stages of tumor growth is often difficult, requires large-scale precision diagnosis, and is often found accidentally during trial laparotomy. Endoscopic ultrasonography is an exemplary non-surgical technique that can be used in the diagnosis of pancreatic cancer. However, reliable detection of pancreatic cancer differentiation from small tumors as well as localized pancreatitis is difficult. The majority of patients with pancreatic cancer are now diagnosed late in life when the tumor has already spread beyond the pancreas and invaded surrounding organs and / or has metastasized to a large scale. Non-Patent Document 1 whose entire text is incorporated herein by reference. Late detection of the disease is common in most patients diagnosed with advanced disease, which often leads to death, and only a few patients are detected in the early stage.

Invasive techniques for diagnosing disorders and diseases associated with GI tubes are inconvenient and put the subject at significant risk. Therefore, there is a need for non-invasive methods and compositions for detecting and identifying target molecules from GI tubes or related organs / tissues. In some embodiments, it is determined whether it is useful as a biomarker associated with a particular feature such as a disease, predisposition to disease, no positive response to a treatment plan or no response to a treatment plan, or a negative response. In order to do so, the target molecule can be evaluated. In addition, biomarkers derived from GI tubes or related organs / tissues can be used to determine if an individual has any of the specific characteristics listed above.

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

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

Some embodiments are methods for assessing the physiological status of a subject, obtaining a gastrointestinal lavage fluid from the subject and detecting target molecules derived from outside the gastrointestinal system in the gastrointestinal lavage fluid. Includes methods including and.

Some embodiments are methods for assessing the physiological status of a subject, obtaining a stool sample from the subject and detecting a target molecule derived from the outside of the gastrointestinal system in the stool sample. Includes methods including and.

In some embodiments, the gastrointestinal lavage fluid is obtained from the subject by partially purifying the subject's gastrointestinal system.

In some embodiments, the gastrointestinal lavage fluid comprises fecal material. In some embodiments, the stool sample comprises a gastrointestinal lavage fluid.

In some embodiments, the target molecule comprises a polypeptide, antibody, bile acid, metabolite or glycan. In some embodiments, the target molecule comprises a biomarker. In some embodiments, the biomarker is associated with a disease, a positive response to treatment, a partial response to treatment, a negative response to treatment or no response to treatment. In some embodiments, the target molecule is associated with cancer or the presence of a cancer predisposition. In some embodiments, the cancer is pancreatic cancer, colorectal cancer, liver cancer or gastric cancer. In some embodiments, the target molecule is derived from the attached gastrointestinal gland. In some embodiments, the accessory digestive glands are salivary glands, pancreas, gallbladder or liver.

Some embodiments include administering a lavage fluid to a subject. In some embodiments, the lavage fluid is orally administered. 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 wash solution is selected from the group consisting of GOLYTELY, HALFLYTELY, NULYTELY, SUPREP, FLEX's PHOPSHO-SODA, magnesium citrate and their general equivalents.

Some embodiments include performing a colonoscopy on a subject.

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

Some embodiments are methods for identifying biomarkers that do not have the test gastrointestinal lavage fluid from multiple test subjects with the desired condition or physiological condition, and neither said condition nor said physiological condition. Obtaining a control gastrointestinal lavage fluid from multiple control subjects, determining the levels of at least 5 target molecules in the test gastrointestinal lavage fluid and the control gastrointestinal lavage fluid, and in the test gastrointestinal lavage fluid for levels in the control gastrointestinal lavage fluid. Includes methods including identifying target molecules that are present at significantly different levels in, thereby identifying biomarkers.

In some embodiments, the gastrointestinal lavage fluid comprises 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 include determining the level of at least 10 target molecules in a test gastrointestinal lavage fluid and a control gastrointestinal lavage fluid. Some embodiments include determining the level of at least 20 target molecules in a test gastrointestinal lavage fluid and a control gastrointestinal lavage fluid. Some embodiments include determining the level of at least 30 target molecules in a test gastrointestinal lavage fluid and a control gastrointestinal lavage fluid. Some embodiments include determining the level of at least 50 target molecules in a test gastrointestinal lavage fluid and a control gastrointestinal lavage fluid. Some embodiments include determining the level of at least 100 target molecules in a test gastrointestinal lavage fluid and a control gastrointestinal lavage fluid.

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

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

In some embodiments, the at least one target molecule is derived from the attached gastrointestinal gland. In some embodiments, the accessory digestive glands are salivary glands, pancreas, gallbladder or liver.

Some embodiments include administering a lavage fluid to a test subject and a control subject. In some embodiments, the lavage fluid is orally administered. 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 wash solution is selected from the group consisting of GOLYTELY, HALFLYTELY, NULYTELY, SUPREP and FLEX's PHOPSHO-SODA, magnesium citrate and their general equivalents.

Some embodiments include performing colonoscopy on test subjects and control subjects.

In some embodiments, the test subject and the control subject are mammals. In some embodiments, the test subject and the control subject are humans.

Some embodiments are methods for identifying biomarkers, in which a test stool sample is obtained from a plurality of test subjects having a desired condition and a control stool sample is obtained from a plurality of control subjects, and the test stool is obtained. Determining the levels of at least 5 target molecules in the sample and control stool sample and identifying target molecules present at significantly different levels in the test stool sample relative to the levels in the control stool sample. Includes methods including identifying biomarkers.

In some embodiments, the stool sample comprises a gastrointestinal lavage fluid.

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

Some embodiments include determining the level of at least 10 target molecules in a test stool sample and a control stool sample. Some embodiments include determining the level of at least 20 target molecules in a stool sample and a control stool sample. Some embodiments include determining the level of at least 30 target molecules in a stool sample and a control stool sample. Some embodiments include determining the level of at least 50 target molecules in a stool sample and a control stool sample. Some embodiments include determining the level of at least 100 target molecules in a stool sample and a control stool sample.

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

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

In some embodiments, the at least one target molecule is derived from the attached gastrointestinal gland. In some embodiments, the accessory digestive glands are salivary glands, pancreas, gallbladder or liver.

In some embodiments, the test subject and the control subject are mammals. In some embodiments, the test subject and the control subject are humans.

Some embodiments are kits for detecting a target molecule in a gastrointestinal lavage fluid, a lavage fluid for oral administration to a subject, a container for collecting the gastrointestinal lavage fluid from the subject, and a gastrointestinal system. Includes a kit containing a drug for detecting externally derived target molecules.

Some embodiments are kits for detecting a target molecule in a stool sample, a wash solution for oral administration to the subject, a container for collecting the stool sample from the subject, and a gastrointestinal system. Includes a kit containing a drug for detecting externally derived target molecules.

Some embodiments include protease inhibitors.

In some embodiments, the target molecule comprises a polypeptide, antibody, bile acid, metabolite or glycan. In some embodiments, the target molecule comprises a biomarker. In some embodiments, the biomarker is associated with a disease, a positive response to treatment or a negative response to treatment.

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

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

FIG. 1 is a diagram showing a graph of the relative abundance of glycan structures derived from various glycoproteins present in the fraction of gastrointestinal lavage fluid. Derivatized glycans were eluted from the C18 reverse phase column on Q-TOF MS with about 20-25% acetonitrile in 0.2% formic acid. Mass spectrometers scanned at m / z 150-2000 per second in MS-only format to obtain derivatized glycan profile data. FIG. 2 is a diagram showing a graph of the relative abundance of compounds including metabolites such as cholic acid present in the fraction of gastrointestinal lavage fluid. Data were acquired on a Waters Q-TOF mass spectrometer using inputs from the LC system and using MassLynx software. MS scanned over a mass range of m / z 100 to m / z 2000 per second.

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

Gastrointestinal lavage is widely used as a lower gastrointestinal (GI) tube preparation for colorectal endoscopy or colon and rectal surgery (eg, the full text of which is incorporated herein by reference, DiPalma JA. et al., (Eg. 1984) Gastroendogy Vol. 86: pp. 856-60). The specific pathophysiology of intestinal disease has been investigated by measuring proteins in GLF (each of which is incorporated herein by reference in its entirety, Evgenikos N et al. (2000) Br J Surg, Vol. 87. : 808-13, Brydon WG, Survey A. (1992) Ranchet 340, 1381-2, Chocolate CP et al. (1993) Gastroenterology 104: 1064-71, Ferguson A et al. (1996) Gut 38. : 120-4, Handy LM et al. (1996) Scand J Gastroenterol Vol. 31: 700-5, and Standley AJ et al. (1996) Gastromentorology 111: 1679-82).

Measurement of fecal protein can be useful for investigating various pathophysiology such as protein-losing enteropathy and mucosal inflammation. However, in some embodiments described herein, feces may be used, because GLF contains less of a substance that interferes with the assay, and in GLF, it passes through the GI tube rapidly. GLF is preferred over feces as a sample for detecting and identifying biomarkers, as it destroys less protein by digestive enzymes and bacterial proteases than fecal 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 can be produced by orally administering a lavage fluid to a subject to allow a large volume of fluid to pass through the intestinal tract, the lavage fluid being a mixture of salts and other such as polyethylene glycol and bisacodyl. May contain substances. The lavage fluid causes the influx of fluid into the colon, which causes the solid to be flushed out. The lavage fluid is commonly used to cause cleansing of the GI tube, as is commonly used in preparation for colonoscopy and other methods used to examine the GI tube. To. These fluids, which are largely flushed from or remain in a clean GI tube, are useful for assessing various diseases due to mouth-to-anal continuity along the GI tube. be. As a result, all organs, including GI tubes that put fluids into GI tubes, are candidates for the methods and compositions provided herein.

GI Tubes and Related Organs / Tissues Some of the methods and compositions provided herein relate to GI tubes and related organs / tissues, including the GI tube and attached gastrointestinal glands. As is well known in the art, GI tubes include upper GI tubes and lower GI tubes. The upper GI tube includes the oral cavity or buccal oral cavity, esophagus, stomach and duodenum. The lower GI tube includes the jejunum, ileum and large intestine and anus. The large intestine includes the appendix, cecum, colon and rectum.

Organs and tissues associated with the GI canal include the outer structure of the GI canal. Examples of such structures include salivary glands such as the submandibular glands, submandibular and submandibular salivary glands, and pancreas, such as the exocrine pancreas, gallbladder, bile ducts and accessory digestive organs such as the liver. Further examples of the GI duct and the structures associated with the outside of the GI duct include the pancreatic duct, the biliary tree and the bile duct.

Gastrointestinal lavage fluid (GLF)
Generally, the lavage fluid can be orally administered to the subject, the oral lavage fluid passes through the subject's GI tube, and the resulting GLF is collected from the subject. As used herein, the term "subject" may include mammals, such as animals such as humans. As mentioned above, GLF provides a cleaner GI tube sampling than a stool / stool sample test. GLF appears to reduce variability associated with food intake, type and digestive status.

Some embodiments described herein are for screening, triage, disease detection, diagnosis, prognosis, treatment for diseases and conditions of the gastrointestinal tract or related organs / tissues for detecting target molecules. Includes response, treatment selection and analysis of GLF for personalized medicine. Some embodiments include analysis of a GLF sample to rule out a particular disease and condition from possible diseases or conditions that the 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 promising treatment plans, and / or assessing response to previous or ongoing treatment plans. Includes GLF analysis.

Methods for Obtaining GLF Some embodiments of the methods and compositions provided herein include obtaining GLF from a subject. Methods of obtaining GLF are well known in the art. During medical and / or diagnostic procedures such as sigmoid colonoscopy, colonoscopy, X-ray examination, preparation for patients undergoing bowel surgery, the bowel and colon are completely cleaned. It is important to be clean. In particular, it is essential that as much fecal material as possible be removed from the colon to allow proper visualization of the intestinal mucosa. This is important prior to diagnostic procedures such as, for example, flexible sigmoid colonoscopy or colonoscopy, and widely performed diagnostic tests to screen patients for colonic disease. In addition, it is important that the intestinal tract is completely lavaged in order to obtain a satisfactory radiograph of the colon. The same condition also applies when the colon is preoperatively prepared for surgery and removal of fecal waste is very important for patient safety. To prepare the colon for endoscopy, current cleansing procedures include erect bipedal colon lavage. Upright gait washing may include orally administering to the subject a washing composition comprising 4 L of polyethylene glycol / electrolyte solution (US Patent Application Publication No. 20070298008, the full text of which is incorporated by reference). Some embodiments include antegrade and prograde washes.

Generally, the oral cleaning composition comprises a solution of electrolytes such as sulfuric acid, hydrogen carbonate, chloride, phosphoric acid or citric acid sodium, potassium and magnesium salts. Some such compositions may also contain polyethylene glycol, which can act as a non-absorbable osmotic agent. Typical compositions include polyethylene glycol with an electrolyte solution and, optionally, compositions containing bisacodyl or ascorbic acid and sulfates such as sodium sulphate, magnesium sulphate or potassium sulphate. In some embodiments, the oral lavage fluid may contain magnesium citrate. In some embodiments, the oral lavage fluid may comprise picosulfate sodium. GOLYTELY (Braintree Labs. Inc.) is an exemplary composition of an oral cleaning solution containing polyethylene glycol with an electrolyte solution. GOLYTELY is formulated according to the following: 59 g of polyethylene glycol, 5.68 g of sodium sulfate, 1.69 g of sodium hydrogen carbonate, 1.46 g of sodium chloride, 0.745 g of potassium chloride and 1 liter of water to make up (see by reference). The full text is incorporated, Davis et al. (1980) Gastronolology Vol. 78: pp. 991-995). Ingestion of GOLYTELY results in a large amount of liquid stool with minimal changes in the subject's water and electrolyte balance. Another example of an oral cleaning composition comprising polyethylene glycol with an electrolyte solution is NULYTELY (Braintree Labs. Inc.). HALFLYTELY is an exemplary oral cleaning composition comprising polyethylene glycol with an electrolyte solution and bisacodyl (Braintree Labs. Inc.). SUPREP is an exemplary oral cleaning composition comprising sulfates such as sodium sulphate, magnesium sulphate or potassium sulphate (Braintree Labs. Inc.). MOVIPLEP is an exemplary composition of an oral wash solution containing polyethylene glycol with an electrolyte solution and ascorbic acid (Salix Pharmaceuticals, Inc.).

Polyethylene glycol is effective as an oral cleaning composition when a large amount of polyethylene glycol is administered in a large volume of diluted salt solution. Usually, about 250-400 g of polyethylene glycol is administered to the subject in about 4 L aqueous electrolyte solution. Oral administration of polyethylene glycol can be used to cause bowel movements for a period of time, eg, overnight. Depending on the required dose, about 10-100 g of polyethylene glycol in 8 ounces of water may be effective. A dose of about 68-85 g of polyethylene glycol may be effective in producing overnight bowel movements without severe diarrhea. The volume of the solution of polyethylene glycol in the isotonic fluid can be an effective amount of osmotic laxative. Capacities of about 0.5 L to about 4 L can be effective. Preferably, the effective capacity is between about 1.5 L and about 2.5 L. Oral administration of 2 L of isotonic solution is effective.

A further example of an oral cleaning composition is a non-phosphate hypertonic solution in combination with an osmotic laxative such as polyethylene glycol (US Patent Application Publication No. 20090258090, the full text of which is incorporated by reference). A mixture of sulfates excluding phosphates, such as one or more of the following sulfates Na ₂ SO ₄ , marriage ₄ and K ₂ SO ₄ , may be effective (eg, SUPREP). Some embodiments include _{over Na} 2 SO ₄ of from about 0.1g~ about 20.0 g, _Na 2 SO ₄ to about 1.0g~10.0g may be useful. The dose of MgSO ₄ about 0.01g~ about 40.0g may be effective. A dose of about 0.1 g to about 20.0 g of Na ₂ SO ₄ as well as a dose of 1.0 to 10.0 g can also be used advantageously. _{Dose of K 2} SO _{4 from} about 0.01 g to about 20.0 g can be effective in resulting in purification, from about 0.1 g to about 10.0 g and from about 0.5 g to about 5.0 g of K. ₂ SO ₄ doses may also be useful. Addition of an osmotic laxative such as polyethylene glycol (PEG) may improve the effectiveness of the salt mixture described above. A dose of about 1.0 g to about 100 g of PEG is effective. A dose of about 10.0 g to about 50 g of PEG is also effective, such as a dose of about 34 g. For ease of administration, the above salt mixture can be dissolved in a convenient volume of water. Volumes of less than 1 liter of water can be well tolerated by most subjects. The mixture may be dissolved in any small volume of water, a volume between 100 and 500 ml is useful. The effective dose may be divided and administered to the patient in two or more doses over an appropriate period. In general, administration of two doses of the equivalent portion of the effective dose, spaced 6 to 24 hours apart, results in satisfactory cleansing. Some embodiments include interruption of normal oral intake prior to administration of the oral wash composition and for a defined period in between.

Some cleaning compositions include laxatives such as bisacodyl. In some embodiments, the laxative can be co-administered to the subject with the wash composition. As will be appreciated, such co-administration was combined with, for example, administration of a laxative up to several hours prior to administration of the wash composition to the subject, administration of the wash composition to the subject. It may include administration of laxatives or administration of laxatives up to several hours after administration of the wash composition to the subject. Examples of laxatives and their effective doses are aloe, 250-1000 mg; bisacodyl, about 5-80 mg; cassanthanol, 30-360 mg; cascara aromatic fluid extract, 2-24 ml; cascara buckthorn bark, 300- 4000 mg; Cascara Sagrada Extract, 300-2000 mg; Cascara Sagrada Fluid Extract, 0.5-5.0 ml; Castor Oil, 15-240 ml; Danslon, 75-300 mg; Dehydrocholic Acid, 250-2000 mg; Phenolphthalein, 30-1000 mg; sennosides A and B, 12-200 mg; and picosulfate, 1-100 mg.

A further example of the cleaning composition is an aqueous solution of concentrated phosphate. Aqueous phosphate concentrates have an osmotic effect on the luminal contents of GI vessels, resulting in intestinal excretion with a large influx of water and electrolytes from the body into the colon. One exemplary composition comprises 480 g / L sodium dihydrogen phosphate and 180 g / L disodium hydrogen phosphate in a stabilized aqueous buffer (PHOSPHO-SODA, CS Fleet of FLEX). Co., Inc.). Subjects typically require a total of 6 ounces (180 ml) to ingest a dose of 2-3 ounces of this composition, separated by 3-12 hour intervals.

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 a toilet insert that captures the fluid. Enzyme inhibitors and denaturants can be used to maintain 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 can be further processed to remove some or all solids and / or bacteria, such as by centrifugation. In some embodiments, the GI tube may not be completely purified by administration of the oral wash composition. For example, a portion of the complete dose of oral cleaning composition required to completely clean a subject's GI tubing may be administered to the subject. In some embodiments, the GLF may contain fecal material. In a further embodiment, the fecal material may include GLF.

Targeted Molecules Some embodiments described herein relate to methods of detecting target molecules in a sample obtained from a GI tube or compositions useful for such detection. As used herein, a "target molecule" includes any molecule that can be detected, measured or identified in a sample obtained from a GI tube. Examples of such a sample include GLF obtained from the subject and a fecal sample obtained from the subject. Examples of target molecules are peptides, polypeptides, proteins, mutant proteins, proteins resulting from selective splicing, post-translationally modified proteins such as glycosylated proteins, phosphorylated proteins and other modified proteins, antibodies. (Eg 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. Examples include molecules such as substances and 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. The target molecule can be derived from the GI tube and the outside of the GI tube, eg, an organ and / or tissue associated with the GI tube, such as an attached gastrointestinal gland. In some embodiments, cell fragments and other by-products (biproducts), such as cells, organisms, including red blood cells, leukocytes and endothelial cells, such as bacteria, protozoans and viruses and viral particles, are GLF or feces. Can be detected in the sample. In some embodiments, the target molecule can be any of the proteins or parts thereof listed in any of Tables 1-10 or parts thereof herein. In some embodiments, some of the proteins listed in any of Tables 1-10 are at least 10, at least 15, or 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 can, from, or can consist essentially of, one of the polypeptides of SEQ ID NOs: 01-804. The polypeptide consisting essentially of one of SEQ ID NOs: 01-804 is an additional amino acid or a substituted amino acid relative to that in SEQ ID NOs: 01-804, such an additional amino acid or a substituted amino acid is detected by the polypeptide. It may be included if it does not prevent it from being possible.

Target molecules also include biomarkers. As used herein, the term "biomarker" is associated with a disease, predisposition to disease, a positive response to a particular treatment plan, a lack of response to a particular treatment plan, or a negative response to a particular treatment plan. Includes any target molecule present in GLF or fecal samples. In some embodiments, biomarkers can be correlated with identification, measurement and / or diagnosis or prognosis of the disease.

In some embodiments, the target molecule is a component of the subject's fluid selected from the group consisting of blood, saliva, gastric fluid, liver secretions, bile, duodenal fluid and pancreatic juice. In some embodiments, the target molecule is expressed in the subject's upper gastrointestinal tract or in the subject's lower gastrointestinal tract. In some embodiments, the target molecule is in a subject selected from the group consisting of buccal oral cavity, esophagus, stomach, gallbladder, gallbladder, duodenum, jejunum, ileum, appendix, cecum, colon, rectum and anal canal. It 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). , Aspartic acid aminotransferase, hemoglobin or eosinophil peroxidase. In some embodiments, the target molecule is a protein found in feces, such as heptaglobulin, hemopexin, α-2-macroglobulin, cadherin-17, carprotectin, carcinonembryogenic antigen. , Metalloproteinase-1 (TIMP-1), S100A12, K-ras or p53. In some embodiments, the target molecule is a protein found in pancreatic fluid, such as anterior gradient-2 (AGR2), insulin-like growth factor binding protein-2, CEACAM6, MUC1, CA19-9, serine. Proteinase-2 (PRSS2) preprotease, pancreatic lipase-related protein-1 (PLRP1), chymotrypsinogen B (CTRB), elastase 3B (ELA3B), tumor rejection antigen (pg96), azlocidine, hepatocellular carcinoma-intestinal-pancreatic / pancreatitis It does not contain related protein I (HIP / PAP-I), matrix metalloproteinase-9 (MMP-9), cancer gene DJ1 (DJ-1) or α-1B-sugar protein precursor (A1BG).

Methods of Characterizing Target Molecules Some embodiments of the methods and compositions provided herein include characterizing a target molecule in a GLF or stool sample. Destituting a 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 include identifying a target molecule comprising a peptide, polypeptide and / or protein, determining its presence or absence, and / or quantifying the target molecule. Such target molecules can be characterized by a variety of methods such as radioimmunoassays as described herein, enzyme binding immunoassays and immunoassays including two antibody sandwich assays. Various immunoassay formats, including competitive and non-competitive immunoassay formats, antigen capture assays and two-antibody sandwich assays, are also useful (Self and Cook, (1996) Curr. Opin. Biotechnol. 7, the full text of which is incorporated by reference). Volume: pp. 60-65). Some embodiments include one or more antigen capture assays. In the antigen capture assay, the antibody is bound to the solid phase and the sample is added such that the antigen, eg, the target molecule in the GLF or fecal sample, is bound by the antibody. After the unbound protein is removed by washing, the amount of bound antigen can be quantified, if desired, for example, using a radioassay (the full text of which is incorporated by reference, Harlow). 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 level of the target molecule in the GLF or fecal sample.

Enzyme-linked immunosorbent assay (ELISA) can 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, with an anti-HMGB1 antibody or with a secondary antibody for use in the methods of the invention. The horseradish peroxidase detection system can be used, for example, with the color-developing substrate tetramethylbenzidine (TMB), which produces a soluble product detectable at 450 nm in the presence of hydrogen peroxide. Other convenient enzyme-bound systems include, for example, alkaline phosphatase detection systems that can be used with the color-developing substrate p-nitrophenyl phosphate to produce soluble products that are readily detectable at 405 nm. Similarly, a β-galactosidase detection system can be used with the color-developing substrate o-nitrophenyl-β-D-galactopyranoside (ONPG) to produce a soluble product detectable at 410 nm, or a urease detection system is urea. -Can be used with substrates such as bromocresol purple (Sigma Immunochemicals). Useful enzyme-bound primary and secondary antibodies can be obtained from several commercially available sources such as Jackson Immuno-Research (West Grove, Pa.) As further described herein.

In certain embodiments, the target molecule in the GLF or fecal sample can be detected and / or measured using chemiluminescence detection. For example, in certain embodiments, a specific antibody against a particular target molecule is used to capture a target molecule present in a biological sample, such as a GLF or fecal sample, to detect the target molecule present in the sample. In order to do so, an antibody that is specific to the target molecule-specific antibody and is labeled with a chemical emission label is used. Any chemiluminescent label and detection system can be used in this method. Chemiluminescent secondary antibodies are commercially available from various sources such as Amersham. Methods of 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 fluorescent dyes include DAPI, fluorescein, Hoechst 33258, R-phycocyanin, B-phycoerythrin, R-phycoerythrin, rhodamine, Texas red and lysamine. In the present invention, a fluorescein or rhodamine-labeled antibody or a fluorescein-or rhodamine-labeled secondary antibody may be useful.

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

The signal from the detectable reagent is, for example, a spectrophotometer for detecting color from a color-developing substrate; a radiation counter for detecting radiation, such as a γ counter for detecting ^{125 I; or a particular radiation counter.} It can be analyzed using a fluorometer for detecting fluorescence in the presence of light of wavelength. When an enzyme-bound assay is used, quantitative analysis of the amount of target molecule uses a spectrophotometer such as EMAX Microplate Reader (Menlo Park, Calif.) According to the manufacturer's instructions. Can be carried out. The assay of the present invention can be performed automatically or by robot control, if necessary, and signals from a plurality of samples can be detected simultaneously.

In some embodiments, a capillary electrophoresis-based immunoassay (CEIA) may be used to detect and / or measure the target molecule, which can be automated as needed. The immunoassays are also described, for example, in Schmalzing and Nashabeh, Electrophoresis, Vol. 18, pp. 2184-93 (1997) and Bao, J. et al., Where the full text is incorporated by reference, respectively. Chromatogr.
B. Biomed. Sci. Volume 699: Can be used with laser-guided fluorescence as described on pages 463-80 (1997). Liposomal immunoassays such as flow injection liposome immunoassays and liposome immunosensors can also be used to detect the target molecule or to determine the level of the target molecule according to certain methods provided herein ( The full text is incorporated by reference, Rongen et al. (1997) J. Immunol. Methods 204: 105-133).

In certain embodiments, sandwich enzyme immunoassays may also be useful. In the two-antibody sandwich assay, the primary antibody binds to the solid phase 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.

In the methods provided herein, quantitative Western blotting may also be used to detect the target molecule or to determine the level of the target molecule. Western blots can be quantified by well-known methods such as scanning densitometry. As an example, a protein sample is electrophoresed on a 10% SDS-PAGE Laemmli gel. For example, a primary mouse monoclonal antibody against the target molecule is reacted with the blot and preliminary slot blot experiments are used to confirm that the antibody binding is linear. A goat anti-mouse horseradish peroxidase-bound antibody (BioRad) was used as the secondary antibody, and signal detection was performed using chemiluminescence, eg, Renaissance chemiluminescence kit (New England Nuclear; Boston, Mass.), Instructions for use by the manufacturer. Implemented using according to the book. An autoradiograph of the blot is analyzed using a molecular dynamics (Sunnyvale, Calif.) And normalized to a positive control. Values are reported, for example, as a ratio between measured values vs. positive controls (concentration measurement index). Such methods are, for example, incorporated herein by reference in their entirety, Parra et al., J. Mol. Vasc. Surg. Volume 28: As described in pp. 669-675 (1998), it is well known in the art.

As described herein, some embodiments for detecting or determining the level of a target molecule include, for example, enzyme-bound immunoadsorption methods, radioimmunoassays and quantitative western analysis. Immunoassays can be useful. Such assays usually 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 proteins having alternative forms of post-translational modification, such as phosphorylated proteins and glycosylated proteins.

Target molecules, such as protein target molecules, can be characterized by a variety of methods. Proteins, polypeptides and peptides are such as protein precipitation, chromatography (eg, reverse phase chromatography, size exclusion chromatography, ion exchange chromatography, liquid chromatography), affinity capture and fractional extensions. It can be isolated by various methods well known in the art.

Isolated proteins can undergo enzymatic digestion or chemical cleavage to yield polypeptide fragments and peptides. Such fragments can be identified and quantified. A particularly useful method for the analysis of polypeptides / peptide fragments and other target molecules is mass spectrometry (US Patent Application No. 20100279382, the full text of which is incorporated by reference). Several mass spectrometric-based quantitative proteomics methods have been developed that identify the proteins contained in each sample and determine the relative abundance of each of the identified proteins across the sample (each by reference). Full text incorporated, Fly et al., Trends Biotechnology. 20: S23-29 (2002), Aeversold, J. Am. Soc. Mass Spectrom. 14: 685-695 (2003), Aeversold, J. Mol. Infect. Dis. Vol. 187, Appendix 2: S315-320 (2003), Patterson and Aeversold, Nat.
Genet. Volume 33, Appendix, pp. 311-223 (2003), Aeversold and Mann, Nature 422: 198-207 (2003), Aeversold, R. et al. And Cravatt, Trends Biotechnology. Volume 20: S1 and 2 (2002), Aeversold and Goodlett, Chem. Rev. Vol. 101, pp. 269-295 (2001), Tao and Aeversold, Curr. Opin. Biotechnol. Volume 14, pp. 110-118 (2003)). Generally, the protein in each sample is labeled to identify the sample of its origin and acquire isotopic discrimination properties that provide the basis for accurate mass spectrometric quantification. Samples with different isotopic discrimination characteristics are then combined and usually analyzed by multidimensional chromatographic tandem mass spectrometry. The resulting collision-induced dissociation (CID) spectrum is then assigned to the peptide sequence and is relative to each of the detected proteins in each sample based on the relative signal intensity of the different isotope-labeled peptides of the same sequence. The target abundance is calculated.

Further techniques for identifying and quantifying target molecules, unlabeled 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, quantitative and statistical analysis. Each sample can be prepared separately and then subjected to a separate LC-MS / MS or LC / LC-MS / MS practice (the full text of which is incorporated by reference, Zhu W. et al., J. of Biomedicine and). Biotech. (2010) Article ID 840518, p. 6). One exemplary technique is the LC-MS, Precision Mass and Time (AMT) tag approach, in which the mass of a peptide is combined with its corresponding chromatograph elution time as a peptide property that uniquely defines the peptide sequence. There is a method called. Using LC (LC-FTICR) MS with Fourier Transform Ion Cyclotron Resonance to obtain chromatograph and high mass accuracy information, AMT tags were stored in a database so far acquired LC-MS /. The peptide sequence can be identified by associating it with the MS sequence information. By utilizing the linear correlation observed between the measured peak area of the peptide and its abundance, these peptides are relatively quantified by the signal intensity ratio of their corresponding peaks compared between MS implementations. (The full text of which is incorporated by reference, Tang, K. et al. (2004) J. Am. Soc. Mass Spectrum. Vol. 15, pp. 1416-1423, and Chelius, D. and Bondarenko, P. V. (2002) J. Protein Res. Volume 1: pp. 317-323). Data obtained from multiple LC-MS runs on each sample can be analyzed using statistical tools such as Student's t-test (the full text of which is incorporated by reference, Wiener, MC et al., 2004). ) Anal. Chem. 76: 6085-6096). Two amplitudes of signal intensity obtained from multiple LC-MS runs at acquisition time and m / z points to detect peptides with statistically significant differences in abundance between samples. Can be compared between samples of.

As will be appreciated, various mass spectrometric systems can be used in methods for identifying and / or quantifying polypeptides / peptide fragments. Ion traps, triple quadrupole and time-of-flight, quadrupole time-of-flight mass spectrometers and Fourier transform ion cyclotron mass spectrometers (FT-ICR-) as mass spectrometers with high mass accuracy, high sensitivity and high resolution. MS). Mass spectrometers typically include a matrix-assisted laser desorption (MALDI) or electrospray ionization (ESI) ion source, but other methods of peptide ionization can also be used. In an ion trap MS, the analyte is ionized by ESI or MALDI and then placed in an ion trap. The trapped ions can then be analyzed separately by MS upon selective release from the ion trap. Fragments can also be made and analyzed in ion traps. Sample molecules such as released polypeptides / peptide fragments can be analyzed, for example, by single-stage mass spectrometry with a MALDI-TOF or ESI-TOF system. Analytical methods of mass spectrometry are well known to those of skill in the art (eg, Yates, J. (1998) Mass Spect. Vol. 33: pp. 1-19, Kinter and Sherman, each of which is incorporated by reference in its entirety. (2000) Protein Analysis and Identification Using Tandem Mass. Spectrometriy, John Willey & Sons, New
York, and Aeversold and Goodlett, (2001) Chem. Rev. Volume 101: see pages 269-295). For high resolution polypeptide fragment separation, liquid chromatography ESI-MS / MS or automated LC-MS / MS utilizing capillary reverse phase chromatography as the separation method can be used (the full text of which is incorporated by reference, Yates). Et al., Methods Mol. Biol. Vol. 112: pp. 553-569 (1999)). Data-dependent collision-induced dissociation (CID) using dynamic exclusion can also be used as a mass spectrometry method (the full text of which is incorporated by reference, Goodlett et al., Anal. Chem. 72: 1112-1118 (2000)). ).

Once the peptides have been analyzed by MS / MS, the resulting CID spectra can be compared against the database for determination of isolated peptide identity. Methods for protein identification using a single peptide have been described so far (Aeversold and Goodlett, Chem. Rev. 101: 269-295, respectively, the full text of which is incorporated by reference, respectively). ), Yates, J. Mass Spec. 33: 1-19 pages (1998), David N. et al., Electrophoresis, 20 volumes 3551-67 pages (1999)). In particular, if the peptide provides the unique distinguishing properties of the parent polypeptide, it may be possible to use one or a few peptide fragments to identify the parent polypeptide from which the fragment was derived. In addition, identification of a single peptide, alone or in combination with knowledge of the site of glycosylation, can be used to identify the parental glycopolypeptide from which the glycopeptide fragment was derived. As will be appreciated, methods involving MS can be used to characterize proteins, fragments thereof and other types of target molecules described herein.

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

Some embodiments include identifying a target molecule containing a glycosylated protein and / or a glycan, determining its presence or absence, and / or quantifying the target molecule. Glycosylated proteins and glycans can be analyzed by a variety of methods well known in the art. Changes in glycosylation can indicate a disease or disease situation. Thus, a particular target molecule may include a particular glycosylated protein and / or glycan. As will be understood, glycans can be constituents of glycoproteins, proteoglycans or other glycan-containing compounds.

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

Methods for Identifying Biomarkers In some embodiments, target molecules detected in GLF or fecal samples are evaluated and they are associated with a particular condition, such as a disease or physiological situation. You can determine if it is a biomarker. Such biomarkers can indicate a particular disease, predisposition to disease, prognosis, positive response to a particular treatment regimen or negative response to a particular treatment regimen. In some embodiments, the presence or absence of a biomarker or its level may be associated with a particular condition, such as a disease or physiological condition. In some embodiments, the presence or absence of a biomarker or its level can be statistically correlated with a particular condition, such as a disease or physiological situation. In some embodiments, the physiological situation may include disease. In some embodiments, the level of biomarker expression in a subject having a condition such as a disease or physiological condition is compared to the level of biomarker expression in a subject having no condition or physiological condition. Allows biomarkers to correlate with specific conditions such as disease or physiological conditions.

In some embodiments, the differential expression of the biomarker in the subject with the condition as compared to the expression of the biomarker in the subject without the condition indicates a condition or physiological condition. As used herein, "differential expression" refers to the difference in the expression level of a biomarker between a subject having a condition such as a disease or a physiological condition and a subject having no condition such as a disease or a physiological condition. Point to. For example, the term "differential expression" can refer to the presence or absence of a biomarker in a subject having a condition such as a disease or physiological condition as compared to a subject having no condition or physiological condition. In some embodiments, differential expression is a subject having a condition such as a disease or physiological condition compared to the level of expression of the biomarker in the subject having no condition such as a disease or physiological condition. It can refer to the difference in the expression level of the biomarker 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 a condition or physiological condition. Here, the ratio is statistically significant. Differential expression between populations can be determined to be statistically significant as a function of p-value. When a p-value is used to determine statistical significance, the biomarker, p-value, is preferably less than 0.2. In another embodiment, the biomarker is differentially expressed when the p-value is less than 0.15, 0.1, 0.05, 0.01, 0.005, 0.0001, etc. Is identified. When determining differential expression based on ratio, the biomarker product has a ratio of the level of expression in the first sample to greater than 1.0 compared to the second sample. When it is less than 0, it is expressed differentially. For example, as a ratio larger than 1.0, for example, a ratio larger than 1.1, 1.2, 1.5, 1.7, 2, 3, 4, 10, 20, etc. can be mentioned. 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 average level of expression in the first population to greater than or less than 1.0 compared to the average level of expression in the second population. It can be expressed differentially 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, and the like, and ratios less than 1.0. For example, ratios less than 0.9, 0.8, 0.6, 0.4, 0.2, 0.1, 0.05 and the like can be mentioned. In another embodiment, the biomarker is differential when the ratio of the level of expression in the first sample to the average of the second population is greater than or less than 1.0. Expression, eg 1.1, 1.2, 1.5, 1.7, 2, 3, 4, 10, 20 greater than or less than 1, eg 0.9, 0.8, 0 The ratios of 0.6, 0.4, 0.2, 0.1 and 0.05 can be mentioned.

In some embodiments, the biomarker is a test GLF or test fecal sample obtained from at least one test subject having a condition or physiological condition and at least one control having no condition or physiological condition. Measuring the level of at least one target molecule in a control GLF or control stool sample obtained from a subject and controlling the level of at least one target molecule in a test GLF or test stool sample, control GLF or control. It can be identified by comparison with the level of at least one target molecule in the stool sample, where a significant difference at the level of at least one target identifies the biomarker. Some embodiments are a test GLF or test fecal sample obtained from a plurality of test subjects having a condition or physiological condition and a control GLF obtained from a plurality of control subjects having no condition or physiological condition. Or it involves measuring and comparing multiple target molecules in a control fecal sample. 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 stool sample is at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, It may be obtained from 95 or 100 test subjects. In some embodiments, the GLF or stool sample is at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, It may be obtained from 95 or 100 control subjects. In some embodiments, the significant difference in the level of the target molecule in the test GLF or test stool sample compared to the control GLF or control stool sample can be statistically significant.

Kits Some embodiments of the methods and compositions provided herein are for detecting target molecules in GLF or stool samples, for determining the presence or absence of target molecules in GLF or stool samples, GLF or It relates to a kit for quantifying a target molecule in a stool sample or for identifying a target molecule in a GLF or stool sample. Some such kits may include a wash composition for oral administration to a subject. In some embodiments, the cleaning solution may contain components such as polyethylene glycol, magnesium sulphate, sodium sulphate, potassium sulphate, magnesium citrate and bisacodyl. In some embodiments, the cleaning solution may optionally contain polyethylene glycol with an electrolyte solution also including bisacodyl or ascorbic acid (eg, GOLYTELY, HALFLYTELY, NULYTELY, MOVIPLEP). In some embodiments, the wash solution may contain a phosphate (eg, FULL's PHOSPHO-SODA). In some embodiments, the wash solution may contain sulfates such as sodium sulphate, magnesium sulphate or potassium sulphate (eg SUPREP). In some embodiments, the wash solution may contain magnesium citrate. In some embodiments, the wash solution may comprise picosulfate sodium.

In some embodiments, the kit may also include a container for collecting GLF and / or fecal samples from the subject. The container for collecting the GLF may include a toilet insert for capturing the GLF or a fecal sample or the like. In some embodiments, the vessel may contain materials for stabilizing and / or storing the target molecule, such as one or more isolated protease inhibitors. In some embodiments, the vessel may contain agents 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 the diagnosis, prognosis of a particular disease. Some embodiments include diseases and disorders associated with the GI tube and associated organs. Exemplary diseases include cancers of the GI duct and related organs, such as gastric cancer, liver cancer, pancreatic cancer. Further examples of the disease include pancreatitis, pancreatic adenocarcinoma, gastrointestinal neuroendocrine tumor, gastric adenocarcinoma, colon adenocarcinoma, hepatocellular carcinoma, cholangiocarcinoma, gallbladder adenocarcinoma, ulcerative colitis and Crohn's disease. Some diseases relate to 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 a portion 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 includes ulcerative colitis (UC), Crohn's disease (CD), uncertain colitis, chronic colitis, discontinuous or patchy disease, ileal inflammation, extracolonial inflammation, granulation according to ruptured crypts. Examples include swelling inflammation, aphthous ulcer, transmural inflammation, microscopic colitis, diverticulitis and vacant colitis. Further examples of the disease include celiac plues, malabsorption disorders and other conditions of the gastrointestinal tract, liver, pancreas and gall bladder.

Some embodiments of the methods and compositions provided herein are for the choice of treatment (often referred to as personalized medicine), the subject's positive response to treatment, the negative response to treatment or treatment. Regarding determining that there is no response. Some such embodiments include determining a patient's partial response to a treatment plan. For example, at the first time point, in a GLF or stool sample obtained from a subject, the presence of biomarkers, the absence of biomarkers or the level of biomarkers can be determined. The presence of biomarkers, the absence of biomarkers or the level of biomarkers can be determined in GLF or stool samples obtained from the subject at a second time point after treatment has begun and / or treatment has been completed. Presence of biomarker in GLF or stool sample obtained from first time point, absence of biomarker or level of biomarker compared to presence of biomarker in GLF or stool sample at second time point, biomarker Absence or differences in biomarker levels may indicate a subject's positive response to treatment, negative response to treatment, partial response to treatment, or no response to treatment. Alternatively, a subject may be given a treatment plan 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 will also be provided to the subject in the future, taking into account the determination of the subject's positive, negative, partial or no response to previous or current treatment regimens. It also includes determining a treatment plan. Accordingly, previous or current treatment plans may be modified based on the 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 level of multiple biomarkers. The presence, absence and / or level of multiple biomarkers is the subject's physiology, such as the likelihood of developing the subject's disease and / or the subject's possible response to a treatment regimen for treating a particular disease. It can be correlated with the likelihood of the situation. Some such methods correlate the presence, absence and / or level of multiple biomarkers of a subject by determining the likelihood that the subject has or develops the disease. A "clinical risk score" can be determined (eg, Sonyung P. et al. (2004) New Eng. J. of Medicine 351: 2817-2286, the full text of which is incorporated herein by reference. Cho C. S. et al. (2008) J. Am. Coll. Surg. Vol. 206: pp. 281-291).

The invention has been described in some detail for the purpose of clarity and understanding, but one of ordinary skill in the art may make various changes in form and detail without departing from the true scope of the invention. Will understand.

(Example 1)
Proteomics Analysis of Sulfate-Based GLF In this analysis, the ability of sulfate-based GLF to support proteomics analysis was evaluated. To identify the target molecule in GLF obtained using a sulfate-based wash composition, SUPREP was administered to 3 human subjects and the resulting protein in GLF was analyzed by mass spectrometry. .. In this example, GLF was collected from the subject as part of the colonoscopy procedure.

Upon collection, a complete protease inhibitor tablet (ROCHE) was added and the sample was rotated at 1000 rpm, 4 ° C. for 30 minutes. The supernatant was rotated again at 14,000 xg for 30 minutes to pellet the bacteria and debris. 1.8 ml of supernatant is precipitated with 6 volumes of acetone, followed by extraction with equal volumes of chloroform, followed by separation on a C-2 reverse phase SPE column (Sep-Pak, Waters). did. The columns were washed with 3 column volumes of 0.1% trifluoroacetic acid (TFA), in 0.1% TFA, 10%, 20% and 30% acetonitrile (ACN), respectively, and in 0.1% TFA. Elution using a 3-column volume of 60% ACN. Samples were dried by centrifugation and 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 obtained on an LTQ-Orbitrap mass spectrometer using inputs from the LC system. Solvent A contained 3% B and 0.2% formic acid in water. Solvent B contained 0.2% formic acid in 3% A and acetonitrile. The solvent was an HPLC grade manufactured by Fisher. For 120 minutes of practice, the starting solvent is 5% B and remains for 7 minutes. The gradient changed to 10% by 13 minutes, 40% by 83 minutes, 90% by 103 minutes, and then decreased from 90% to 5% by 111 minutes. It was then rebalanced for the next injection. Three injections were performed for each sample to determine reproducibility.

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

The database search uses the RefSeq database (http://www) with mass accuracy of bioclassification identified as human (homo humans), mass accuracy of (MS) 10 ppm for parent ion and 0.6 Da for fragment ion (MS / MS). .Ncbi.nlm.nih.gov/RefSeq/) was performed using a Masscot search engine (Matrix Science, UK) and "not enzyme" was selected. A search without enzyme specificity was performed due to the presence of digestive enzymes in the sample that could modify or cleave the peptide being tested. The RefSeq database was complemented 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 show 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 a search of this database without enzyme specificity; a score of 40 is p. <Conforms to 0.01. Standard Mascot scoring was used in which only the highest score of each peptide detected was added, even if sampled between MS / MS multiple times. For all data included, the scores were all> 40 for at least one sample per protein lineage. For further confidence, the number of significant peptides was also reported and the minimum criterion of at least two peptides was selected. Very few had less than 3 peptides. All significant peptides counted corresponded to different sequences (individual peptides) from their respective proteins. The score and the number of significant peptides are reported in the form x / y (where x is the score and y is the number of significant peptides in the formula). If no protein is detected in a particular sample, it is listed as "ND". The protein was reported as a protein name and provided with a "gi" number as defined by NCBI's protein database. The sequences contained within each of the "gi" numbers in the NCBI database listed throughout the present application are incorporated herein by reference. When a protein is named in a pre-protein or other immature form, the mature form of the protein is equally implied, including modifications such as removal of the signal sequence and addition of post-translational modifications. In all cases, the protein was named by its gene-derived sequence to provide consistency.

Table 1 lists examples of the most abundant proteins identified in GLF obtained from three distinct patients, defined as patients 3, 4 and 6, shown in the above format. 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 1485397840) that may be associated with colon cancer and other cancers. Antibodies and putative glycosylation proteins were also identified.

In another experiment, SUPREP was administered to the subject according to the manufacturer's guidelines, and the resulting GLF was self-collected by the subject in a collection container placed in the toilet immediately prior to colonoscopy. Was done. The GLF proteome was analyzed by MS as described above. Table 2 summarizes the results showing the Mascot scores of the most abundant species in existence. The results showed some urinary contamination. Similar proteomics profiles were observed for samples collected during colonoscopy. Table 2 shows that a number of different proteins were identified in the GLF collected by the subject. The identified proteins included DMBT1, pancreatic proteins and antibodies, consistent with the data in Table 1.

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

(Example 2)
Proteomics Analysis of Polyethylene Glycol-Based GLF In this analysis, the ability of polyethylene glycol-based GLF to support proteomics analysis was evaluated. In order to identify the target molecule in GLF obtained using the polyethylene-based cleaning composition, the polyethylene glycol-based cleaning composition was administered to two human subjects, and the protein in the obtained GLF was administered. It was analyzed by mass spectrometry as described in Example 1. Removal of polyethylene glycol was largely achieved by chloroform extraction of the cleaning solution. A number of different proteins were identified in GLFs obtained from these subjects receiving polyethylene glycol-based cleaning compositions. Examples of the most abundant identified proteins identified are shown in Table 3, consistent with those observed in the previous tables.

In another experiment, subjects were administered a PEG-based wash composition and the resulting GLF was self-collected in a collection container placed in a 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 as well as the corresponding Mascot scores and the number of significant peptides for each protein are listed in Table 4. A larger protein list provided evidence of urinary contamination. Similar proteomics profiles were observed for samples collected during colonoscopy.

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

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

(Example 3)
Proteomics Analysis of Magnesium Citrate-Based GLF In this analysis, the ability of magnesium citrate-based GLF to support proteomics analysis was evaluated. In order to identify the target molecule in GLF obtained from a human subject to which the magnesium citrate-based cleaning composition was administered, the magnesium citrate-based cleaning composition was administered to the subject and the GLF was subjected to colon endoscopy. Collected from subjects as part of the microscopic 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 above 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 Concentration In this analysis, samples obtained using GLF in combination with SSL-7 Concentration were used. The ability to detect IgA-1 and IgA-2 antibodies was evaluated. Staphylococcus
Using the affinity for aureus superantigen-like protein 7 (SSL-7), a human subject is administered a sulfate-based cleaning composition (SUPREP) to identify the target molecule in GLF and has an SSL-7 affinity. Proteins were concentrated at each GLF using sex beads. GLF was collected from subjects as part of the colonoscopy procedure.

IgA-1 and IgA-2 were specifically isolated using SSL-7 affinity beads. To 1 ml of sample, 20 μl of SSL-7 agarose (Invitrogen, San Diego, CA) was added and incubated overnight on rollers at 4 ° C. The test tube was rotated at 1,000 xg for 2 minutes to pellet the beads and discard the supernatant. The beads were washed 4 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. The 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, WI). Mass spectrometry and database search 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 the previous examples, the antibodies are present in the GLF and their enrichment and analysis is possible using affinity reagents and therefore specific analysis of this subproteome in the GLF. Enables. The most abundant antibody was IgA. IgA has been consistently reported to be present in the intestinal tract.

The above 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 Concentration This analysis detects IgA and IgM antibodies in samples obtained using GLF in combination with Protein L Concentration. Evaluated ability. To identify the target molecule in GLF using its affinity for protein L, a human subject is administered a sulfate-based wash composition (SUPREP) and protein L-affinitive beads are used to attach the protein in each GLF. Concentrated. GLF was collected from subjects as part of the colonoscopy procedure.

Protein L affinity beads were used to isolate antibodies containing the κ light chain. To 1 ml of sample, 20 μl of protein L agarose (Santa Cruz Biotechnology, Santa Cruz, CA) was added and incubated overnight on a roller at 4 ° C. The test tube was rotated at 1,000 xg for 2 minutes to pellet the beads and discard the supernatant. The beads were washed 4 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. The 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, WI). Table 8 summarizes the most abundant identified proteins present in GLF with affinity for protein L and their corresponding Mascot scores and the number of significant peptides. As expected, IgA and associated chains derived from the antibody were detected again. Since protein L is not completely specific for IgA antibody, IgM antibody (gi number 193806374) was also detected.

The above analysis demonstrates that IgA and IgM antibodies can be detected in samples obtained using GLF in combination with protein L enrichment.

(Example 6)
Detection of Bacterial Proteins in Samples Obtained Using GLF This analysis evaluated the ability of GLFs to facilitate detection of bacterial proteins. To identify target molecules associated with bacteria in GLF, two human subjects were administered a sulfate-based wash composition (SUPREP) and the resulting GLF was subjected to colonoscopy procedures. Collected from the subject as part. 100 μl of each GLF was seeded in Super Optimal Bros (SOB) medium and incubated overnight at 37 ° C. with 220 rpm shaking. The pellet was dissolved in a bead beater in 8M urea and the lysate was diluted to 2M urea with 50 mM ammonium bicarbonate / 10 mM tris (2-carboxyethyl) phosphine and sequenced grade trypsin (Promega, Madison, WI). Digested using. Data were obtained using a 120 minute run on the Orbitrap MS system as described above. Create an MGF search file and use the RefSeq database (http://www) with a mass accuracy of 10 ppm for parent ions (MS) and 0.6 Da for fragment ions (MS / MS) identified as eubacteria. .Ncbi.nlm.nih.gov/RefSeq/) was searched using the Masscot search engine (Matrix Science, UK). The most abundant identified proteins present in GLF associated with bacteria and their corresponding Mascot scores and the number of significant peptides are summarized in Table 9. In the sample 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 above analysis demonstrates that proteins of bacterial origin can be detected in samples obtained using GLF.

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

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

(Example 8)
Proteomics Analysis of Fecal Samples This analysis evaluated the ability of fecal samples to support proteomics analysis. No wash composition was administered to the human subject to identify the target molecule in the stool sample. Fecal samples were collected from the subject using a collection container placed in the toilet during normal defecation. A small amount of stool (stool) sample was homogenized in 0.1% TFA and then centrifuged at 13000 xg. The protein was precipitated with 6 volumes of acetone, resuspended in 0.1% TFA, extracted with equal volume of chloroform, and then treated in an SPE column, as described in Example 1. did. The most abundant identified proteins and their corresponding Mascot scores and the number of significant peptides are summarized in Table 11. Proteins, mostly of pancreatic origin, were detected in the sample, indicating that feces, after discovery in GLF, are the source for the detection of these biomarker proteins. However, the sample also contained some other non-human material that made the analysis much more limited, especially for the discovery of biomarkers.

The above analysis demonstrates that a large number of target molecules can be detected in fecal samples.

(Example 9)
Detection of Glycans in Samples Obtained Using GLF In this analysis, the ability of glycans to be detected in samples obtained using GLF was evaluated. GLF was collected from human subjects to identify and analyze target molecules, including glycans, in GLF. 1.8 mL of GLF was added to 12 mL of ice-cold acetone and incubated for 1 hour to pellet the protein. The sample was 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 overnight in a shaker at 37 ° C. with 2 μL of PNGase F. 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. For dried glycans, use 4-ABEE (ethyl 4-aminobenzoate) and add 25 μL of derivatized solution (90:10 MeOH: HAc containing 35 mM ABEE and 100 mM 2-PB) 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 placed in SpeedVac for a short time to remove any residual ether. The labeled glycans were then performed by HPLC and eluted from the Agilent C8 reverse phase column with between 20-25% acetonitrile. This fraction was vacuum dried, redissolved in 50 μL of 0.1% TFA and performed on Waters Q-TOF LC-ESI-MS for glycan analysis. The derivatized glycans were eluted with about 20-25% acetonitrile in 0.2% formic acid from a C18 reverse phase column on Q-TOF MS. Mass spectrometers scanned at m / z 150-2000 per second in MS-only format to obtain derivatized glycan profile data. FIG. 1 summarizes these results and represents a graph of the relative abundance of glycan structures derived from various glycoproteins present in the gastrointestinal lavage fluid fraction. As shown in FIG. 1, the glycan structure derived from some glycoproteins contains certain modifications associated with chain termination. These modifications may be due to bacterial activity present in the GLF sample, as it is known that bacteria can digest and consume glycans from proteins. However, such modifications may also be associated with the disease, especially cancers in which abnormal glycosylation is associated with the disease.

The above analysis demonstrates that large numbers of glycans can be detected in samples obtained using GLF.

(Example 10)
Detection of Metabolites in Samples Obtained Using GLF This analysis evaluated the ability of metabolites to be detected in samples obtained using GLF. To identify and analyze target molecules, including metabolites, in GLF, human subjects were administered a magnesium citrate-based cleaning composition and GLF obtained for metabolites such as cholic acid and other bile acids. Was analyzed. The resulting GLF was collected from the subject as part of the colonoscopy procedure.

3 ml of GLF was centrifuged at maximum rate 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 of 0.1% TFA.

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

The above analysis demonstrates that metabolites can be detected in samples obtained using GLF.

The term "comprising" is synonymous herein with "inclusion," "contining," or "characterized by." , Comprehensive or unconstrained, and does not exclude additional, unlisted elements or method steps.

It is understood that all numbers representing the amounts of components, reaction conditions, etc. used herein are modified in all examples by the term "about". Thus, unless otherwise indicated, the numerical parameters shown herein are approximations that may vary depending on the desired properties sought to be obtained. At a minimum, it does not seek to limit the application of the equivalent principle of any claims in any application claiming priority to this application, and each numerical parameter is a number of significant digits and ordinary. It must be interpreted with the rounding approach in mind.

The above description discloses some methods and materials of the present invention. The present invention is susceptible to modifications in methods and materials as well as changes in manufacturing methods and equipment. Such modifications will be apparent to those skilled in the art from consideration or implementation thereof of the present invention disclosed herein. As a result, the invention is not intended to be limited to the specific embodiments disclosed herein, but is intended to cover all modifications and modifications within the true scope and intent of the invention. ..

References Each of the following references is incorporated herein by reference in its entirety.

All references cited herein, including but not limited to published and unpublished applications, patents and references, are hereby incorporated by reference in their entirety. It forms part of this specification. As long as the publications and patents or patent applications incorporated by reference conflict with the disclosures contained herein, the specification supersedes and / or supersedes any such conflicting material. It shall be.

Claims (1)

The invention described in the specification.

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