Method, device, and kit for population-screening for cancer, cancer recurrence and precancerous conditions in symptom-free individuals

Abstract

To provide a test which can be performed on the public, including those who do not currently present symptoms or otherwise display a need for diagnostic testing, as part of a general population screening practice.SOLUTION: The present disclosure relates to a screening test method, device, and kit for carbohydrates found in a biological sample and associated conditions including cancerous and precancerous conditions. Specifically, the method tests abnormal carbohydrates in a biological sample using reagents of galactose oxidase and Schiff's reagents. The screening test method, device, and kit provide expanded testing capabilities across a range of known conditions and in an otherwise healthy population. This disclosure further relates to use of the device or kit for initial evaluation for cancerous and precancerous conditions in people without obvious signs and symptoms, and cancer recurrence in a point-of-care facility or at home.SELECTED DRAWING: None

Description

[Technical Field]

[0001] [CROSS-REFERENCE TO RELATED APPLICATIONS] This application is a continuation-in-part of U.S. Non-provisional Patent Application No. 16 / 937,051, filed July 23, 2020, and a continuation-in-part of U.S. Non-provisional Patent Application No. 16 / 936,998, filed July 23, 2020. This application also claims priority to and the benefit of U.S. Provisional Patent Application No. 62 / 893,484 and U.S. Provisional Patent Application No. 62 / 893,477, filed August 29, 2019, the disclosures of which are incorporated herein by reference in their entireties.

[0002] The present invention relates to a screening test for pre-cancerous conditions in otherwise healthy populations and / or individuals, and to a kit containing the components necessary to perform the test. [Background technology]

[0003] Cancer is a major global public health problem. Despite the development of pharmaceuticals for cancer treatment, early detection and prevention remain the greatest hope for combating this human tragedy. U.S. Patent No. 4,857,457 and Shamsuddin et al., Human Pathology, 19:7-10, 1988 (both of which are incorporated herein by reference in their entireties) report a colon cancer test that can detect elevated risk of colon cancer using rectal mucus. A freeze-dried cellulose membrane filter was pre-impregnated with an enzyme phosphate buffer solution, and then the mucus was reacted with the enzyme galactose oxidase by contacting the pre-impregnated cellulose membrane filter with a Metricel membrane filter containing a mucus sample for 1 to 2 hours. The membrane filter containing the mucus was then washed with distilled water for 1 minute, reacted with basic fuchsin for 15 minutes, washed with tap water for 10 minutes, and air-dried.

[0004] This basic procedure, while simple, is currently used to detect colon cancer and precancerous conditions. However, it has since been discovered that the disclosed screening procedure, i.e., the use of the marker disaccharides employed therein as predictors of colon cancer, is not limited to cancer of the colon. Rather, as described, for example, in U.S. Patent Nos. 5,162,202 and 5,348,860 to Shamsuddin (incorporated herein by reference in their entireties), the disaccharide markers may be present in other sites throughout the body where similar precancerous or cancerous traits are found. Additionally, the tests described therein are time-consuming.

[0005] Most cancers in humans are believed to be the result of exposure to one or more environmental carcinogens via the air we breathe, the food we ingest, the water and beverages we ingest, etc. Carcinogens are excreted via the large intestine, or the kidneys and bladder, or the lungs, the skin (through sweating), etc. Thus, carcinogen(s) and / or their metabolite(s) can be expected to cause changes in cancerous and precancerous organs, in addition to simultaneously causing corresponding changes in these organs. A precancer or precancerous condition is a stage or disease that can progress to cancer and therefore renders an otherwise healthy, asymptomatic individual highly susceptible to (showing high-risk symptoms of) cancer later on. Thus, the presence or threat of a cancerous or precancerous condition in an individual's body, including but not limited to the large intestine, can be accurately detected by the methods of the present invention, initially using sampling of rectal mucus, followed, if necessary, by sampling other bodily fluids such as breast, prostate secretions, semen, cervical and vaginal mucus, sputum, bronchial or alveolar secretions until the precise location of the cancerous or precancerous condition is identified, without the prior risk of false negatives that limits the value of this approach as a screening test for the general population, particularly as a field test. The invention disclosed herein has the unexpected advantage of accurately and reliably detecting cancer and precancerous conditions in any organ in individuals who do not exhibit signs or symptoms of the cancerous or precancerous condition.

[0006] Furthermore, the disclosure provided herein may help save lives by providing a routine screening process for people who have no obvious signs or symptoms of pre-cancerous conditions. As the old adage goes, "an ounce of prevention is better than a pound of cure," the present disclosure will not only reduce mortality and morbidity rates, but also financially, significantly reduce national healthcare costs for patients and healthcare providers alike worldwide. Summary of the Invention [Problem to be solved by the invention]

[0007] A general objective of the present disclosure is to provide a test that can be administered to the general public, including those who are not currently symptomatic or otherwise indicate a need for diagnostic testing, for example, as part of a general population screening method. The screening tests provided herein allow for rapid testing and results, facilitating their use in large-scale testing programs that do not require specialized diagnostic laboratories, reporting the results of the test to the tested individual(s) (or their physicians) before the individual leaves the testing site, and / or allowing for additional rectal mucus samples to be obtained if a negative result is determined to be a false negative.

[0008] Another object is to provide such a kit.

[0009] It is yet another object of the present invention to provide a kit for carrying out the screening test of the present invention in which the key components are stable over time.

[0010] Yet another object is to provide a method of performing a screening test using the kit of the present invention that can be completed at the point of care or in the subject's home quickly enough to provide test results while the tested individual waits.

[0011] Upon further study of this specification and the appended claims, further objects and advantages of this invention will become apparent to those skilled in the art. [Means for solving the problem]

[0012] The present disclosure relates to a screening test method, device, and kit for cancerous and precancerous conditions. Specifically, the method tests for abnormal carbohydrates in mucus or body fluids using a galactose oxidase reagent and a Schiff's reagent. The screening test method, device, and kit provide improved accuracy and minimize handling procedures. The present disclosure further relates to the use of the device or kit in a medical facility for the early evaluation of cancerous and precancerous conditions.

[0013] In one aspect, a method for screening for cancer and precancerous conditions and lesions comprises the steps of applying mucus or body fluid to a test strip or membrane pre-embedded with galactose oxidase, oxidizing marker carbohydrates in the mucus or body fluid containing marker carbohydrates by reacting with the galactose oxidase, and activating a reservoir containing a Schiff's reagent solution adjacent to the test strip or membrane to contact the test strip or membrane, wherein the Schiff's reagent solution is not initially in contact with the test strip or membrane.

[0014] In some embodiments, the method for screening for cancerous and precancerous conditions and lesions includes one or more additional steps selected from applying water onto the test strip or membrane before applying the mucus or body fluid to the test strip or membrane, removing the mucus or body fluid by washing before activating the container containing the Schiff's reagent solution, rinsing the test strip or membrane with tap water after contacting the Schiff's reagent solution with the test strip or membrane, drying the test strip or membrane, and / or evaluating the color of the test strip or membrane.

[0015] In another aspect, a device for treating cancer and precancerous conditions and lesions includes a test strip or membrane pre-embedded with galactose oxidase and a container containing a Schiff's reagent solution, wherein the Schiff's reagent solution is initially out of contact with the test strip or membrane and can be activated to contact the test strip or membrane after the marker carbohydrate has been oxidized by galactose oxidase. The mechanism for activating the carbohydrate Schiff's reagent solution (bringing it into contact with the test strip or membrane after the marker carbohydrate has been oxidized by galactose oxidase) is not limited. In one aspect, the mechanism can be a twist valve, a breakable barrier, or the like, where the twist valve is opened or the barrier is broken before the Schiff's reagent solution contacts the test strip or membrane.

[0016] In another aspect, the present disclosure provides a test kit for cancerous and precancerous conditions, comprising a test strip or membrane pre-embedded with galactose oxidase and a container containing a Schiff's reagent solution. In one aspect, the galactose oxidase pre-embedded test strip or membrane further contains a dried culture medium that can activate the galactose oxidase upon addition of water onto the test strip or membrane. In another aspect, the pre-embedded galactose oxidase of the test strip or membrane is microencapsulated.

[0017] In one aspect, a method for screening for cancer and precancerous conditions and lesions comprises the steps of applying mucus or body fluid to a test strip or membrane pre-embedded with a Schiff's reagent, oxidizing the marker carbohydrates in the mucus or body fluid containing the marker carbohydrates by reacting with galactose oxidase, and activating adjacent Schiff's reagents embedded in the test strip or membrane to contact the test strip or membrane.

[0018] In some embodiments, the method for screening for cancer and precancerous conditions and lesions includes one or more additional steps selected from applying water onto the test strip or membrane before applying the mucus or body fluid to the test strip or membrane, removing the mucus or body fluid by washing before activating the Schiff's reagent, rinsing the test strip or membrane with tap water after contacting the Schiff's reagent with the sample, drying the test strip or membrane, and / or evaluating the color of the test strip or membrane.

[0019] In another aspect, a device for cancer and precancerous conditions and lesions comprises a test strip or membrane pre-embedded with Schiff's reagent and a reservoir containing galactose oxidase, wherein the Schiff's reagent can be activated and contacted with the sample after the marker carbohydrate has been oxidized by the galactose oxidase. The mechanism for activating the Schiff's reagent solution (contacting the sample after the marker carbohydrate has been oxidized by the galactose oxidase) is not limited. In one aspect, the mechanism can be by adding a pore-forming agent.

[0020] In another aspect, the present disclosure provides a kit for testing for cancerous and precancerous conditions, comprising a test strip or membrane pre-embedded with Schiff's reagent and a container containing a galactose oxidase reagent solution. In one aspect, the test strip or membrane pre-embedded with Schiff's reagent further contains a dried culture medium that can activate the galactose oxidase upon addition of water onto the test strip or membrane. In another aspect, the Schiff's reagent in the test strip or membrane is microencapsulated.

[0021] In another aspect, the present invention provides a screening method for rapidly testing a subject for a precancerous condition when the subject has no obvious signs or symptoms of a cancerous or precancerous condition, the method comprising, in step (a), obtaining a biological sample from an individual. In step (b), a portion of the sample is assayed for the presence of glycoproteins containing at least one carbohydrate selected from the group consisting of β-D-Gal-(1→3)-D-GalNAc, Fuc-α-1→2-Gal-β-(1→4)-Fuc-α-1→3-GlcNAc, Fuc-α-1→2-Gal-β-(1→4)-Fuc-α-1→3-GlcNAc-β-(1→3)-Gal-β-(1→4)-GlcNAc, and Fuc-α-1→2-Gal-β-(1→4)-Fuc-α-1→3-GlcNAc-β-(1→3)-Gal-β-(1→4)-Fuc-α-1→3-GlcNAc. Assaying the biological sample can include briefly subjecting the sample to oxidizing conditions capable of selectively oxidizing only the cyclic sugar moieties of any marker carbohydrates present on glycoproteins in the sample at their hydroxyl-bearing ring carbon atoms to form aldehyde sugar moieties, followed by visualization of any aldehyde sugar moieties formed with a Schiff base bleaching dye. Next, in step (c), a portion of the biological sample can be assayed for the presence of any glycoproteins in the sample by subjecting the sample to the oxidizing action of an oxidizing agent that oxidizes the sugar moieties of any glycoproteins in the sample to aldehyde sugar moieties, followed by visualization of any aldehyde sugar moieties formed. The presence of aldehyde sugar moieties formed can confirm the adequacy of sampling, while the absence of aldehyde sugar moieties can establish that a negative test result was due to sampling error. In step (d), the method can include retesting the individual in the same manner as above using a new biological sample if the initial sample assayed negative in steps (b) and (c). Finally, in step (e), the method may include diagnosing the asymptomatic patient as having cancer or a precancerous condition based on visualization of the color change of the Schiff base dye.

[0022] The assay may involve adsorbing the biological sample to a water-insoluble substrate that captures proteins, followed by washing the substrate to remove non-immobilized components of the sample from the substrate. The insoluble substrate may be a membrane filter.

[0023] Selective oxidation of glycoproteins allows assaying of sugar moieties of marker carbohydrates by selectively oxidizing the primary hydroxyl groups of any galactose moieties to aldehyde groups, and the oxidized glycoproteins are then assayed for oxidation of adjacent galactose moieties. Galactose moieties can be oxidized using galactose oxidase, and the oxidized adjacent galactose moieties can be visualized with basic fuchsin. Any aldehyde sugar moieties in the sample can be further oxidized with periodate and visualized with basic fuchsin.

[0024] The above method can be performed simultaneously on multiple biological samples from multiple individuals as part of a field screening for cancer.

[0025] Step (c) may be performed simultaneously with step (b) on a different portion of the same biological sample, and if negative results are obtained in both steps (b) and (c), another biological sample may be taken from the individual immediately thereafter, and steps (a), (b) and (c) may be repeated.

[0026] Prior to assay, the biological sample may be adsorbed to a water-insoluble substrate that captures proteins, and the substrate may be washed to remove non-immobilized components of the biological sample from the substrate. The sample is then assayed in step (a) by selectively oxidizing any glycoproteins in the sample, thereby oxidizing the primary hydroxyl groups of the galactose sugar moieties of any carbohydrates therein to aldehyde groups, which can then be visualized with basic fuchsin. Samples that test negative in this assay can be further oxidized with periodate, which allows the aldehyde sugar moieties in the oxidized sample to be visualized with basic fuchsin.

[0027] The insoluble substrate can be a membrane filter, and galactose oxidase can be used to oxidize the galactose sugar moieties. An agent for oxidizing the galactose moieties can be present in the insoluble substrate or applied directly to the insoluble substrate after the sample has been applied to the insoluble substrate.

[0028] In an embodiment, the insoluble substrate can be a membrane filter, the galactose sugar moieties can be oxidized using galactose oxidase, and the galactose oxidase is applied directly to the membrane filter after the biological sample is applied to the membrane filter.

[0029] Disclosed is a method for screening for pre-cancerous conditions in organs other than the large intestine in a human when the human has no obvious signs or symptoms of a cancerous or pre-cancerous condition. The method may comprise assaying a sample of a proteinaceous secretion or fluid other than rectal mucus associated with the organ for the presence in the sample of a marker carbohydrate selected from the group consisting of β-D-Gal-(1→3)-D-GalNAc, Fuc-α-1→2-Gal-β-(1→4)-Fuc-α-1→3-GlcNAc, Fuc-α-1→2-Gal-β-(1→4)-Fuc-α-1→3-GlcNAc-β-(1→3)-Gal-β-(1→4)-GlcNAc and Fuc-α-1→2-Gal-β-(1→4)-Fuc-α-1→3-GlcNAc-β-(1→3)-Gal-β-(1→4)-Fuc-α-1→3-GlcNAc.

[0030] Proteinaceous secretions may be adsorbed to a water-insoluble substrate that captures the protein prior to assay, and the substrate may then be washed to remove non-immobilized components of the sample from the substrate. Samples can be assayed for marker carbohydrates by selectively oxidizing any glycoproteins in the sample using galactose oxidase, thereby selectively oxidizing the primary hydroxyl groups of any galactose sugar moieties of any marker carbohydrates therein to aldehyde groups. The oxidized galactose moieties can then be visualized with basic fuchsin. Portions of samples that test negative in this assay can be further oxidized with periodate, allowing the aldehyde sugar moieties in the oxidized samples to be visualized with basic fuchsin.

[0031] The portion of the sample of proteinaceous secretion that is oxidized with periodate may be oxidized simultaneously with a different portion of the same sample that is oxidized with galactose oxidase.

[0032] The human may be female and the biological sample may be any or all of vaginal mucus, endocervical mucus, nipple aspirate, sputum, bile duct fluid, rectal mucus, and / or pancreatic duct aspirate. The human may be male and the biological sample may be any or all of prostate secretions, semen, sputum, bile duct fluid, rectal mucus, and / or pancreatic duct aspirate. [Brief explanation of the drawings]

[0033] [Figure 1] Figure 1 shows the principle of the reactions involved in the kits and methods of the present invention. In Figure 1, the marker Gal-GalNAc reacts with galactose oxidase (GO) to generate two adjacent aldehydes at the C6 position, which are then conjugated with basic fuchsin, imparting a magenta color. [Figure 2] 1 shows one embodiment of the method of the claimed invention in which samples are reacted on a slide to identify cancerous or precancerous conditions. In samples from subjects without any cancer or precancer, the test panel is colorless, while typically a magenta color (ranging from pink to purple) indicates marker disaccharides specific for cancer and precancerous conditions and lesions. [Figure 3] 1 shows one embodiment of the method of the claimed invention in which samples are reacted on a slide to identify cancerous or precancerous conditions. In samples from subjects without any cancer or precancer, the test panel is colorless, while typically a magenta color (ranging from pink to purple) indicates marker disaccharides specific for cancer and precancerous conditions and lesions. DETAILED DESCRIPTION OF THE INVENTION

[0034] The following is a detailed description presented to aid those skilled in the art in practicing the present disclosure. Those skilled in the art may make modifications and variations to the embodiments described herein without departing from the spirit or scope of the present disclosure. All publications, patent applications, patents, figures and other references mentioned herein are expressly incorporated herein by reference in their entirety.

[0035] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the disclosure.

[0036] Where a range of values ​​is presented, it is understood that each intervening value, to the tenth of the unit of the lower limit, between the upper and lower limits of that range, and any other stated or intervening value within that stated range, is encompassed within the disclosure unless the context clearly dictates otherwise (e.g., in the case of a group having a large number of carbon atoms, where each number of carbon atoms included within a range is recited). The upper and lower limits of these smaller ranges may be independently included in the smaller ranges, subject to any specifically excluded limit in the stated range, and are also encompassed within the disclosure. Where the stated range includes one or both of the limits, ranges excluding either of those included limits are also included in the disclosure.

[0037] All numerical values ​​in the detailed description and claims herein are modified by the stated value being "about" or "approximately" to account for experimental error and variations that would be expected by one of ordinary skill in the art.

[0038] The following terms are used to describe the present disclosure. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present disclosure.

[0039] As used in this specification and the appended claims, the non-specifying articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article, unless the context clearly indicates otherwise. By way of example, "an element" means one element or more than one element.

[0040] The term "and / or," as used in the specification and claims, is understood to mean "either or both" of the elements so conjunctively, i.e., elements present conjunctively in some cases, and elements present disjunctively in other cases. Multiple elements listed with "and / or" are to be construed in the same manner, i.e., "one or more" of the elements so conjunctively. Other elements other than the elements specifically identified by the "and / or" clause may optionally be present, whether related or unrelated to the elements specifically identified. Thus, as a non-limiting example, "A and / or B," when used in conjunction with open-ended language such as "comprising," can refer in one embodiment to A only (optionally including elements other than B); in another embodiment to B only (optionally including elements other than A); in yet another embodiment to both A and B (optionally including other elements), etc.

[0041] As used in the specification and claims, "or" is understood to have the same meaning as "and / or" as defined above. For example, when separating listed items, "or" or "and / or" is interpreted as inclusive, that is, the inclusion of at least one, but also two or more, of a plurality of elements or a list of elements, and optionally additional unlisted items. Only terms clearly indicating the contrary, such as "only one of" or "exactly one of," or, when used in the claims, "consisting of," refer to the inclusion of exactly one element of a plurality of elements or a list of elements. In general, the term "or" as used herein will be interpreted as indicating exclusive alternatives (i.e., "one or the other, but not both") only when followed by terms of exclusion, such as "either," "one of," "only one of," or "exactly one of."

[0042] In the claims and the foregoing specification, all transitional phrases such as "comprising," "including," "carrying," "having," "containing," "involving," "holding," "composed of," and the like, are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases "consisting of" and "consisting essentially of" shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.

[0043] The phrase "at least one," as used herein and in the claims, in reference to a list of one or more elements, is understood to mean at least one element selected from any one or more of the elements in the list of elements, but does not necessarily include at least one of each and every element specifically listed in the list of elements, and does not exclude any combination of elements in the list of elements. Again, this definition allows for the optional presence of elements other than those specifically identified in the list of elements to which the phrase "at least one" refers, whether or not related to the specifically identified elements. Thus, as a non-limiting example, "at least one of A and B" (or, equivalently, "at least one of A or B," or, equivalently, "at least one of A and / or B") can refer in one embodiment to at least one A (optionally including more than one) and no B (optionally including elements other than B); in another embodiment to at least one B (optionally including more than one) and no A (optionally including elements other than A); in yet another embodiment to at least one A (optionally including more than one) and at least one B (optionally including more than one) (optionally including other elements); and so on.

[0044] As used herein and in the claims, the phrase "marker carbohydrate" or "marker saccharides" should be understood to mean carbohydrates that can provide information about cancer and precancer using the methods described herein. Marker carbohydrates include, but are not limited to, β-D-Gal-(1→3)-D-GalNAc, Fuc-α-1→2-Gal-β-(1→4)-Fuc-α-1→3-GlcNAc, Fuc-α-1→2-Gal-β-(1→4)-Fuc-α-1→3-GlcNAc-β-(1→3)-Gal-β-(1→4)-GlcNAc, and Fuc-α-1→2-Gal-β-(1→4)-Fuc-α-1→3-GlcNAc-β-(1→3)-Gal-β-(1→4)-Fuc-α-1→3-GlcNAc.

[0045] As used in this specification and claims, the terms "mucous" and "body fluid" are intended to be interchangeable and are intended to be broadly interpreted as any fluid or mucus derived from the human body that contains marker carbohydrate(s).

[0046] As used herein and in the claims, the terms "embedded," "impregnated," and "pre-embedded" are intended to be interchangeable. In certain embodiments, the reagents of the present invention can be embedded directly into a membrane or test strip by binding or immobilizing the reagent on the surface of the membrane or test strip, or by loading the reagent into the pores of a porous membrane via a coating. In still other embodiments, the reagents of the present invention can be encapsulated in capsules or microcapsules and then embedded, coated, or impregnated into the reagent or test strip. In still other embodiments, the reagents can be used as an eluent in a lateral flow assay or lateral flow test strip. In certain embodiments, a fluid containing Schiff's reagent can be used as an eluent in a lateral flow assay or lateral flow test strip. In another specific embodiment, a fluid containing galactose oxidase can be used as an eluent in a lateral flow assay or lateral flow test strip.

[0047] The background art and theory used in this disclosure for detecting the presence of marker carbohydrates in the mucus or body fluids of individuals being tested for cancerous or precancerous conditions is disclosed in U.S. Patent Nos. 4,857,457, 5,162,202, and 5,348,860, as well as "Usefulness of Galactose Oxidase-Schiff Test in Rectal Mucus for Screening of Colorectal Malignancy ANTICANCER RESEARCH 21:1247-1256 (2001)." All of these references are incorporated herein by reference. The reactions used in the methods and kits of the present invention are shown in FIG. 1.

[0048] The present invention improves upon the assays described above with respect to minimizing false positives as a result of sampling or procedural errors, as well as for convenience.

[0049] Also, in any particular method described herein that includes more than one step or act, unless the context indicates otherwise, it should be understood that the order of the method steps or acts is not necessarily limited to the order in which the method steps or acts are recited.

[0050] The term "patient" or "subject" is used throughout this specification to describe an animal, preferably a human or domestic animal, to which treatment, including prophylactic treatment, with a composition according to the present disclosure is administered. For treatment of an infection, condition, or disease state specific to a particular animal, such as a human patient, the term patient refers to the particular animal, including domestic animals such as dogs or cats, or livestock such as horses, cows, sheep, etc. Generally, in this disclosure, the term patient refers to a human patient unless otherwise specified or suggested by the context of the use of the term.

[0051] "Having no obvious signs or symptoms" or "not exhibiting symptoms" includes otherwise healthy subjects who exhibit minimal or no symptoms normally associated with cancer or precancer. In certain embodiments, the term "having no obvious signs or symptoms" includes otherwise healthy individuals who have a genetic predisposition to certain cancers or a family history of such cancers (e.g., falling into the "highly susceptible to later cancer" category). In certain embodiments, the term "having no obvious signs or symptoms" includes otherwise healthy individuals who are at increased risk for certain cancers based on lifestyle, environmental factors, or other social determinants (e.g., smokers, individuals who work with radiation or hazardous materials, individuals exposed to carcinogens via groundwater, individuals taking medications that increase the risk of cancer). In certain embodiments, the term "having no obvious signs or symptoms" includes otherwise healthy individuals who have previously been treated for a cancerous condition that has been eliminated or has gone into remission. In such embodiments, the methods of the present invention can be used to detect recurrence of a cancerous condition. In certain embodiments, the term "no obvious signs or symptoms" includes individuals who exhibit symptoms not solely attributable to cancer or precancer. Such symptoms include, but are not limited to, fatigue, loss of appetite, weight loss, cough, shortness of breath, swollen lymph nodes, indigestion, nausea, and pain. In certain embodiments, an individual is considered to have no obvious signs or symptoms of lung cancer if they exhibit minimal or no symptoms of rib or chest pain, chronic or persistent cough, shortness of breath, and / or wheezing. In certain embodiments, an individual is considered to have no obvious signs or symptoms of breast cancer if they exhibit minimal or no signs or symptoms of breast lumps, breast tenderness, nipple discharge, and / or changes in breast or nipple shape. In certain embodiments, an individual is considered to have no obvious signs or symptoms of colon cancer if they exhibit minimal or no symptoms of changes in bowel habits, changes in stool consistency, blood in the stool, and / or persistent abdominal discomfort.In certain embodiments, an individual is considered to have no overt signs or symptoms of prostate cancer if they experience minimal or no symptoms of: frequent urination, difficulty in starting or maintaining a steady stream of urine, dribbling, excessive urination or the urge to urinate, urinary retention, urinary leakage, and / or bone pain. In certain embodiments, an individual is considered to have no overt signs or symptoms of endometrial cancer if they experience minimal or no symptoms of: pelvic pain, discomfort or pain during intercourse, abnormal menstrual periods or spotting, abnormal vaginal bleeding, and / or abnormal vaginal discharge. In certain embodiments, an individual is considered to have no overt signs or symptoms of ovarian cancer if they experience minimal or no symptoms of: abdominal or pelvic pain, changes in bowel habits, abdominal bloating, ascites, and / or an abdominal lump. In certain embodiments, an individual is considered to have no overt signs or symptoms of pancreatic cancer if they exhibit minimal or no symptoms of abdominal or mid-back pain, ascites, abnormally dark urine, and / or yellowing of the skin or eyes. In certain embodiments, a subject is considered to have no overt signs or symptoms of head and neck cancer if they do not exhibit any of the following: swelling or non-healing sores, red or white patches in the mouth, lumps, masses, or masses in the head or neck with or without pain, persistent sore throat, bad breath not explained by hygiene, hoarseness or altered voice, nasal obstruction or persistent nasal congestion.

[0052] Specific embodiments of the techniques used in the present invention to detect the presence of marker carbohydrates in the rectal mucus of symptomatic individuals (exhibiting signs and symptoms of colon and intestinal cancer) are disclosed in U.S. Pat. No. 4,857,457 and U.S. Pat. No. 5,348,860, which are incorporated herein by reference in their entireties.

[0053] The present invention provides a reliable screening tool for the detection of a wide range of cancers, for example, of the lung, rectum, colon, stomach, kidney, gallbladder, liver, pancreas, prostate, testis, breast, head and neck, cervix, uterus, and ovaries. The present invention also provides a reliable screening tool for the detection of precancerous conditions or elevated risk, for example, when an individual exhibits high-risk symptoms (e.g., falls into a "highly susceptible to later cancer" category). For example, "highly susceptible to later cancer" patients may include individuals who do not develop signs and symptoms of cancer or precancerous conditions but may be affected by certain lifestyle- or work-related externalities, such as smoking, alcohol or drug use, unhealthy diets, sedentary, dangerous, or harmful work or living environments. In these cases, patients may not have obvious signs or symptoms of cancer or precancerous conditions at the time, but may still exhibit a propensity for cancer or precancerous conditions. However, the described screening tests, kits, and methods can be used for general screening, for example, in patients who are not in high-risk categories or who otherwise have no known symptoms of cancer or precancerous conditions. Ideally, the described screening tests, kits and methods would be beneficial to make part of annual health checkups or regular doctor visits for patients in the target population, allowing for much earlier detection and intervention. As used herein, the target population may generally be people over the age of 30, although there is no reason why younger individuals could not be screened.

[0054] In certain embodiments, the present invention provides reliable screening tools for the detection of a wide range of cancers, e.g., of the lung, rectum, colon, lymph nodes, stomach, kidney, gallbladder, liver, pancreas, prostate, testes, breast, head and neck, cervix, uterus, and ovaries. Precancerous conditions include, for example, conditions in which an individual has no obvious signs or symptoms. In other words, the present invention provides tools that can detect cancer or precancer even when the condition is not indicated to be present. This includes individuals who have a genetic predisposition to certain cancers or a family history of such cancers (e.g., falling into the "highly susceptible to later cancer" category). However, the described screening tests, kits, and methods can be used for general screening, e.g., in patients who are not in high-risk categories or who otherwise have no known symptoms of cancer or precancer.

[0055] In some embodiments, if the screening tool of the invention indicates evidence of a cancerous or precancerous condition, the invention further provides for initiating treatment for the cancerous or precancerous condition or reinstating previously discontinued treatment for the cancerous condition. In some embodiments, if the screening tool of the invention does not indicate evidence of a cancerous or precancerous condition, the invention further provides for screening the individual again after a predetermined period of time.

[0056] In certain embodiments, the present invention provides screening tools for various biological samples, biological matrices, and biological fluids. The term "biological sample" refers to any solution or extract containing a molecule or mixture of molecules, including at least one biomolecule, derived from a biological source (e.g., human or animal), that is subject to extraction or analysis. Biological samples are intended to include crude or purified, e.g., isolated or commercially obtained, samples. Biological samples may include, but are not limited to, inclusion bodies, biological fluids, biological tissues, biological matrices, embedded tissue samples, cells (e.g., one or more types of cells), and cell culture supernatants. Specific examples include plasma, urine, cerebrospinal fluid, synovial fluid, and other biological fluids, including extracts of tissues such as liver tissue, muscle tissue, brain tissue, and heart tissue.

[0057] The term "biological matrix" is intended to include anything that cells contain or make up, such as bone, inclusion bodies, blood components, cell debris such as cell lysates, and the like.

[0058] The term "biological fluid," as used herein, is intended to include bodily fluids obtained from biological sources. Exemplary biological fluids include, but are not limited to, blood, plasma, urine, cerebrospinal fluid, mucosal tissue secretions, tears, interstitial fluid, saliva, sputum, synovial fluid, semen, and breast secretions. In specific embodiments, the biological fluid is a protein-containing or proteinaceous biological fluid. In yet other embodiments, the biological fluid is sputum, saliva, nipple aspirate, prostate massage secretions, pancreatic and biliary duct fluid, rectal mucus, or endocervical mucus. In certain embodiments, the biological fluid is not rectal mucus.

[0059] Specifically, marker carbohydrates or sugars in mucus or body fluid samples are detected by selective oxidation of glycoproteins in the mucus or body fluid sample using galactose oxidase or an equivalent oxidizing agent, which oxidizes the primary hydroxy groups of only the galactose moieties of the sugars present on the glycoprotein to aldehyde groups. The resulting aldehyde groups can then be visualized with Schiff's reagent, e.g., basic fuchsin, which produces a magenta color.

[0060] Galactose moiety marker carbohydrates or saccharides are selectively oxidized to aldehyde sugar moieties by galactose oxidase rapidly at room temperature, e.g., in less than about 15 minutes, e.g., about 5 to 10 minutes, and even more rapidly at elevated temperatures up to the inactivation temperature of the enzyme. When using this enzyme, any ratio of enzyme to substrate and vehicle suitable for enzyme activation known in the art can be used.

[0061] The oxidized sample, with or without first removing or inactivating the galactose oxidase, is then treated with a reagent that visualizes or allows visualization of the aldehyde sugar moieties thus generated, such as fuchsin, rosaniline, magenta, or other Schiff base decolorizing dyes.

[0062] The objects, features and advantages of the present invention are achieved in one aspect by providing a rapid, reliable with respect to false negatives, and commercially viable method for detecting the presence of a precancerous or cancerous condition in humans. The present invention employs a testing method comprising obtaining a mucus or body fluid sample, assaying the sample to detect the presence therein of at least one of the marker carbohydrates β-D-Gal-(1→3)-D-GalNAc, Fuc-α-1→2-Gal-β-(1→4)-Fuc-α-1→3-GlcNAc, Fuc-α-1→2-Gal-β-(1→4)-Fuc-α-1→3-GlcNAc-β-(1→3)-Gal-β-(1→4)-GlcNAc, and Fuc-α-1→2-Gal-β-(1→4)-Fuc-α-1→3-GlcNAc-β-(1→3)-Gal-β-(1→4)-Fuc-α-1→3-GlcNAc, and optionally diagnosing the presence and extent of precancer or cancer based on the amount of marker carbohydrate(s) detected in the mucus or body fluid.

[0063] Use of the galactose oxidase strip test for primary screening. This technique utilizes the ability of galactose oxidase to selectively oxidize both the C-6 hydroxyl group of galactose moieties of glycoproteins in mucus, e.g., galactose and n-acetylgalactosamine residues of β-D-Gal-(1→3)-D-GalNAc, to D-galactohexodialdose. The presence of aldehyde sugar moieties in this oxidized product provides evidence of the marker carbohydrate in the glycoprotein. Their presence can be detected using Schiff's reagents, e.g., basic fuchsin.

[0064] A sample is obtained and applied to the scored side of a membrane filter (e.g., Metricel membrane filter 0.45 μm, Gelman Sciences, Inc., Ann Arbor, Michigan 48106). An appropriate amount (depending on the size of the filter paper) of galactose oxidase is applied directly to the filter. After an incubation period of approximately 5–10 minutes, usually at room temperature, the membrane is washed with deionized water for 1 minute, then placed in Schiff's reagent for 1 minute, followed by a 1-minute wash with tap water. Excess water is shaken off, and the membrane is allowed to air dry or oven-dried. A bright magenta coloration of the mucosal smear upon complete drying indicates a positive test.

[0065] Typical results show no false negatives and far fewer false positives than conventional blood tests for colon cancer. Samples that produce negative results are then tested for glycoprotein content in accordance with the present invention by oxidation with periodate followed by retreatment with Schiff's reagent. The development of a magenta color confirms that the negative result is a biological negative and not a false negative due to sampling error. The entire procedure takes less than 30 minutes, allowing for convenient acquisition of a second sample from an individual whose first sample produces a false negative.

[0066] preparation Storage-stable Schiff's reagent solution Dissolve 1.0 g of basic fuchsin in 200.0 ml of hot distilled water and heat to boiling. Cool to 50°C, add 20.0 ml of 1N HCl, cool further, and add 1.0 g of sodium metabisulfate. Refrigerate in the dark until the solution turns straw-colored (approximately 48 hours). Next, add 5 g of activated charcoal, stir thoroughly, and remove the charcoal by filtration. The clear filtrate is Schiff's reagent, which is storage stable for many months, e.g., at least one year. Furthermore, the resulting magenta color is deeper than that obtained with conventionally prepared Schiff's reagent.

[0067] Galactose oxidase test strips The present disclosure provides test strips or membranes pre-embedded with galactose oxidase. A separate galactose oxidase solution is not required. The galactose oxidase in the test strip or membrane may be encapsulated and may be activated by moisture or contact with water. The amount of galactose oxidase in the test strip or membrane is not limited, so long as it is sufficient to oxidize the marker carbohydrate in the sample.

[0068] In one aspect, the test strips or membranes of the present disclosure can be designed to have different fluid uptake amounts to accommodate different amounts of marker carbohydrates in different mucus or body fluids. For example, a strip or membrane for testing rectal mucus can have a different fluid uptake than a strip or membrane for testing breast secretions. In another aspect, the amount of galactose oxidase in the test strip or membrane can be varied based on the concentration of the marker carbohydrate in different mucus or body fluids. In another aspect, the test strip or membrane further contains a dried culture medium that can activate galactose oxidase when water is added to the test strip or membrane.

[0069] Galactose oxidase test strip device The present disclosure provides a device comprising a test strip or membrane pre-embedded with galactose oxidase and a container containing a Schiff's reagent solution, wherein the Schiff's reagent solution is not initially in contact with the test strip or membrane and can be activated to contact the test strip or membrane after the marker carbohydrate has been oxidized by the galactose oxidase.

[0070] The mechanism for activating the Schiff's reagent solution (contacting it with the test strip or membrane after the marker carbohydrate has been oxidized by galactose oxidase) is not limited. In one embodiment, the mechanism can be a twist valve, a breakable barrier, or the like, where the Schiff's reagent solution contacts the test strip or membrane after opening the twist valve or breaking the barrier. In another embodiment, the mechanism for activating the Schiff's reagent solution is manually transferring the Schiff's reagent solution onto the test strip or membrane. In one embodiment, the device of the present invention includes all components in a single unit to minimize sampling or procedural errors.

[0071] In one embodiment, the galactose oxidase pre-embedded test strip or membrane further contains a dried culture medium, which can activate the galactose oxidase when water is added onto the test strip or membrane. In another embodiment, the pre-embedded galactose oxidase of the test strip or membrane is microencapsulated. In yet another embodiment, the pre-embedded galactose oxidase of the test strip or membrane can be activated by adding a few drops of water onto the test strip or membrane, or by adding mucus or body fluid onto the test strip or membrane.

[0072] In one aspect, the present disclosure relates to the use of the device to screen for cancerous or pre-cancerous conditions, including but not limited to the type of cancerous or pre-cancerous condition, given that marker carbohydrates are present in mucus or bodily fluids of cancerous or pre-cancerous conditions of, for example, the rectum, colon, blood, lymph nodes, stomach, kidney, gallbladder, prostate, testicles, breast, cervix, and ovary.

[0073] In certain embodiments, the galactose oxidase pre-embedded test strip or membrane further contains a pre-embedded Schiff's reagent, in which the pre-embedded galactose oxidase is activated prior to activation of the pre-embedded Schiff's reagent, such that reaction of the sample with galactose oxidase occurs prior to reaction with the Schiff's reagent.

[0074] Galactose oxidase test strips with embedded Schiff's reagent The present disclosure provides test strips or membranes pre-embedded with a Schiff's reagent for reaction of a sample with galactose oxidase. The Schiff's reagent in the test strip or membrane may be encapsulated and may be activated by swelling with water or contact with a pore-forming agent. The amount of Schiff's reagent in the test strip or membrane is not limited, so long as it is sufficient to react with marker carbohydrates in the sample after oxidation by galactose oxidase.

[0075] In one aspect, the test strips or membranes of the present disclosure can be designed to have different fluid uptake amounts to accommodate different amounts of marker carbohydrates in different mucus or body fluids. For example, a strip or membrane for testing rectal mucus can have a different fluid uptake than a strip or membrane for testing breast secretions. In another aspect, the amount of galactose oxidase in the test strip or membrane can be varied based on the concentration of the marker carbohydrate in different mucus or body fluids. In another aspect, the test strip or membrane further contains a dried culture medium that can activate galactose oxidase when water is added to the test strip or membrane.

[0076] Galactose oxidase test strip device with embedded Schiff's reagent The present disclosure provides a device comprising a test strip or membrane pre-embedded with a Schiff's reagent and a reservoir containing a galactose oxidase reagent solution, the galactose oxidase reagent solution not initially in contact with the test strip or membrane, wherein the sample and galactose oxidase reagent solution are added to the test strip and allowed to react prior to activation of the Schiff's reagent.

[0077] In one embodiment, the test strip or membrane pre-embedded with Schiff's reagent further contains a dried culture medium, which can be activated upon addition of a sample onto the test strip or membrane. In another embodiment, the pre-embedded Schiff's reagent of the test strip or membrane is microencapsulated. In yet another embodiment, the pre-embedded Schiff's reagent of the test strip or membrane can be activated by adding a few drops of water onto the test strip or membrane, or by adding mucus or body fluid onto the test strip or membrane. In another embodiment, the pre-embedded Schiff's reagent of the test strip can be activated by adding a pore-forming agent to the strip before, after, or simultaneously with the addition of the sample.

[0078] In one aspect, the present disclosure relates to the use of the device to screen for cancerous or pre-cancerous conditions, without limitation of type, by considering marker carbohydrates present in mucus or bodily fluids of cancerous or pre-cancerous conditions of, for example, the rectum, colon, blood, lymph nodes, stomach, kidney, gallbladder, prostate, testicles, breast, cervix, and ovary.

[0079] In certain embodiments, the test strip or membrane pre-embedded with Schiff's reagent further contains pre-embedded galactose oxidase, which is activated prior to activation of the Schiff's reagent, such that reaction of the sample with the galactose oxidase occurs prior to reaction with the Schiff's reagent.

[0080] Embedding and Encapsulation As discussed above, in certain embodiments, galactose oxidase is pre-embedded in the test strip or membrane. In some embodiments, galactose oxidase is directly embedded, absorbed, coated, or impregnated into the membrane or test strip. In certain embodiments, galactose oxidase is encapsulated before being embedded, absorbed, coated, or impregnated into the membrane or test strip.

[0081] In embodiments in which the galactose oxidase is encapsulated or microencapsulated, the encapsulating material may be made of one or more polymers to provide controlled, sustained, or immediate release of the reagent.

[0082] As discussed above, in certain embodiments, galactose oxidase is pre-embedded in the test strip or membrane. In some embodiments, the Schiff's reagent is directly embedded, absorbed, coated, or impregnated in the membrane or test strip. In certain embodiments, the Schiff's reagent is encapsulated before being embedded, absorbed, coated, or impregnated in the membrane or test strip.

[0083] In embodiments in which the Schiff's reagent is encapsulated or microencapsulated, the encapsulating material may be made of one or more polymers to provide controlled, sustained, or immediate release of the reagent.

[0084] In certain embodiments, the encapsulating material can be prepared according to the methods of Caruso (Phys. Chem. Chem. Phys., 2011,13, 4782), Sukhishvili (Chem. Mater., 2006, 18 (2), 328), US Patent Application Publication No. 20150164805, EP 2213280, or Schwendeman (J Control Release. 2014;196:60).Materials used to prepare encapsulating materials include emulsifiers, materials with different melting points, materials with different hydrophilic / lipophilic balances (HLB), phospholipids, fatty acids, plant sterols, sorbitan esters, beeswax, carnauba wax, paraffin, stearates, shellac, cellulose derivatives, maltodextrin, starch, gum, cellulose, polypyrrole, polycarbonate, cetyltrimethylammonium halides, silanes, diblock copolymers, triblock copolymers, such as poly(ethylene oxide)-block-poly(ethylene oxide) copolymers designated as P123 (PEO20PPO70PEO20) and F127 (PEO106PPO70-PEO106). (propylene oxide)-block-poly(ethylene oxide), alginate, chitosan, xanthan gum, polysaccharides, polysaccharide hydrogels, poly(lysine), poly(acrylic acid), agarose, PEG, poly(hydroxyethyl methacrylate-methyl methacrylate), poly(acrylic acid-co-acrylamide), poly(allylamine hydrochloride), poly(styrenesulfonic acid sodium salt), poly(diallyldimethylammonium chloride), poly(ethyleneimine), N-hydroxysuccinimide-PEG, maleimide-PEG conjugated phospholipids, paraffin, cyclodextrin, carboxymethylated polysaccharides, polycaprolactone, humic substances, Span 60, cholesterol, N-trimethylchitosan chloride, poly(methyl methacrylate), poly(2-hydroxyethyl methacrylate), poly(N-isopropylacrylamide), poly(N-isopropylmethacrylamide), poly(Nn-propylacrylamide), carboxymethyl cellulose, plastics, gold, molecular weight cut-off filters, organic / inorganic hybrid materials, metal oxides, plastics, silica including SBA-15 (PD 50 Å to 89 Å) and MCM-41, ceramics, clays, smectic clays, and niosomes.

[0085] In certain embodiments, the encapsulating material has or is later modified to present functional groups that can subsequently react with the membrane or test strip using standard synthetic reactions. For example, in certain embodiments, the encapsulating material may have aminoalkyl, ester, amide, or carbamate functional groups that can react with active groups on the membrane or test strip, resulting in immobilization of the reagent. Many standard coupling methods are known in the literature, including, but not limited to, March (Advanced Organic Chemistry, 3rd Edition, Wiley, New York, 1985), Odian (The Principles of Polymerization, 2nd Edition, Wiley, New York, 1981), and Bioconjugate Techniques (Hermanson, G.T., Bioconjugate Techniques; Academic Press: San Diego, 1996).

[0086] Other methods of encapsulation include, but are not limited to, those disclosed in U.S. Patent Application Publication No. 20160075976 or U.S. Patent No. 6,258,870, each of which is incorporated herein by reference.

[0087] polymer materials Thermoplastic polymers suitable for incorporation as an encapsulating material include, but are not limited to, polylactides, polyglycolides, polycaprolactones, polyanhydrides, polyamides, polyurethanes, polyesteramides, polyorthoesters, polydioxanones, polyacetals, polyketals, polycarbonates, polyorthocarbonates, polyphosphazenes, polyhydroxybutyrates, polyhydroxyvalerates, polyalkylene oxalates, polyalkylene succinates, poly(malic acid) polymers, polymaleic anhydrides, poly(methyl vinyl) ethers, poly(amino acids), chitin, chitosan, and copolymers, terpolymers, or combinations or mixtures of the above materials.

[0088] Examples of biodegradable polymers and oligomers suitable for use in the compositions and methods of the present invention include, but are not limited to, poly(lactide); poly(glycolide); poly(lactide-co-glycolide); poly(lactic acid); poly(glycolic acid); and poly(lactic-co-glycolic acid); poly(caprolactone); poly(malic acid); polyamides; polyanhydrides; polyamino acids; polyorthoesters; polyetheresters; polycyanoacrylates; polyphosphazines; polyphosphoesters; polyesteramides; polydioxanone; polyacetals; polyketals; polycarbonates; polyorthocarbonates; degradable polyurethanes; polyhydroxybutyrates; polyhydroxyvalerates; polyalkylene oxalates; polyalkylene succinates; chitin; chitosan; oxidized cellulose; and copolymers, terpolymers, blends, combinations, or mixtures of any of the above materials.

[0089] As used herein, "hydrophobic" refers to a polymer that is not substantially soluble in water. As used herein, "hydrophilic" refers to a polymer that may be water-soluble or that has an affinity for absorbing water, but that will draw water into the device, typically not when covalently bonded to a hydrophobic moiety as a copolymer.

[0090] Hydrophilic polymers suitable for use herein can be obtained from a variety of commercial, natural, or synthetic sources known in the art. Suitable hydrophilic polymers include polyanions, including anionic polysaccharides such as alginate; agarose; heparin; polyacrylates; polymethacrylates; ethylene maleic anhydride copolymer (half ester); carboxymethyl amylose; carboxymethyl cellulose; carboxymethyl dextran; carboxymethyl starch; carboxymethyl chitin / chitosan; carboxycellulose; 2,3-dicarboxycellulose; tricarboxycellulose; carboxy gum arabic; carboxy carrageenan; carboxy pectin; carboxy tragacanth gum; carboxy xanthan gum; carboxy guar gum; carboxy starch; pentosan polysaccharides. Examples of hydrophilic polymers include, but are not limited to, cellulose sulfate, curdlan, inositol hexasulfate, β-cyclodextrin sulfate, hyaluronic acid, chondroitin-6-sulfate, dermatan sulfate, dextran sulfate, heparin sulfate, carrageenan, polygalacturonate, polyphosphate, polyaldehyde-carbonate, poly-1-hydroxy-1-sulfonate-propene-2, copolystyrene maleate, mesoglycan, sulfopropylated polyvinyl alcohol, cellulose sulfate, protamine sulfate, phosphoguar gum, polyglutamic acid, polyaspartic acid, polyamino acids, and any derivatives or combinations thereof. Those skilled in the art will recognize other hydrophilic polymers that are also within the scope of the present invention.

[0091] Various water-soluble polymers suitable for use herein include, but are not limited to, poly(alkylene glycols), polyethylene glycol ("PEG"); propylene glycol; ethylene glycol / propylene glycol copolymers; carboxymethyl cellulose; dextran; polyvinyl alcohol ("PVOH"); polyvinylpyrrolidone; poly(alkyleneamines); poly(alkylene oxides); poly-1,3-dioxolane; poly-1,3,6-trioxane; ethylene / maleic anhydride copolymers; polyamino acids; poly(n-vinylpyrrolidone); polypropylene oxide / ethylene oxide copolymers; polyoxyethylated polyols; polyvinyl alcohol succinate; glycerin; ethylene oxide; propylene oxide; poloxamers; alkoxylated copolymers; water-soluble polyanions; and any derivatives or combinations thereof. In addition, the water-soluble polymer may be of any suitable molecular weight and may be branched or unbranched.

[0092] Depending on the desired softness and flexibility of the encapsulating material, the rate and extent of reagent release, degradation rate, etc., the amount and type of polymer can be varied to achieve the desired results.

[0093] Encapsulation Shell and Impregnation Layer In certain embodiments, the polymer becomes porous over time under certain conditions, thereby forming an encapsulating shell that can control release. The pores can be formed by swelling of the polymer shell or by dissolution or degradation of the shell.

[0094] The mass, volume and thickness of the polymer in each encapsulating shell / sphere can also be varied to tailor the release rate of the incorporated reagent.

[0095] The use of the term shell / sphere, as used herein, is not limiting with respect to the shape of the encapsulating material. The shape of the material is generally spherical, although conical shells, tubular shells, oval shells, cylindrical rods, and the like can be made and used. In certain embodiments, the material may be amorphous or irregular. In certain other embodiments, the encapsulating material may be coated or adhered to the surface of a scaffolding or chromatographic material. In such embodiments, the encapsulating material may take the shape of the material to which it adheres. In certain embodiments where the chromatographic or scaffolding material is porous, the encapsulating material may or may not penetrate the pores of the underlying material.

[0096] In certain other embodiments, a polymer can be impregnated with the reagent and coated onto the surface of a membrane or test strip. In such embodiments, the reagent is blended or mixed with the polymer such that the reagent is embedded, encapsulated, or impregnated into the polymer matrix. In such embodiments, the encapsulating material does not form a discreet encapsulating shell, but instead, the encapsulating material containing the reagent is coated as a layer onto the membrane or test strip and may optionally be covalently or ionically bonded thereto.

[0097] In certain embodiments, the encapsulating material may be wax, hydrogel, silicone rubber, or trehalose glass. While the use of hydrogel, silicone rubber, and trehalose glass are particularly suited to impregnating the polymeric material with reagents, any suitable polymer may be used in such embodiments.

[0098] In yet other embodiments, the reagents may be loaded into the pores of the porous membrane or test strip material, in which case the porous material may be coated with one or more polymeric encapsulating materials as described herein after the reagents have been loaded into the pores of the porous material.

[0099] Induction of release The encapsulated galactose oxidase reagent can be released from the encapsulating material by a variety of means known to those skilled in the art. In certain embodiments of the present invention, such release can be induced by contacting the encapsulating material with a pore-forming agent. In other embodiments of the present invention, release can be induced by a physical or chemical change. For example, but not limited to, release can be induced by a change in temperature, pH, ionic charge, counterion charge, or counterion atom. Similarly, release can be induced by contacting the encapsulating material with a solvent, including, but not limited to, an organic solvent, an aqueous solvent, an aliphatic solvent, an aromatic solvent, an oxygenated solvent, or a halogenated solvent, or water.

[0100] Generally, the release rate of the reagents will be determined by one of skill in the art based on the membrane or test strip used. In certain embodiments, the desired release rate is immediate, while in other embodiments, the release rate is controlled so that the reagents are released over a period of time. In certain embodiments, the reagents are released during the course of the test / workflow so that about 100% of the reagents are released by about 100% of the sample being introduced. In other embodiments, the reagents are released during the course of the test / workflow so that about 100% of the reagents are released by about 90% of the sample being introduced, about 80% of the sample being introduced, about 75% of the sample being introduced, about 50% of the sample being introduced, or about 25% of the sample being introduced.

[0101] Pore-forming agents Other additives can be advantageously used to further control the desired release rate of the reagent for a particular test / workflow protocol. For example, if the thermoplastic polymer liquid composition is too impermeable to water, a pore-forming agent can be added to generate additional pores in the matrix. Any suitable water-soluble material can be used as the pore-forming agent. These agents can be either soluble in the liquid composition or simply dispersed therein. These agents can dissolve, diffuse, or disperse from both the solidified polymer matrix and the formed polymer system, resulting in the generation of pores and microporous channels within the matrix and system. The amount of pore-forming agent in the composition (and, if appropriate, the size of dispersed particles of such pore-forming agent) directly affects the size and number of pores within the polymer system.

[0102] Other factors can also affect the size and / or diameter of the pores formed within the polymer system. For example, the amount of organic solvent and the rate at which the polymer system solidifies can both affect the porosity of the polymer system. While generally microporous matrices without a decomposed core and skin can be produced according to the present invention, typically, without the use of an additional pore-forming agent, polymer systems formed from the liquid composition will consist of a surface skin and an inner core. The surface skin is typically less porous, or even relatively non-porous, compared to the inner core. The inner core may have pores with diameters ranging from about 10 μm to 1000 μm. The use of an additional pore-forming agent results in the core and skin having substantially uniform pore sizes, both of which have pores in the range of 10 μm to 1000 μm.

[0103] The concentration of pore-forming agent relative to the thermoplastic polymer in the composition will vary depending on the degree of pore formation desired. Generally, this concentration will range from about 0.01 g to 1 g of pore-forming agent per gram of polymer. If the agent is soluble in the liquid composition, the mixing or dispersion of the agent in the liquid composition and its coagulation when the thermoplastic polymer solidifies will determine the size of the pores obtained when the agent dissolves from the polymer matrix.

[0104] Pore-forming agents include any pharmaceutically acceptable organic or inorganic substance that is substantially miscible with water and body fluids and dissipates from the formed matrix into aqueous media or body fluids, or into water-immiscible substances that rapidly degrade into water-soluble substances. The pore-forming agent may be soluble or insoluble in the polymer liquid composition of the present invention. It is further preferred that the pore-forming agent in the liquid composition of the present invention be miscible or dispersible in organic solvents to form a uniform mixture. Suitable pore-forming agents include, for example, sugars such as sucrose and dextrose, salts such as sodium chloride and sodium carbonate, and polymers such as hydroxypropyl cellulose, carboxymethyl cellulose, polyethylene glycol, and polyvinylpyrrolidone. The size and range of pores can be varied widely by varying the molecular weight and proportion of the pore-forming agent incorporated into the polymer system.

[0105] Other excipient materials, such as sucrose, dextrose, sodium chloride, sorbitol, lactose, polyethylene glycol, mannitol, fructose, polyvinylpyrrolidone, or suitable combinations thereof, can be added to the device to vary the porosity. Additionally, the active agent can be dispersed in oils (e.g., sesame oil, corn oil, vegetable oil), or mixtures thereof with phospholipids (e.g., lecithin), or medium-chain triglycerides (e.g., Miglyol 812) to obtain an oily suspension.

[0106] Test Method The present disclosure provides a method for rapidly detecting the expression of a marker carbohydrate in a subject using a test strip or membrane having a pre-embedded galactose oxidase and Schiff's reagent solution, or galactose oxidase and pre-embedded Schiff's reagent.

[0107] The present disclosure also provides a method for rapidly testing for cancerous or pre-cancerous conditions in humans using a test strip or membrane having a pre-embedded galactose oxidase and Schiff's reagent solution, or galactose oxidase and pre-embedded Schiff's reagent.

[0108] In one aspect, the method comprises the steps of applying mucus or body fluid to a test strip or membrane pre-embedded with galactose oxidase, oxidizing the marker carbohydrates in the mucus or body fluid containing the marker carbohydrates by reacting them with the galactose oxidase, and activating a reservoir containing a Schiff's reagent solution adjacent to the test strip or membrane to contact the test strip or membrane, wherein the Schiff's reagent solution is not initially in contact with the test strip or membrane.

[0109] In one aspect, the method comprises reacting the mucus or body fluid with galactose oxidase, contacting the oxidized sample with a test strip or membrane pre-embedded with Schiff's reagent, and activating the Schiff's reagent solution in the test strip or membrane and contacting it with the sample.

[0110] In some embodiments, the method includes one or more steps selected from applying water onto the test strip or membrane before applying the mucus or body fluid to the test strip or membrane, removing the mucus or body fluid by washing before activating the container containing the Schiff's reagent solution, rinsing the test strip or membrane with tap water after contacting the Schiff's reagent solution with the test strip or membrane, drying the test strip or membrane, and / or evaluating the color of the test strip or membrane.

[0111] In some embodiments, the method includes one or more steps selected from applying water onto the test strip or membrane before applying the mucus or body fluid to the test strip or membrane, removing the mucus or body fluid by washing before activating the Schiff's reagent, rinsing the test strip or membrane with tap water after contacting the Schiff's reagent with the sample, drying the test strip or membrane, and / or evaluating the color of the test strip or membrane.

[0112] In one embodiment, the time for oxidizing the marker carbohydrate in mucus or body fluid by reacting it with galactose oxidase is not particularly limited. For example, the oxidation time can be 3 to 30 minutes, 5 to 20 minutes, 7 to 15 minutes, or 8 to 12 minutes. In another embodiment, the time for contacting the Schiff's reagent solution with the test strip or membrane is not particularly limited. For example, the contact time can be 0.2 to 10 minutes, 0.5 to 5 minutes, 0.8 to 3 minutes, or 1 to 2 minutes.

[0113] In another embodiment, the mechanism for activating the Schiff's reagent solution (contacting it with the test strip or membrane after the marker carbohydrate has been oxidized by galactose oxidase) is not limited. In one embodiment, the mechanism can be a twist valve, a breakable barrier, or the like, where the Schiff's reagent solution contacts the test strip or membrane after opening the twist valve or breaking the barrier. The material of the breakable barrier is not limited, and the breakable barrier can be made of plastic, glass, or any material suitable for liquid containers.

[0114] Test kit The ready-to-use test kit is packaged in a conventional manner in a cardboard box and includes (a) a capped vial containing sufficient storage-stable basic fuchsin to saturate (b) twice, prepared according to the following recipe, (b) a strip of membrane filter (Metricel membrane filter 0.46 μm, Gelman Sciences, Inc., Ann Arbor, Michigan), (c) a storage-stable form of galactose oxidase impregnated into the membrane filter strip and contained in a sealed, capped bottle in the kit, in an amount sufficient to oxidize a marker carbohydrate in the sample, (d) periodic acid, and (e) a color chart for comparison with and interpretation of test results.

[0115] For field testing purposes, the kit contains multiple, e.g., 5, 10, 50, 100 or more strips of membrane filters with proportionally increasing amounts of galactose oxidase, buffer, and basic fuchsin solution.

[0116] The present disclosure provides a screening kit for rapidly detecting the expression of marker carbohydrates in a subject using a test strip or membrane pre-embedded with galactose oxidase and a Schiff's reagent solution.

[0117] The present disclosure further provides a screening kit for rapidly testing for a cancerous or precancerous condition in humans, the screening kit comprising a test strip or membrane pre-embedded with galactose oxidase and a container containing a solution of Schiff's reagent.

[0118] In one embodiment, the galactose oxidase pre-embedded test strip or membrane further contains a dried culture medium that can activate the galactose oxidase upon addition of water onto the test strip or membrane, hi another embodiment, the pre-embedded galactose oxidase of the test strip or membrane is microencapsulated.

[0119] The present disclosure also provides a screening kit for rapidly detecting the expression of marker carbohydrates in a subject using a test strip or membrane having galactose oxidase and a pre-embedded Schiff's reagent.

[0120] The present disclosure further provides a screening kit for rapidly testing for a cancerous or precancerous condition in humans, the screening kit comprising a test strip or membrane having galactose oxidase and a pre-embedded Schiff's reagent.

[0121] In one embodiment, the pre-embedded Schiff's reagent of the test strip or membrane is microencapsulated.

[0122] In one aspect, the present disclosure relates to the use of the test kit for screening for cancerous or precancerous conditions, without limitation of the type of cancerous or precancerous condition, considering marker carbohydrates present in body fluids of cancerous or precancerous conditions of, for example, the rectum, colon, blood, lymph nodes, stomach, kidney, gallbladder, prostate, testicles, breast, cervix, and ovary.

[0123] A population that does not show obvious signs or symptoms of cancer or precancerous conditions In certain embodiments, the present invention provides a tool for monitoring individuals who have been afflicted with cancer and treated for disease recurrence. Studies have shown that 32 of 53 colon cancer patients (60%) were positive for the marker after tumor resection. Of these 32 patients who remained positive after surgery, 5 (16%) experienced tumor recurrence within one year. Therefore, monitoring patients after surgery using markers and tests allows physicians to identify recurrent cancer early and manage it appropriately.

[0124] In another embodiment, 21 out of 28 individuals without signs and symptoms of lung cancer were found to be positive, of which 15 patients were identified to have lung cancer upon further workup. Therefore, monitoring individuals who test positive is very important in identifying the disease at a very early stage.

[0125] Comparative and correlative studies of the pathogenesis and pathogenesis of colon cancer in animal models and human disease have led to the conceptualization of the "field effect" theory and the identification of markers expressed early in carcinogenesis. Acquisition of this body of knowledge has led to the development of a simple rectal mucus test for colon cancer screening, which can be adapted to screen for other epithelial cancers. The marker, galactose-N-acetylgalactosamine (Gal-GalNAc), is expressed in rectal mucus from patients with colon cancer or precancerous lesions and is detected by a 1-minute color reaction after enzymatic oxidation (10 minutes). This test's high sensitivity, specificity, positive and negative predictive values, and cost-effectiveness make it an excellent tool in strategies for the early detection and, ultimately, control of colon cancer. This marker is similarly expressed in breast, lung, prostate, uterine, and pancreatic cancers, making it a potentially useful general cancer screening test. Its high accuracy (e.g., in contrast to fecal occult blood testing) and cost-effectiveness (e.g., compared to chest x-rays) will reduce the number of unnecessary colonoscopies and other expensive procedures, thereby reducing societal health care costs.

[0126] Gal-GalNAc expression was determined in 133 tissue samples from 81 breast, ovarian, pancreatic, gastric, and endometrial cancers, as well as 52 normal controls. None of the 52 normal tissues (except for 15 gastric cases) showed Gal-GalNAc expression. In contrast, 100% of breast (19 of 19), ovarian (15 of 15), and pancreatic (6 of 6) adenocarcinomas, as well as 94.1% (16 of 17) gastric carcinomas and 91.7% (11 of 12) uterine adenocarcinomas, expressed Gal-GalNAc. Furthermore, 62 of 65 prostate cancers expressed this marker (95.4% sensitivity), whereas none of the 35 benign prostatic hyperplasias showed expression (100% specificity). Normal epithelium and their secretions in the vicinity ("in the field") of cancers in the breast, bronchus, endometrium, and pancreatic duct also expressed Gal-GalNAc; in contrast, normal tissues obtained from cancer-free individuals were completely unreactive. Therefore, it was concluded that the tumor marker Gal-GalNAc, recognized by the galactose oxidase-Schiff sequence, is highly expressed not only by various adenocarcinomas, but also by seemingly normal-appearing epithelia and their secretions in the vicinity of, or even away from, cancers.

[0127] Adenocarcinoma is a mucus-producing tumor. Surprisingly, it has been found that the above markers are also expressed in the sputum of individuals with cancers that are not adenocarcinoma. These cancers, such as squamous cell carcinoma, small cell undifferentiated carcinoma (also known as oat cell carcinoma), and large cell undifferentiated carcinoma, do not produce mucus. The presence of the above markers in sputum samples from these non-mucus-secreting cancers would not be expected or understood by those skilled in the art.

[0128] In one study, 113 of 123 (91.9%) squamous cell carcinoma patients and 16 of 19 (84.2%) small cell undifferentiated carcinoma patients unexpectedly expressed these markers in sputum.

[0129] TIFF2022173151000001.tif41170

[0130] Similarly, in head and neck cancers, the proportion of non-mucin-producing squamous cell carcinomas is very high, as opposed to mucin-producing adenocarcinomas. The saliva of people with cancer or precancerous lesions expresses the above markers.

[0131] With respect to the kit embodiment of the present invention, it has surprisingly been discovered that, without adopting the commonly accepted method for preparing Schiff's reagents, in which the prepared reagent is mixed with activated charcoal and then refrigerated, solutions that are shelf-stable at room temperature for several months can be readily and reproducibly prepared. This method results in a stronger color reaction than that obtained with conventional Schiff's reagents. According to the method of the present invention, the Schiff's base is first refrigerated in the absence of activated charcoal, e.g., at about 0°C to 15°C, preferably about 0°C to 10°C, until its color fades to a straw color, e.g., for one, two, or more days, usually about 48 hours. After the solution fades to a straw color, it is treated with activated charcoal or a similar surfactant adsorbent, e.g., at room temperature for about several minutes to several hours or days, e.g., with stirring. After removing the charcoal, e.g., by filtration, the Schiff's reagent is filled into vials or bottles of a volume appropriate for the number of tests for which the kit is designed, e.g., 1, 5, 10, 50, 100, or more tests. Although the screening methods and kits disclosed herein are described using Schiff's reagents, it will be understood by those skilled in the art that in some embodiments, substances that function in substantially the same or similar manner as Schiff's reagents may also be used to stain, color, or otherwise label samples without departing from the scope of the present disclosure.

[0132] In another aspect of the invention, the detection method is used to field test a plurality of individuals for any cancerous or precancerous condition. When this step is performed, preferably only those samples that assay negative for the marker saccharide are further tested for any saccharides. Alternatively, each sample can be split into two, one assayed for the marker saccharide and the other assayed for any other saccharides that can be oxidized to an aldehyde sugar moiety, allowing for a simultaneous diagnostic procedure and false-negative test, thereby further reducing the time required before a biologically negative test result can be reported.

[0133] Furthermore, with regard to the speed aspect of the present invention, assays for marker sugars can be performed much more quickly than was evident from U.S. Patent No. 4,857,457 and the inventors' publication in Human Pathology, supra, and, as can be seen from the examples below, the entire testing procedure, including false negative testing, can be completed in less than 30 minutes, e.g., about 15-20 minutes, after sample collection, thereby allowing the examining physician or laboratory to report the results of the test (or collect another sample in the event of a negative biological result) before the individual leaves the testing site.

[0134] Marker sugars in a sample are detected by selective oxidation of glycoproteins in a mucus sample using galactose oxidase or an equivalent oxidizing agent, which oxidizes the primary hydroxyl groups of only the galactose moieties of the sugars present on the glycoprotein to aldehyde groups. The resulting aldehyde groups can then be visualized with a Schiff's reagent, such as basic fuchsin, which produces a magenta color. Samples can be rectal mucus, sputum, nipple aspirate, endocervical mucus, prostate massage secretions, pancreatic duct or biliary secretions.

[0135] Galactose moiety marker sugars are selectively oxidized to aldehyde sugar moieties by galactose oxidase rapidly at room temperature, e.g., in less than about 15 minutes, e.g., about 5 to 10 minutes, and even more rapidly at elevated temperatures up to the inactivation temperature of the enzyme. When using this enzyme, any ratio of enzyme to substrate and vehicle suitable for enzyme activation known in the art can be used.

[0136] The oxidized sample, with or without first removing or inactivating the galactose oxidase, is then treated with a reagent that visualizes or allows visualization of the aldehyde sugar moieties thus generated, such as fuchsin, rosaniline, magenta, or other Schiff base decolorizing dyes.

[0137] Marker carbohydrates can be assayed by selectively oxidizing the galactose moieties therein with galactose oxidase, followed by visualization of the resulting aldehyde sugars with basic fuchsin. Marker carbohydrates can also be assayed using, for example, an agglutination inhibition test using approximately stoichiometric amounts of peanut agglutinin, quince, wheat germ agglutinin, or other suitable lectin to detect the presence of biotinylated lectin and detect the complex upon reaction with avidin conjugated with another enzyme, such as horseradish peroxidase or alkaline phosphatase. Similarly, a marker can be detected using a primary antibody against the marker, followed by reaction with an enzyme-conjugated secondary antibody and dye detection according to standard ELISA assays.

[0138] Marker saccharides in biological fluid samples can also be detected by agglutination of sensitized beads.

[0139] The objects, features and advantages of the present invention are achieved in one aspect by providing a rapid, reliable with respect to false negatives, and commercially viable method for detecting the presence of a precancerous or cancerous condition in humans. The present invention employs a testing method comprising obtaining a sample of rectal mucus, assaying the sample to detect the presence therein of at least one of the marker carbohydrates β-D-Gal-(1→3)-D-GalNAc, Fuc-α-1→2-Gal-β-(1→4)-Fuc-α-1→3-GlcNAc, Fuc-α-1→2-Gal-β-(1→4)-Fuc-α-1→3-GlcNAc-β-(1→3)-Gal-β-(1→4)-GlcNAc, and Fuc-α-1→2-Gal-β-(1→4)-Fuc-α-1→3-GlcNAc-β-(1→3)-Gal-β-(1→4)-Fuc-α-1→3-GlcNAc, and optionally diagnosing the presence and extent of precancer or cancer based on the amount of the marker carbohydrate(s) detected in the rectal mucus. Except for the modifications thereto made by the present invention which make the testing procedure rapid, reliable with respect to false negatives, and commercially viable, the testing method is that disclosed in U.S. Pat. No. 4,857,457 and prior U.S. patent application Ser. No. 07 / 228,468, filed Aug. 5, 1988, the disclosures of which are incorporated herein by reference.

[0140] In one embodiment, the assay can be performed by reacting a body fluid with a precise amount of peanut agglutinin or other specific binding moiety such as a lectin (PNA, JAC, ACA, SNA, SRL, WGA, galectin-3, etc.) and / or any suitable antibody against a saccharide, followed by detecting the presence of unbound moiety. The reactive moiety can be immobilized on a water-insoluble support, such as a membrane filter or solid beads made of latex, plastic, glass, etc. To increase the sensitivity of the method, the reactive moiety can first be biotinylated by conventional methods.

[0141] The complex can be detected directly or indirectly by any of a variety of suitable techniques, e.g., immunologically, enzymatically, redox-based. Currently preferred is the formation of a complex with avidin conjugated to a suitable marker, e.g., fuchsin or other dyes, radioactive labels, fluorescent dyes such as fluorescein isothiocyanate or rhodamine B, or luminescent dyes such as luciferol, luminol, or biotin.

[0142] The presence of the disaccharide β-D-Gal-(1→3)-GalNAc is readily detected by agglutination of sensitized beads, such as glass, agarose, polystyrene, or latex, coated with PNA, JAC, SRL, WGA, ACA, SNA, galectin-3, etc. A preferred method for detecting the presence of the conjugate is by selectively oxidizing the galactose moiety of the sugar to an aldehyde disaccharide, e.g., using galactose oxidase, and thereby detecting the presence of the oxidized aldehyde sugar moiety.

[0143] In one embodiment, the assay test detects specific biochemical changes in subjects associated with, for example, colon cancer or precancerous conditions that result in the production of the disaccharide β-D-Gal-(1→3)-D-GalNAc, also known as the T or TF antigen, which is absent in the body fluids of normal individuals but present in the rectal mucus of individuals with at least some cancerous and precancerous conditions. Shamsuddin et al. have developed various techniques for detecting this sugar moiety in a simple, low-cost manner (although these are not as rapid as the methods used in the present invention, or allow the use of storage-stable fuchsin, or eliminate false negatives), which can be used to screen individuals for a wide range of colon diseases, including cancer.

[0144] Peanut agglutinin (PNA), a lectin, specifically binds to T antigens and causes agglutination of T antigen-activated RBCs. Taking advantage of these properties of PNA, a simple inhibition assay was first developed in which T antigens in a body fluid sample bind to PNA, preventing the PNA from reacting with RBCs and thus preventing red blood cell agglutination. This test is very simple and rapid. Using a microtiter plate, a large number of samples can be screened in a short time. The galactose oxidase test can be easily performed on a strip of membrane filter.

[0145] Since not all cancerous and precancerous conditions produce all of the marker carbohydrates identified herein in the same proportions, in one embodiment of the present invention, two or more of the assay tests described herein are used to test multiple samples of proteinaceous body fluids from individuals being screened for a particular cancerous or precancerous condition.

[0146] In addition to cancerous conditions, the tests used in the methods of the present invention can detect other diseases of the colon, including those that carry a high risk of cancer, such as polyps, fistulas, Crohn's disease, and ulcerative colitis.

[0147] In one embodiment, biological fluids are tested for marker carbohydrates as a first step screening test for the presence of any cancerous or precancerous condition. If the screening test is positive, other proteinaceous bodily secretions that are easily accessed by non-invasive methods are tested as a second step, a screening method that targets cancer or precancer in specific organs.

[0148] Additional assays can be developed using other properties such as immobilization of PNA or other suitable lectins to water-insoluble supports, immunological detection of glycoconjugates, or oxidation of the sugar moiety and detection with dyes, radiochemicals, etc. The use of antibodies against this sugar moiety in immunoassays allows for accurate estimation and monitoring of this moiety in rectal mucus and other body fluids. In immunoassays, antibodies can be tagged with radioactive fluorescent or other suitable labels for quantitative or semi-quantitative detection.

[0149] Avidin (67,000 MW), a glycoprotein, has an extremely high affinity for the vitamin biotin. Biotin molecules can be attached to various proteins (biotinylation), allowing avidin to be conjugated with various markers, such as enzymes, dyes, heavy metals, and radioisotopes. Avidin has four binding sites for biotin, allowing many biotin molecules to be incorporated onto a given protein. This amplification principle can be useful for detecting minute amounts (i.e., ng / mL or even pg / mL) of marker disaccharides in glycoproteins of rectal mucus obtained during digital rectal examination. Mucus glycoproteins containing specific disaccharides bind strongly to lectins immobilized on a solid phase. The matrix formed by the biotinylated lectin and enzyme-avidin D conjugate binds to the remaining disaccharides on the immobilized glycoprotein lectin, and the reaction is amplified by a suitable substrate.

[0150] Without further elaboration, it is believed that one skilled in the art can, using the preceding description, utilize the present invention to its fullest extent. The following preferred specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.

[0151] In the above and following examples, all temperatures are set forth uncorrected in degrees Celsius, and all parts and percentages are by weight unless otherwise specified. The entire disclosures of all applications, patents and publications, cited above and below, and of corresponding applications, if any, are hereby incorporated by reference.

[0152] In all tests described herein, sterile gloves must be worn, forceps, scissors and all work surfaces must be meticulously cleaned, and membrane filters must be handled with forceps and must not be contaminated (even by saliva). [Example]

[0153] Example 1 - A mucosal scraping sample is collected from a subject. The scraping sample is mixed with distilled water or reverse osmosis water and applied to a test strip pre-embedded with galactose oxidase at a concentration of 100 U / mL. The sample is left on the test strip for 10 minutes, after which the test strip is rinsed with additional distilled water. In a separate container, Schiff's reagent is activated in solution, and then the solution is added to the test strip. The sample is left in contact with Schiff's reagent for 1 minute, after which the test strip is rinsed with water and dried in open air or in an oven. Figure 2 illustrates the test sample processing. A color change (from white or colorless to magenta) indicates the presence of the carbohydrate marker.

[0154] Example 2 - A mucosal scraping sample is collected from a subject. The scraping sample is mixed with distilled water or reverse osmosis water and a solution of galactose oxidase is added at a concentration of 100 U / mL. The sample is left in contact with the galactose oxidase for 10 minutes, after which the sample is added to a test strip pre-embedded with Schiff's reagent. The test strip is rinsed with additional distilled water, after which the Schiff's reagent is activated and left in contact with the sample for 1 minute, after which the test strip is rinsed with water and dried in open air or in an oven. Figure 3 illustrates the test sample processing. A color change (from white or colorless to magenta) indicates the presence of the carbohydrate marker.

[0155] Example 3 - Following the procedure described above for the galactose oxidase strip test, rectal mucus was tested from 382 individuals who had known cancerous or precancerous conditions of the colon or other body sites, or who did not exhibit any obvious signs or symptoms of cancer or precancerous conditions. The term "other body sites" includes, but is not limited to, the uterus, cervix, kidneys, head and neck, ovaries, breasts, lymph nodes, stomach, testes, prostate, lungs, gallbladder, liver, and pancreas.

[0156] TIFF2022173151000002.tif53170

[0157] The data in the first row is the number of individuals tested positive / total number of individuals in each category. Note that only 5% of apparently healthy individuals elicited a positive response.

[0158] According to the present invention, mucus samples from each individual were obtained by digital rectal examination using a gloved finger lubricated with saline or other common lubricants used in such procedures. The mucus on the test finger was rubbed onto a protein-retaining membrane filter, reacted with galactose oxidase for 10 minutes at room temperature (25°C), washed with deionized distilled water, reacted with basic fuchsin for 1 minute, and then washed with tap water for 1 minute. The presence of a cancerous or precancerous condition was indicated by pink, magenta, or purple coloration of the sample area. Each sample that tested negative in this test (by the absence of magenta coloration) was then reacted with periodic acid for 5 minutes, washed with deionized distilled water, reacted again with basic fuchsin for 1 minute, and washed with tap water. A positive reaction, resulting in a pink, purple, or magenta color, indicates the actual presence of mucus glycoproteins but is negative for the presence of marker carbohydrates therein. The absence of pink, magenta, or purple coloration indicates a sampling error and the individual should be tested again with a new mucus sample.

[0159] In another independent study using the above procedure, the presence of 9 / 11 cases of stomach cancer and 4 / 4 cases of liver, gallbladder, common bile duct, and pancreatic cancer was indicated by positive tests using rectal mucus, galactose oxidase, and basic fuchsin; similarly, in another study, 12 / 18 patients with stomach, pancreatic, and liver cancer produced positive test results. In other studies, rectal mucus from individuals with ovarian, breast, or stomach cancer tested positive for marker proteins.

[0160] Contemplated equivalents of the method of the present invention are its use as a primary screening test using prostatic fluid, ejaculated semen, breast secretions, vaginal mucus, sputum, pancreatic and biliary secretions for the detection of cancerous or precancerous conditions of the prostate, breast and cervix, and / or ovaries, lungs, pancreas, liver and bile ducts, respectively, and the use of another galactose moiety-specific oxidizing agent in place of galactose oxidase.

[0161] Example 4 - Hemagglutination Inhibition Test - This standard hemagglutination inhibition test, such as that described in US Pat. No. 4,857,457, can be used to test for marker carbohydrates used in the present invention.

[0162] Typical results from this test are no false negatives and far fewer false positives than conventional fecal occult blood tests (about 1 / 3 vs. about 95). Testing samples for glycoprotein content in accordance with the present invention confirms the negative test results of appropriate mucus sampling procedures.

[0163] Example 5 - Latex Agglutination Test - In this test, 500 μL of suspended latex beads (15.8 μm diameter, Sigma Chemical Co., St. Louis, Mo.) are centrifuged at 3000 RPM for 15 seconds and the supernatant is decanted. 500 μg of lectin, e.g., PNA (Vector Laboratories Ltd., Burlingame, Calif.), is dissolved in 500 μL of carbonate buffer (pH 9.6) and added to the latex bead pellet. The pellet is resuspended and incubated at 25°C for 2 hours with occasional gentle shaking to resuspend the beads and allow for more uniform binding. After incubation, the sample is centrifuged at 3000 RPM for 15 seconds, the supernatant is decanted, and the pellet is resuspended in PBS (pH 7.4). All unbound PNA is washed away by repeating the previous step three times. The final pellet is resuspended and diluted 10-fold with PBS.

[0164] For testing mucus samples collected during a digital rectal examination, 10 μL of mucus in PBS is added to an equal volume of latex beads and placed on a glass slide. The slide is read after 5 minutes of incubation at 25° C. Agglutination of the beads, indicating the presence of the marker disaccharide, is read as a positive for cancer status, while the absence of agglutination after 5 minutes indicates the absence of the disaccharide and therefore a cancer-free status, or a sampling error. The latter possibility is eliminated by assaying the mucus sample for glycoprotein content according to the present invention.

[0165] Example 6 - Biotinylated Lectin Avidin-Enzyme Assay - Plant lectins, such as PNA, JAC, ACA, SNA, SRL, and WGA, are dissolved in carbonate buffer (pH 9) to a final concentration of 100 ng / ml and used to coat microtiter wells. 10 ng of lectin in 100 μL of buffer is added to each well and incubated at 37°C for 2 hours. After rinsing the wells with phosphate-buffered saline (PBS) at pH 7.4, 100 μL of test mucus (dissolved in PBS) is added to the microtiter wells and the mixture is incubated at 37°C for 1 hour. The wells are then washed three times with PBS, and 100 μL of unbound mucus is removed. Biotinylated lectin (1 μg / mL) is incubated for an additional hour at 37°C to bind to any remaining marker carbohydrates. The wells are washed three times with PBS to wash away unbound biotinylated lectin. Avidin-D-alkaline phosphatase (Vector Corporation, Burlingame, Calif.) is then added to the wells (100 μL / well, 50-fold dilution) and incubated for 1 hour at 37° C. After washing twice with PBS and three times with bicarbonate buffer (pH 9.8), the substrate p-nitrophenyl phosphate (1 mg / mL) is added to the wells (100 μL / well). The absorbance at 405 nm is read after 30 minutes of incubation at 37° C. Mucus from patients with known cancer yields positive results, while mucus from patients without cancer yields negative results.

[0166] In another example, samples were applied to nitrocellulose filters and treated with 3% BSA-PBS for 1 hour. The filters were then incubated with peroxidase-conjugated peanut lectin (PNA, Sigma, St. Louis, MO) at a concentration of 1 μg / ml in 0.1% BSA-PBS for 1 hour, with 10 mL of PNA solution applied to each filter. The filters were washed three times (10 minutes each) with 0.1% BSA-PBS and once with PBS alone. Color development was then performed at room temperature for 10 minutes using 0.6 mg / ml (PBS) chloronaphthol (Sigma) in the presence of 0.15% H2O2 as a substrate. A purple precipitate indicated positive staining. Staining was assessed using a computer-assisted image analysis program to assess PNA positivity.

[0167] Example 7 - Immunoassay using anti-Gal-GalNAc antibody - A monoclonal antibody against Gal-GalNAc (also known as TF antigen) is applied to a Nunc 96-well plate. After washing with TBS containing 0.1% Tween 20 and blocking with this solution containing 2% bovine serum albumin, 100 μL of sample is added and incubated for 4 hours at room temperature. After washing with TBS containing 0.1% Tween 20, biotinylated asialofetuin (a standard source of TF) is added. The washed wells are incubated with avidin / peroxidase complex, and peroxidase activity is measured by incubation in 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) liquid substrate system. Absorbance is read at 405 nm using an ELISA plate reader. The concentration of Gal-GalNAc(TF) is determined by interpolation of absorbance values ​​against a standard curve generated using different dilutions of biotinylated asialofetuin.

[0168] The above examples can be repeated with similar success by substituting the generically or specifically described reactants and / or operating conditions of the invention for those used in the above samples.

[0169] Incorporation by Reference The entire contents of all patents, published patent applications and other references cited herein are expressly incorporated herein by reference in their entirety.

[0170] equivalent Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments and methods described herein which equivalents are intended to be encompassed by the claims appended hereto.

[0171] It is understood that the detailed examples and embodiments described herein are provided by way of example only and for illustrative purposes only, and are not intended to limit the present disclosure in any way. Various modifications or variations therein will be suggested to those skilled in the art and are considered to be within the spirit and scope of this application and the scope of the appended claims. For example, the relative amounts of ingredients may be altered to optimize a desired effect, additional ingredients may be added, and / or similar ingredients may be substituted for one or more of the described ingredients. Further advantageous features and functionality associated with the systems, methods, and processes of the present disclosure will be apparent from the appended claims. Moreover, those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments of the disclosure described herein. Such equivalents are intended to be encompassed by the appended claims.

Claims

1. 1. A screening method for rapidly testing a subject for a cancerous or precancerous condition, wherein the subject does not exhibit any obvious signs or symptoms of the cancerous or precancerous condition, the method comprising: (b) A portion of a biological sample obtained from an individual is subjected to a step of isolating β-D-Gal-(1→3)-D-GalNAc, Fuc-α-1→2-Gal-β-(1→4)-Fuc-α-1→3-GlcNAc, Fuc-α-1→2-Gal-β-(1→4)-Fuc-α-1→3-GlcNAc-β-(1→3)-Gal-β-(1→4)-GlcNAc, and Fuc-α-1→2-Gal-β-(1→4)-Fuc-α-1→3-GlcNAc-β-(1→3)-Gal-β-(1→ 4) assaying for the presence of glycoproteins containing at least one carbohydrate selected from the group consisting of -Fuc-α-1→3-GlcNAc by briefly subjecting the sample to oxidizing conditions capable of selectively oxidizing only the cyclic sugar moiety of any marker carbohydrate present on glycoproteins in the sample at the ring carbon atom bearing its hydroxy group to form an aldehyde sugar moiety, followed by visualization of any aldehyde sugar group thereby formed with a Schiff base dye; (c) also assaying a portion of the biological sample for the presence of any glycoproteins in the sample by subjecting the sample to the oxidizing action of an oxidizing agent that oxidizes sugar moieties of any glycoproteins in the sample to aldehyde sugar moieties, followed by visualization of any aldehyde sugar moieties thereby produced, wherein the presence of aldehyde sugar moieties thereby formed confirms the adequacy of sampling and the absence of aldehyde sugar moieties thereby formed in the sample establishes that a negative test result was due to sampling error; (d) if the initial sample assays negative in steps (b) and (c), retesting the individual in the same manner using a new biological sample; a change in color of the Schiff base dye indicates that the asymptomatic patient has cancer or a precancerous condition; method.

2. The method described in claim 1, wherein the assay includes a step of adsorbing the biological sample to a water-insoluble substrate that captures proteins, and then a step of washing the substrate to remove non-immobilized components of the sample from the substrate.

3. The method described in claim 2, wherein the insoluble substrate is a membrane filter.

4. 2. The method of claim 1, wherein the sugar moieties of the marker carbohydrate are assayed by selectively oxidizing the glycoprotein to selectively oxidize the primary hydroxy group of any galactose moiety thereof to an aldehyde group, whereby the oxidized glycoprotein is subsequently assayed for oxidation of adjacent galactose moieties.

5. 5. The method of claim 4, wherein the galactose moieties are oxidized using galactose oxidase.

6. 5. The method of claim 4, performed simultaneously on multiple biological samples obtained from multiple individuals as part of a field screening for cancer.

7. The method of claim 5, wherein the oxidized adjacent galactose moieties are visualized with basic fuchsin.

8. 2. The method of claim 1, wherein step (c) is performed simultaneously with step (b) on a different portion of the same biological sample, and if negative results are obtained in both steps (b) and (c), steps (b) and (c) are repeated on another portion of the biological sample taken from the individual shortly thereafter.

9. 9. The method of claim 8, wherein any aldehyde sugar moieties in the sample further oxidized with periodate are visualized with basic fuchsin.

10. 2. The method of claim 1, wherein the biological sample is adsorbed to a water-insoluble substrate that captures proteins prior to assay, the substrate is then washed to remove non-immobilized components of the biological sample from the substrate, and the sample is assayed by selectively oxidizing any glycoproteins in the sample, thereby oxidizing the primary hydroxy groups of the galactose sugar moieties of any carbohydrates therein to aldehyde groups, whereby the oxidized galactose moieties are subsequently visualized with basic fuchsin, and samples that test negative in the assay are further oxidized with periodate, whereby the aldehyde sugar moieties in the oxidized samples are visualized with basic fuchsin.

11. The method of claim 10, wherein the insoluble substrate is a membrane filter and the galactose sugar moiety is oxidized using galactose oxidase.

12. The method of claim 10, wherein the agent for oxidizing the galactose moieties is present in an insoluble substrate or is applied directly to the insoluble substrate after the sample has been applied to the insoluble substrate.

13. 13. The method of claim 12, wherein the insoluble substrate is a membrane filter, the galactose sugar moieties are oxidized using galactose oxidase, and the galactose oxidase is applied directly to the membrane filter after the biological sample is applied to the membrane filter.

14. 1. A method for screening for a cancerous or precancerous condition in an organ other than the large intestine in a human, the human being exhibiting no obvious signs or symptoms of a cancerous or precancerous condition, the method comprising: detecting in said sample a proteinaceous secretion or proteinaceous fluid other than rectal mucus associated with said organ, the detection of β-D-Gal-(1→3)-D-GalNAc, Fuc-α-1→2-Gal-β-(1→4)-Fuc-α-1→3-GlcNAc in said sample; , Fuc-α-1→2-Gal-β-(1→4)-Fuc-α-1→3-GlcNAc-β-(1→3)-Gal-β-(1→4)-GlcNAc, and Fuc-α-1→2-Gal-β-(1→4)-Fuc-α-1→3-GlcNAc-β-(1→3)-Gal-β-(1→4)-Fuc-α-1→3-GlcNAc.

15. 15. The method of claim 14, wherein the proteinaceous secretions are pre-adsorbed onto a water-insoluble protein-capturing substrate, the substrate is subsequently washed to remove non-immobilized components of the sample from the substrate, the sample is assayed for marker carbohydrates by selectively oxidizing any glycoproteins in the sample using galactose oxidase to selectively oxidize the primary hydroxy groups of any galactose sugar moieties of any marker carbohydrates therein to aldehyde groups, whereby the oxidized galactose moieties are subsequently visualized with basic fuchsin, and a portion of the sample that tests negative in the assay is further oxidized with periodate, whereby the aldehyde sugar moieties in the oxidized sample are visualized with basic fuchsin.

16. 16. The method of claim 15, wherein the portion of the sample of proteinaceous secretion that is oxidized with periodate is a different portion of the same sample that is oxidized with galactose oxidase, and is oxidized simultaneously therewith.

17. 15. The method of claim 14, wherein the human is a female and the sample is vaginal or endocervical mucus.

18. 15. The method of claim 14, wherein the human is a female and the sample is nipple aspirate fluid.

19. 15. The method of claim 14, wherein the human is a male and the sample is prostatic secretion or semen.

20. The method of claim 14, wherein the sample is sputum.

21. The method of claim 14, wherein the sample is saliva.

22. 15. The method of claim 14, wherein the sample is bile duct fluid or pancreatic duct aspirate.

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