CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 62/825,848 filed March 29, 2019, which is incorporated herein by reference.

STATEMENT OF FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under grant numbers CA 152756 and CA 88130 awarded by the National Institutes of Health. The government has certain rights in the invention.

SEQUENCE LISTING

[0003] The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on March 13, 2020, is named CEMIP patent_ST25.txt and is 3,194 bytes in size.

TECHNICAL FIELD

[0004] This invention now describes methods for detecting cancer biomarker proteins or their fragments in a cell free plasma fraction of a blood sample.

BACKGROUND

[0005] Gastrointestinal cancers affect millions of patients per year. For example, over 15,000 new cases of esophageal cancer were diagnosed in 2010, and there were nearly as many deaths from this cancer alone. Similarly, about 21,000 new cases of stomach cancer were diagnosed in 2010, and over 10,000 deaths resulted from stomach cancer. The occurrence of colorectal cancer (i.e., cancer of the colon or rectum) is even higher. Approximately 40% of individuals with colorectal cancer die. As with other cancers, these rates can be decreased by improved methods for diagnosis. Although methods for detecting colorectal cancer exist, the methods are not ideal. Generally, a combination of endoscopy, isolation of cells (for example, via collection of cells/tissues from a fluid sample or from a tissue sample), and/or imaging technologies are used to identify cancerous cells and tumors. There are also a variety of specific tests conducted for colorectal cancer, but these have limitations. For example, colon cancer may be detected with digital rectal exams (i.e., manual probing of rectum by a physician), which are relatively inexpensive, but are unpleasant and can be inaccurate. Fecal occult blood testing (i.e., detection of blood in stool) is nonspecific because blood in the stool has multiple causes. Colonoscopy and sigmoidoscopy (i.e., direct examination of the colon with a flexible viewing instrument) are both uncomfortable for the patient and expensive. Double-contrast barium enema (i.e., taking X-rays of barium-filled colon) is also an expensive procedure, usually performed by a radiologist.

[0006] An increasingly common method for diagnosing cancer is the use of cancer biomarkers, such as protein cancer biomarkers. However, detection of protein cancer biomarkers in biological samples such as blood can be difficult due to the low levels of biomarkers and other complicating factors. Previous work by the inventors has demonstrated that the CEMIP protein is made by human colorectal cancer cells. They have also shown that in mice bearing tumor xenografts of human CCSP1 producing cells, that the cells secrete CEMIP protein into the blood circulation where it can be detected. However, a blood test for accurately detecting human CEMIP protein in a human blood fraction for use in detecting individuals with colon cancer had not yet been developed.

SUMMARY [0045] Methods of detecting a protein or protein fragment in the blood of a subject will now be described. These methods of specifically collecting and processing a blood sample enable detection of very low levels of the protein. In particular, the blood sample collected in a container containing an anti -coagulant buffer comprising a citrate buffer. A cell free plasma fraction is then obtained from the collected blood sample. Then an assay, such as an immunoassay can be performed on the cell free plasma faction enabling detection of the protein in the blood without cellular interference. Serial centrifugation may be used to obtain the cell free plasma fraction of the plasma fraction with antibodies reactive to the protein or protein fragment. CEMIP, a colon cancer biomarker is detectable in blood samples in patients having colorectal cancer by use of these methods

BRIEF DESCRIPTION OF DRAWINGS

[0007] The present invention may be more readily understood by reference to the following figures, wherein:

[0008] Figure 1 is an immunoprecipitation/western blot demonstrating detection of CEMIP in Media Collected from a Doxycycline-Inducible CEMIP- V5/6xHis Expressing HeLa Cell Line.

[0009] Figure 2 is an immunoprecipitation/western blot demonstrating detection of CEMIP in plasma collected from xenograft athymic mice implanted with HeLa A11-4 cells.

[0010] Figures 3 A and 3B are Western blot (3 A) and Immunoprecipitation/Western blot (3B) images demonstrating detection of human CEMIP by anti-CEMIP antibodies IP5, IP6, and IP14.

[0011] Figure 4 is a schematic representation of tagged CEMIP structure and general region of ELISA antibody recognition.

[0012] Figure 5 is a graph demonstrating the results from testing CEMIP antibody pairs in sandwich ELISA against 10 ng/ml recombinant CEMIP. [0013] Figure 6 is a bar graph showing the results from testing CEMIP antibody pairs for detection of CEMIP.

[0014] Figure 7 is a graph showing results of a CEMIP ELISA of single spun Patient SERUM set A.

[0015] Figure 8 is a graph showing results of CEMIP ELISA of single spun patient PLASMA set B.

[0016] Figures 9A-9C provide graphs show the results of CEMIP ELISA in double sup patient PLASMA set C. FIG9A is a scatter plot showing results of CEMIP ELISA of double spun patient PLASMA set C. FIG 9B-C show the statistical analysis of the scatter plot data.

[0017] Figures 10A-10C provide graphs showing results of CEMIP ELISA of double spun patient SERUM set C.

[0018] Figures 11A-B are scatter plot graphs providing a comparison of CEMIP levels in matched serum and plasma samples of set C.

[0019] Figures 12A and 12B provide a scatter plot graph and summary table showing the results of CEMIP ELISA of double spun patient plasma levels based on ethnicity.

[0020] Figure 13 provides a scatter plot showing results of CEMIP ELISA of double spun patient plasma levels based on gender.

[0021] Figures 14A-C show the results of CEMIP ELISA of double spun patient plasma levels in a set of samples also analyzed for methylated DNA markers. FIG 16A is a scatter plot showing results of CEMIP ELISA. FIG 16B-C show the statistical analysis of the scatter plot data.

[0022] Figures 15A and 15B provide a scatter plot (A) of percent VIM that is Methylated at 6+ CpGs in same sample subset as FIG 14 and a statistical analysis of the scatter plot data (B). [0023] Figures 16A and 16B provide a scatter plot of percent UnUpl46 that is Methylated at 6+ CpGs in Sample Subset also Analyzed for CEMIP Expression and VIM (A) and a statistical analysis of the scatter plot data (B).

[0024] Figure 17 is a scatter plot showing CEMIP plasma levels from different collection vacutainers, set D.

[0025] Figure 18 is scatter plot of CEMIP levels in different collection vacutainers, set D vs set C.

[0026] Figure 19 is a scatter plot of CEMIP levels in different collection vacutainers, set E comparing EDTA, Sodium Citrate and ACD-A (anticoagulant citrate dextrose solution A).

[0027] Figure 20 is a scatter plot of CEMIP levels of set E and set F in different collection vacutainers containing either EDTA, Sodium Citrate and ACD-A.

[0028] Figure 21 is a scatter plot of CEMIP levels of sets D, E and F in different collection vacutainers containing either EDTA, Sodium Citrate and ACD-A.

[0029] Figures 22A and 22B represents analysis of CEMIP levels in set G. FIG 22A is a scatter plot of CEMIP levels in set G in different collection vacutainers containing either EDTA, Sodium Citrate and ACD-A and a combination of EDTA/ACD-A or EDTA/Sodium Citrate. FIG 22B is a paired t-test statistical analysis of the scatter plot data.

[0030] Figures 23A and 23B represents analysis of CEMIP levels in set G with different dilution parameters. FIG 23A is a scatter plot of CEMIP levels in set G in different collection vacutainers containing either EDTA, Sodium Citrate and ACD-A and a combination of EDTA/ACD-A or EDTA/Sodium Citrate. FIG 23B is a paired t-test statistical analysis of the scatter plot data. [0031] Figures 24A and 24B represents analysis of CEMIP levels in samples spun one or twice during plasma collection. FIG 24A is a scatter plot of CEMIP levels under different anti coagulation butters and number of spins (lx or 2x) and FIG 24B is statistical analysis of the scatter plot data.

[0032] Figure 25 is a scatter plot of CEMIP levels when SPS and EDTA are added to vacutainer.

[0033] Figures 26A and 26B provide bar graphs represent CEMIP expression as a function of processing time after blood. (FIG 26A) and data expressed as percent of one hour time point (FIG 26B).

[0034] Figures 27A and 27B provide bar graphs represent CEMIP expression in a sample spiked with 60 ng/ml purified CEMIP as a function of processing time after blood (FIG 27A) and data expressed as percent of one hour time point (FIG 27B).

[0035] Figure 28 provides a flow chart of the steps involved in sample preparation.

[0036] Figure 29 provides a scatter plot comparison of CEMIP levels prepared according to the flow chart of FIG 28.

[0037] Figure 30 provides a scatter plot showing each data point graphed as a plot along with bars that denote the mean and standard errors of the mean (SEM) for the test protocol sample set.

[0038] Figure 31 provides a bar graph showing the mean and standard error of the mean for each test protocol.

[0039] Figure 32 provides a bar graph showing the mean and standard error of the mean for each test protocol with the mean of test protocol number 1 normalized to a value of 1.0.

DETAILED DESCRIPTION

[0040] The invention provides methods of detecting a cancer biomarker protein or fragment thereof in the blood of a subject. The steps of the method including providing a blood sample from the subject, the blood sample collected in a container containing an anti -coagulant buffer comprising a citrate buffer to form a first mixture; obtaining from the first mixture a cell free plasma fraction; and performing an immunoassay on the plasma fraction with antibodies reactive to the cancer biomarker protein or fragment thereof. A positive immunoassay reaction indicates the presence of the cancer biomarker protein or fragment thereof in the blood of the subject. Methods of diagnosing colorectal cancer in a subject by using the method to detect the protein CEMIP, and kits for carrying out such a diagnosis, are also provided.

Definitions

[0041] 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 these exemplary embodiments belong. The terminology used in the description herein is for describing particular exemplary embodiments only and is not intended to be limiting of the exemplary embodiments. As used in the specification and the appended claims, the singular forms“a,”“an,” and“the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.

[0042] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0043] The term "about" as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or 110%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

[0044] As used herein, the term "diagnosis" can encompass determining the likelihood that a subject will develop a disease, or the existence or nature of disease in a subject. The term diagnosis, as used herein also encompasses determining the severity and probable outcome of disease or episode of disease or prospect of recovery, which is generally referred to as prognosis). "Diagnosis" can also encompass diagnosis in the context of rational therapy, in which the diagnosis guides therapy, including initial selection of therapy, modification of therapy ( e.g ., adjustment of dose or dosage regimen), and the like.

[0045] As used herein, the term“prognosis” refers to a prediction of the probable course and outcome of a disease, or the likelihood of recovery from a disease. Prognosis is distinguished from diagnosis in that it is generally already known that the subject has the disease, although prognosis and diagnosis can be carried out simultaneously. In the case of a prognosis for colon cancer, the prognosis categorizes the relative severity of the colon cancer, which can be used to guide selection of appropriate therapy for the colon cancer.

[0046] As used herein, the terms "treatment," "treating," and the like, refer to obtaining a desired pharmacologic or physiologic effect. The effect may be therapeutic in terms of a partial or complete cure for a disease or an adverse effect attributable to the disease. "Treatment," as used herein, covers any treatment of a disease in a mammal, particularly in a human, and can include inhibiting the disease or condition, i.e., arresting its development; and relieving the disease, i.e., causing regression of the disease.

[0047] As used herein, the terms "peptide," "polypeptide" and "protein" are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least four amino acids, unless specified otherwise, and no limitation is placed on the maximum number of amino acids that can comprise the sequence of a protein or peptide. Polypeptides include any peptide or protein comprising four or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. "Polypeptides" include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. The polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.

[0048] The term antibody, as used herein and unless further limited, refers to single chain, two-chain, and multi-chain proteins and glycoproteins belonging to the classes of polyclonal, monoclonal, chimeric and hetero immunoglobulins; it also includes synthetic and genetically engineered variants of these immunoglobulins. The term "antibody fragment" includes Fab, Fab', F(ab')2, and Fv fragments, as well as any portion of an antibody having specificity toward a desired target epitope or epitopes.

[0049] Antibodies are grouped into classes, also referred to as isotypes, as determined genetically by the constant region. Human constant light chains are classified as kappa (CK) and lambda (CX) light chains. Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. The IgG class is the most commonly used for therapeutic purposes. In humans this class comprises subclasses IgGl, IgG2, IgG3, and IgG4. In mice this class comprises subclasses IgGl, IgG2a, IgG2b, IgG3. IgM has subclasses, including, but not limited to, IgMl and IgM2. IgA has several subclasses, including but not limited to IgAl and IgA2. Thus, "isotype" as used herein is meant any of the classes or subclasses of immunoglobulins defined by the chemical and antigenic characteristics of their constant regions. The known human immunoglobulin isotypes are IgGl, IgG2, IgG3, IgG4, IgAl, IgA2, IgMl, IgM2, IgD, and IgE.

[0050] The term antigen, as used herein, refers to a molecule or a portion of a molecule capable of being bound by an antibody which is additionally capable of inducing an animal to produce an antibody capable of binding to an epitope of that antigen. An antigen can have one or more than one epitope. The specific reaction referred to above is meant to indicate that the antigen will react, in a highly selective manner, with its corresponding antibody and not with the multitude of other antibodies which can be evoked by other antigens.

[0051] The term epitope, as used herein, refers to that portion of any molecule capable of being recognized by, and bound by, an antibody. In general, epitopes consist of chemically active surface groupings of molecules, for example, amino acids or sugar side chains, and have specific three- dimensional structural characteristics as well as specific charge characteristics. The epitopes of interest for the present invention are epitopes comprising amino acids.

[0052] The term monoclonal antibody, as used herein, refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and/or bind the same epitope, except for possible variants that may arise during production of the monoclonal antibody, such variants generally being present in minor amounts. In contrast to polyclonal antibody preparations that typically include different antibodies directed against different epitopes, each monoclonal antibody is directed against a single epitope on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they are uncontaminated by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies.

[0053] As used herein, the phrase "specifically binds" refers to antibody binding to a target structure, wherein the antibody binds a target structure, or subunit thereof, but does not bind to a biological molecule that is not a target structure. Antibodies that specifically bind to a target structure, or subunit thereof, do not cross-react with biological molecules that are outside the target structure family. An antibody specific for CEMIP can be an antibody or antibody fragment capable of binding to that specific protein with a specific affinity of between 10 ⁸ M and 10 ¹¹ M. In some embodiments, an antibody or antibody fragment binds to a selected antigen with a specific affinity of greater than 10 ⁷ M, 10 ⁸ M, 10 ⁹ M, 10 ¹⁰ M, or 10 ^U M, between 10 ⁸ M - 10 ^U M, 10 ⁹ M - 10 ¹⁰ M, and 10 ¹⁰ M - 10 ¹¹ M. In a preferred aspect, specific activity is measured using a competitive binding assay as set forth in Ausubel FM, (1994). Current Protocols in Molecular Biology. Chichester: John Wiley and Sons ("Ausubel"), which is incorporated herein by reference.

Methods of Detecting a Cancer Biomarker Protein

[0054] In one aspect, the present invention provides a method of detecting a cancer biomarker protein or a fragment thereof in the blood of a subject. The method includes providing a blood sample from the subject, the blood sample being collected in a container containing an anti- coagulant buffer comprising a citrate buffer to form a first mixture; obtaining from the first mixture a cell free plasma fraction; and performing an immunoassay on the plasma fraction with antibodies that specifically bind to the cancer biomarker protein or a fragment thereof, wherein a positive immunoassay reaction indicates the cancer protein or a fragment thereof is present in the blood of the subject. The methods described herein can detect any cancer biomarker protein in blood. The methods are particularly beneficial for detecting cancer biomarker proteins in blood wherein detection of the cancer biomarker protein is otherwise undetectable due to interference from the cellular fraction of a blood sample or undetectable due to lysis of the cellular fraction of a blood sample.

[0055] The present invention provides a method of detecting a cancer biomarker protein or a fragment thereof. The cancer biomarker protein is one that can be found in the blood of some subjects, and in particular in the blood of subject having cancer. The main proteins found in blood are albumin and globulin; however, a wide variety of different proteins are also found in lower levels in the blood. The proteins can have a size from 50 to 1000 amino acids, with an average size of about 500 amino acids. In some embodiments, the proteins have a size from 100 to 900 amino acids, while in further embodiments the proteins have a size from 200 to 800 amino acids, 300 to 700 amino acids, or 400 to 600 amino acids.

[0056] Cancer biomarker proteins have been identified for a variety of different types of cancer. Specific examples of cancers that do not limit the definition of cancer can include melanoma, leukemia, astrocytoma, glioblastoma, retinoblastoma, lymphoma, glioma, Hodgkin's lymphoma, and chronic lymphocytic leukemia. Examples of organs and tissues that may be affected by various cancers include pancreas, breast, thyroid, ovary, uterus, testis, prostate, pituitary gland, adrenal gland, kidney, stomach, esophagus, rectum, small intestine, colon, liver, gall bladder, head and neck, tongue, mouth, eye and orbit, bone, joints, brain, nervous system, skin, blood, nasopharyngeal tissue, lung, larynx, urinary tract, cervix, vagina, exocrine glands, and endocrine glands. Alternatively, a cancer can be multicentric or of unknown primary site (CUPS).

[0057] A wide variety of protein cancer biomarkers are known to those skilled in the art. See Tan et al ., Mass Spectrom Rev., 31(5):583-605 (2012). For example, protein biomarkers are known for pancreatic cancer (Loosen et al., Tumour Biol., 39(6): 1010428317692231 (2017), lung cancer (H, Cho JY, Adv Clin Chem., 72: 107-70 (2015)), and ovarian cancer (Dochez et al ., J Ovarian Res. 2019, 27; 12(1) (2019)). Additional protein cancer biomarkers are being discovered using microarrays. Hu etal ., Proteomics Clin Appk, 9(l-2):98-l 10 (2015). In some embodiments, the protein is a biomarker for colon cancer. See Loktionov, World J Gastrointest Oncol., 12(2): 124-148 (2020); Grady WM, Markowitz SD, Dig Dis Sci., 60(3):762-72 (2015); or Das et al ., Biomed Pharmacother., 87:8-19 (2017). In some embodiments, the protein is cell migration- inducing and hyaluronan-binding protein precursor (CEMIP; also known as KIAA1199) (NP 061159.1, SEQ ID NO: 1). CEMIP is a secreted protein that his highly expressed by human colon cancers. (Fink et al. Oncotarget, 6(31):30500-15 (2015)).

[0058] In some embodiments, the method can be used to detect CEMIP analog proteins. Analogs may be made through substitution of conserved amino acids. A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta- branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a predicted non-essential amino acid residue in a CEMIP protein is preferably replaced with another amino acid residue from the same side chain family. Alternatively, in another embodiment, mutations can be introduced randomly along all or part of a CEMIP coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for CEMIP biological activity to identify mutants that retain activity. Following mutagenesis of the nucleotide sequence for CEMIP, the encoded protein can be expressed recombinantly and the activity of the protein can be determined.

[0059] A "non-essential" amino acid residue is a residue that can be altered from the wild-type sequence of CEMIP without abolishing or, more preferably, without substantially altering a biological activity, whereas an "essential" amino acid residue results in such a change. For example, amino acid residues that are conserved among the polypeptides of the present invention are predicted to be particularly unamenable to alteration.

[0060] In some embodiments, the method can be used to detect CEMIP homologs. By "homologs" what is meant is peptides closely corresponding to the sequences identified for CEMIP, including sequences from other mammalian species that are substantially homologous at the overall protein (i.e., mature protein) level to human CEMIP, so long as such homologous peptides retain their respective known activities. Various levels of homology, from 55% to 99%, are described herein. For example, preferred levels of homology include 75%, 85%, 90%, and 95%.

[0061] In some embodiments, the method includes detection of a fragment of a cancer biomarker protein. The protein fragment may include a protein that is less than the full length of the normal cancer biomarker protein, but still retains antigenic regions of the protein that can be detected by an immunoassay. Protein fragments include portions of proteins that lack about 10, 25, 50, 100, 150, 200, or 250 amino acids compared to the original protein. Preferably, the protein fragment is at least 10, 25, 50, 100, 150, 200 or 250 or more amino acids in length.

[0062] The cancer biomarker protein or a fragment thereof is detected in a blood sample from a subject. A“subject,” as used herein, can be any animal, and may also be referred to as the patient. Preferably the subject is a vertebrate animal, and more preferably the subject is a mammal, such as a domesticated farm animal (e.g., cow, horse, pig) or pet (e.g., dog, cat). In some embodiments, the subject is a human. In some embodiments, the subject is a subject having an increased risk of having cancer (e.g., colorectal cancer). A subject can have an increased risk due to a variety of factors, such as a genetic predisposition or exposure to environmental hazards, or can have an increased risk due to displaying one or more symptoms associated with cancer.

[0063] The method includes the step of providing a blood sample from the subject. The methods described herein are usable on any blood sample to be tested. A blood sample, as used herein, refers to a whole blood sample. The blood sample may be fresh or stored (e.g. blood or blood fraction stored in a blood bank). The blood sample may be expressly obtained for the assays of this invention or a sample obtained for another purpose which can be subsampled for use in the methods of this invention. The blood sample collection may include blood drawn into a container (e.g., a BD vacutainer blood drawing tube 369714), or equivalent blood drawing tube, or blood drawing tube with size proportionate equivalent amount of anti -coagulant buffer. In some embodiments, the blood sample is obtained from the subject using a 21 gauge or larger needle.

[0064] In some embodiments, the method includes discarding the first 2 or more milliliters of blood before collecting the sample for CEMIP analysis. In further embodiments, the method includes mixing the blood with the anticoagulant buffer by gentle inversion 3 to 5 or more times immediately after drawing the sample. In additional embodiments, the method includes maintaining the sample at room temperature from the time of blood draw from the patient through the time of preparing all plasma fractions and up to the time of freezing the final plasma fraction. In yet further embodiments, the method inclues addition to the blood drawing tube or to any or each step of plasma preparation of 1 mM PGE1 (Prostaglandin El), or of 0.1 - 10 mM or more PGE1, or of a biologically equivalent PGE1 analog or mimetic (e.g., misoprostol).

[0065] After a blood sample has been provided, the blood sample is collected in a container containing an anti -coagulant buffer comprising a citrate buffer to form a first mixture. The container can be any self-contained reaction container, such as a test tube or the wells included on a well plate such as that used in an enzyme-linked immunosorbent assay (ELISA). The anti coagulant buffer is a buffer designed to decrease coagulation of the blood, and is typically based on a citrate buffer. Preferably, the pH of the anticoagulant buffer is from 4.0 to 6.5, from 4.5 to 6.1, from 4.8 to 5.2, or from 4.9 to 5.1. Citrate buffers are prepared using a aqueous solution including a citric acid salt so that they include a mixture of a weak acid and its conjugate base. Generally the citrate buffer is comprised of anhydrous citric acid, sodium citrate, and other substituents.

[0066] In some embodiments, the citrate buffer includes anhydrous citric acid (CeHsCh) and sodium citrate dihydrate in a gm:gm ratio of 0.33 : 1.0, or from 0.16: 1.0 to 0.66: 1.0. In further embodiments, the citrate buffer comprises anhydrous citric acid, sodium citrate dihydrate and dextrose monohydrate, and the concentration of dextrose monohydrate is within the range of 1% and 4%. Other substituents that may be included in the anti-coagulant buffer include dextrose, dextrose monohydrate, or other formulation of dextrose. The concentration of dextrose monohydrate can be in the range from 1 to 4%, from 2 to 3%, from 2.3 to 2.7%, or from 2.4 to 2.5%. In some embodiments, the citrate buffer may also comprise theophylline, adenosine, dipyridamole, anhydrous citric acid, citric acid, sodium citrate dextrose, sodium phosphate and adenine.

[0067] In some embodiments, the citrate buffer comprises from about 2.5% to about 5% sodium citrate, or from about 3% to about 4% sodium citrate. In further embodiments, the buffer comprises 3.2% sodium citrate, or 3.8% sodium citrate, or between about 3.2%-3.8% sodium citrate, or between about 3.2%-4.0% sodium citrate, or between about 2.5-5.0% sodium citrate wherein the citrate buffer comprises from 0.2% to 1.5% anhydrous citric acid, 1% to 4% sodium citrate dihydrate, and dextrose monohydrate. In cases where sodium citrate is a part of the citrate buffer, the anticoagulant citrate buffer and blood is mixed 1 part buffer plus nine parts blood or the anticoagulant citrate buffer and blood is mixed 1 part buffer plus any of 1 part blood up to 10 parts blood.

[0068] In yet another embodiment, the citrate buffer comprises anhydrous citric acid, sodium citrate dihydrate and dextrose monohydrate, and the ratio of citrate buffenblood is an amount selected from the group consisting of: about 1.5:8.5, about 1.5: 17, about 1 :6 and about 1 :8. In some cases the citrate buffer comprises a ratio of anhydrous citric acid: sodium citrate dihydrate is between about 0.16: 1.0 and about 0.66: 1.0. In some embodiments, the citrate buffer is comprised of anhydrous citric acid, sodium citrate dihydrate, and dextrose monohydrate, or chemically equivalent formulation, and the sodium citrate dihydrate is between 1% -4%, or between 1.9%- 2.5%, or between 2.1-2.3%, and the anhydrous citric acid is between 0.2%-1.5%, or between 0.5%-0.9%, or between 0.7%-0.8%. [0069] In some embodiments, the citrate buffer may be comprised of 3.2% -3.8% sodium citrate, plus any of theophylline, and/or adenosine, and/or dipyridamol. The buffer may be comprised of sodium citrate, theophylline, adenosine, and dipyridamole, termed CTAD.

[0070] In some embodiments, citrate buffer comprises an acid-citrate-dextrose solution A (ACD-A). ACD-A is comprised of 7.3 gm of anhydrous citric acid (CeHsCb), 22 gm sodium citrate dihydrate, 24.5 gm dextrose monohydrate (C6Hi206*H20), and sufficient water to make 1000 ml. This provides a solution of 2.2% sodium citrate dehydrate, 0.73% anhydrous citric acid, and 2.45% dextrose monohydrate. The ACD-A solution is at pH 4.98, or at pH 4.5-5.5. The ACD-A buffer and blood is mixed 1.5 part buffer plus 8.5 part blood or is mixed 1.5 part buffer plus any of 1 part blood up to 17 parts blood.

[0071] In other embodiments, the citrate buffer comprises an acid-citrate-dextrose solution B (ACD-B). ACD-B is comprised of 4.4 gm of anhydrous citric acid (CeHsCh), 13.2 gm sodium citrate dihydrate, 14.7 gm dextrose monohydrate (C6Hi206*H20), and sufficient water to make 1000 ml. This provides is a solution of 1.32% sodium citrate dehydrate, 0.44% anhydrous citric acid, and 1.47% dextrose monohydrate. The ACD-B solution is at pH 5.6, or at pH 5.1-6.1. The ACD-B butter and blood are mixed as 1 part buffer plus 6 parts blood or mixed 1 part buffer plus any of 1 part blood up to 8 parts blood.

[0072] The citrate buffer may comprise Citrate Phosphate Dextrose Adenine Solution, (CPDA-1) in which 63 mL of CPDA-1 solution is comprised of 2 g Dextrose (monohydrate), 1.66 g Sodium Citrate (dihydrate), 188 mg Citric Acid (anhydrous), 140 mg Monobasic Sodium Phosphate (monohydrate) and 17.3 mg Adenine USP.

[0073] The anti-coagulant buffer may comprise additional components in addition to the citrate buffer; however the anticoagulant should not contain EDTA in any form or SPS. EDTA refers to ethylenediaminetetraacetic acid, and K2EDTA refers to the potassium salt of EDTA (i.e., ethylenediaminetetraacetic acid dipotassium salt). SPS refers to Sodium Polyanetholesulfonate.

[0074] Generally, it is preferable that the time between collection the blood sample and preparation of the cell free plasma fraction is not greater than 72 hours. Of course, cell free plasma fraction production can take place immediately after the sample is drawn or can occur at any time between collection of the blood sample and 72 hours later.

[0075] After the first mixture has been prepared, a cell free plasma fraction is obtained from the first mixture (i.e., the mixture of blood and the anti-coagulant buffer). Plasma is the component of blood that holds the cells. It is about 95% water by volume, and contains important dissolved proteins, glucose, clotting factors, electrolytes, hormones, and oxygen. A cell free plasma fraction is one in which the cells normally present in blood have substantially all been removed. Preferably, the cell free plasma fraction is substantially free of evidence of hemolysis, such as a pink color or other positive heme indicator. In some embodiments, the sample is maintained at about room temperature until the immunoassay is performed.

[0076] The step of obtaining a cell free plasma fraction may include centrifugation. This centrifugation step may include more than one centrifugation process, or serial centrifugation. Centrifugation to separate plasma from the formed elements of the blood may include centrifugation at greater than 133 x g, at greater than 400 x g, greater than 800 x g, or greater than 1600 x g, centrifugation at 1650 x g. Centrifugation may occur for 5 minutes, for greater than 5 minutes, for 10 minutes, or for greater than 10 minutes to separate plasma from the formed elements of the blood.

[0077] In some cases, when two serial centrifugation steps are required, the first step is from about 5 minutes to about 20 minutes at from about 60 x g to about 5,000 x g and the second step from about 2 minutes to about 30 minutes at from about 200 x g to about 22,000 x g. Alternatively, the two serial centrifugation steps include a first step of about 10 minutes at about 130 x g, and a second step of about 20 minutes at about 460 x g.

[0078] The methods of preparation of a cell free plasma fraction may include processing the sample through two serial centrifugation step, the first centrifugation at lower (time x g-force) to separate the plasma portion from most of the cellular elements of the blood, followed by a second centrifugation at equal or increased (time x g-force) to remove any remaining platelet or cellular elements from the plasma. In some embodiments using a serial centrifugation process, the first centrifugation is from 5 minutes to 20 minutes at from 60 x g to 5000 x g and the second centrifugation is for a duration of 2 minutes to 30 minutes at from 200 x g to 22,000 x g. In particular, the first centrifugation step is for a duration of 10 minutes at 133 x g and the second centrifugation is for 20 minutes at 455 x g.

[0079] The method also includes the step of performing an immunoassay on the plasma fraction with antibodies that specifically bind to the cancer biomarker protein or a fragment thereof. A positive immunoassay reaction indicates the presence of the cancer biomarker protein or a fragment thereof in the blood of the subject. An immunoassay is a biochemical test that measures the presence or concentration of an antigenic substance (e.g., a cancer biomarker protein) in a solution through the use of an antibody that specifically binds to the antigenic substance. Immunoassays come in many different formats and variations. Types of immunoassays include competitive, homogenous immunoassays, competitive heterogeneous immunoassays, one-site noncompetitive immunoassays, and two-site, noncompetitive immunoassays. See Goldys, Ewa, “Fluorescence Applications in Biotechnology and Life Sciences,” Wiley-Blackwell. p. 311, 2012. [0080] In some embodiments, the immunoassay is a two-site, noncompetitive immunoassay. A two-site, noncompetitive immunoassay uses two antibodies; a capture antibody and a detection antibody, both of which specifically bind to the protein. The capture antibody functions to capture the protein from solution, while the detection antibody includes a detection label which allows the captured protein to be detected.

[0081] In some embodiments, the two-site noncompetitive immunoassay comprises the steps of:

a) providing an immobilized capture antibody on a solid support, wherein the capture antibody comprises an antibody that specifically binds to the cancer biomarker protein (e.g., CEMIP); b) incubating the cell free plasma fraction with the capture antibody;

c) washing the immobilized capture antibody to remove unbound cancer biomarker protein; d) incubating with a detection antibody;

e) washing to remove unbound antibody;

f) detecting the presence of the detection antibody, wherein, detection of the detection antibody is a positive immunoassay reaction indicating the presence of the cancer biomarker protein (e.g., CEMIP) in the blood sample.

[0082] In some embodiments, the cancer biomarker protein is CEMIP protein and the immunoassay uses antibodies that specifically bind to CEMIP.

[0083] The immunoassay can use polyclonal or monoclonal antibodies. In some embodiments, the antibodies of the immunoassay are monoclonal antibodies. These antibodies include the capture antibodies and the detection antibodies. In further embodiments, the antibodies are directed to specific antigenic regions of the cancer biomarker protein. For example, where the cancer biomarker protein is CEMIP, the antibodies can be directed to epitopes of CEMIP selected from the group consisting of: SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4. SEQ ID NO: 2 is amino acids 340-543 of CEMIP, SEQ ID NO: 3 is amino acids 29-151 of CEMIP, and SEQ ID NO: 4 is amino acids 205-235 of CEMIP.

[0084] The immunoassay methods also includes various wash steps that are carried out at the completion of several of the steps of the method. For example, wash steps are carried out after initial incubation of the biological sample with the capture antibody, after activating the fibrinogen, and after contacting the immobilized capture antibody with a detection antibody. The wash steps are carried out using an aqueous solution to remove excess sample, reagents, or antibody so that subsequent steps can be carried out without inference from these materials. Methods and conditions for wash steps in an immunoassay are known to those skilled in the art.

[0085] The detection antibody includes a detection label. Examples of detection labels include enzymes, radioisotopes, and fluorescent compounds. Methods for detecting the detection label vary depending on the nature of the detection label, and include, for example, detection of fluorescence, luminescence, chemiluminescence, absorbance, reflectance, transmittance, birefringence or refractive index (e.g., surface plasmon resonance, ellipsometry, a resonant mirror method, a gating coupler waveguide method or interferometry). Optical methods include microscopy (both confocal and non-confocal), imaging methods and non-imaging methods. Electrochemical methods include voltammetry and amperometry methods. Radio frequency methods include multipolar resonance spectroscopy. Detection can be either quantitative or qualitative.

Diagnosing Colorectal Cancer [0086] Another aspect of the invention provides a method of diagnosing colorectal cancer in a subject. The method of diagnosis includes providing a blood sample from the human patient, the blood sample collected in a container containing an anti-coagulant buffer comprising a citrate buffer to form a first mixture; obtaining from the first mixture a cell free plasma fraction; performing an immunoassay on the plasma fraction with antibodies that specifically binds to the colon cancer biomarker protein, wherein a positive immunoassay reaction indicates the presence of the colon cancer biomarker protein in the blood of the subject and indicates a positive diagnosis of colorectal cancer (CRC). The steps of detecting the colon cancer biomarker protein include all of the embodiments of the methods of detecting a protein described herein. In some embodiments, the colon cancer biomarker protein is CEMIP.

[0087] The term "colon" as used herein is intended to encompass the right colon (including the cecum), the transverse colon, the left colon, and the rectum. The terms "colorectal cancer" and "colon cancer" are used interchangeably herein to refer to any cancerous neoplasia of the colon, including the rectum.

[0088] In certain aspects, the invention relates to methods for determining whether a patient is likely or unlikely to have a colon neoplasia. A colon neoplasia is any cancerous or precancerous growth located in, or derived from, the colon. The colon is a portion of the intestinal tract that is roughly three feet in length, stretching from the end of the small intestine to the rectum. Viewed in cross section, the colon consists of four distinguishable layers arranged in concentric rings surrounding an interior space, termed the lumen, through which digested materials pass. In order, moving outward from the lumen, the layers are termed the mucosa, the submucosa, the muscularis propria and the subserosa. The mucosa includes the epithelial layer (cells adjacent to the lumen), the basement membrane, the lamina propria and the muscularis mucosae. In general, the "wall" of the colon is intended to refer to the submucosa and the layers outside of the submucosa. The "lining" is the mucosa.

[0089] Precancerous colon neoplasias are referred to as adenomas or adenomatous polyps. Adenomas are typically small mushroom-like or wart-like growths on the lining of the colon and do not invade into the wall of the colon. Adenomas may be visualized through a device such as a colonoscope or flexible sigmoidoscope. Several studies have shown that patients who undergo screening for and removal of adenomas have a decreased rate of mortality from colon cancer. For this and other reasons, it is generally accepted that adenomas are an obligate precursor for the vast majority of colon cancers.

[0090] When a colon neoplasia invades into the basement membrane of the colon, it is considered a colon cancer, as the term "colon cancer" is used herein. In describing colon cancers, this specification will generally follow the so-called "Dukes" colon cancer staging system. The characteristics that describe a cancer are generally of greater significance than the particular term used to describe a recognizable stage. The most widely used staging systems generally use at least one of the following characteristics for staging: the extent of tumor penetration into the colon wall, with greater penetration generally correlating with a more dangerous tumor; the extent of invasion of the tumor through the colon wall and into other neighboring tissues, with greater invasion generally correlating with a more dangerous tumor; the extent of invasion of the tumor into the regional lymph nodes, with greater invasion generally correlating with a more dangerous tumor; and the extent of metastatic invasion into more distant tissues, such as the liver, with greater metastatic invasion generally correlating with a more dangerous disease state.

[0091] In some embodiments, the method of diagnosing a subject for colon cancer also includes evaluating the status of an additional marker for CRC selected from the group consisting of: VIM or UnUpl46; combining a positive immunoassay reaction indicating the presence of CEMIP and a positive assay for the presence of the additional marker to diagnose CRC in the human patient.

[0092] In some embodiments, the method of diagnosis further includes the step of providing treatment for subject who have been identified as having colon cancer. Methods of cancer treatment include cryoablation, thermal ablation, radiotherapy, chemotherapy, radiofrequency ablation, electroporation, alcohol ablation, high intensity focused ultrasound, photodynamic therapy, administration of monoclonal antibodies, and administration of immunotoxins. Chemotherapeutic agents used for treatment of colon cancer include fluorouracil, capecitabine and oxaliplatin. The type of treatment provided can vary depending on the stage and severity of the colon cancer, as would be understood by those skilled in the art. For example, surgical removal of the cancer is typically recommended if the cancer is found at an early stage.

Kits for Detecting CEMIP

[0093] Another aspect of the invention provides a kit for determining the presence of the colon cancer biomarker CEMIP in a blood sample. The kit includes a capture anti-CEMIP antibody, a detection anti-CEMIP antibody; a container to which the capture anti-CEMIP antibody is bound; detection means for detecting the detection antibody; instructions to perform an immunoassay with the two anti-CEMIP antibodies and a cell free plasma fraction of the blood sample that has been obtained by mixing the blood sample with a citrate buffer and centrifugation of the blood and citrate buffer mixture; and evaluate a positive immunoassay reaction as indicating the presence of CEMIP in the blood sample. In some embodiments, at least one of the capture or detection antibodies is IP5 or IP6. Preferably, the kit also includes the components for providing the anti- coagulant buffer described herein. More preferably, the components of the anti-coagulant buffer are provided either as an aqueous buffer, or one that can be rapidly prepared by addition of an appropriate amount of water.

[0094] The kit also includes reagents, buffers, and the like for carrying out an immunoassay (e.g., ELISA), which are known to those of ordinary skill in the art. Kits for treatment include anti-CEMIP antibodies, or effective fragments thereof, together with a pharmaceutically acceptable carrier for administration of the antibodies. The antibody or fragment thereof that specifically binds to CEMIP can be any of the antibodies described herein. For example, in some embodiments the antibodies are monoclonal detection and capture antibodies.

[0095] The kits are typically provided in a package which contains all elements, optionally including instructions. Instructions may be in any form, including paper or digital. The instructions may be on the inside or the outside of the package. The instructions may be in the form of an internet address which provides the detailed manipulative or analytic techniques. The package may be divided so that components are not mixed until desired.

[0096] Components of the kits of the present invention may be in different physical states. For example, some components may be lyophilized and some in aqueous solution. Some may be frozen. Individual components may be separately packaged within the kit. Other useful tools for performing the methods of the invention or associated testing, therapy, or calibration may also be included in the kits, including buffers, enzymes, chemiluminescence reagents, PMAT reagents, gels, plates, detectable labels, vessels, etc. Kits may-include tools for collecting suitable samples, such as a 21 gauge blood collection needle.

[0097] Examples have been included to more clearly describe particular embodiments of the invention and its associated cost and operational advantages. However, there are a wide variety of other embodiments within the scope of the present invention, which should not be limited to the particular examples provided herein. The invention is not limited to the exact details shown and described, for variations obvious to one skilled in the art will be included within the invention defined by the claims.

EXAMPLES

[0098] Monoclonal antibodies against CEMIP were identified and were used to develop western blot assays, immunoprecipitation assays, and ELISA assays for CEMIP detection. However, initial studies comparing CEMIP protein levels in serum or in plasma of humans with and without colon cancer showed high background levels of CEMIP protein in the serum and plasma of control normal individuals, with substantial overlap in the levels of CEMIP protein detected in blood fractions of normal individuals and of individuals with colon cancer. The inventors hypothesized that CEMIP protein detected in the blood of normal individuals might be generated artifactually during the process of drawing blood or in preparing blood fractions of plasma and serum through CEMIP protein release from formed elements of the blood, and that this could be prevented through methods to minimize cellular activation during blood drawing and processing. They therefore worked to develop a method of handling and processing blood samples that would minimize the level of CEMIP generated and detected in blood or blood fractions from normal individuals, without interfering with the ability to detect CEMIP as assayed in simulated colon cancer samples in which recombinant CEMIP was spiked into blood samples from normal individuals. In the examples below we lay out the steps through which a method meeting these criteria has been successfully developed.

Example 1 : Detection of CEMIP protein secreted into in the cell culture media [0099] FIG 1 shows detection of CEMIP protein secreted into in the cell culture media from HeLa cell clone A11-4 that bears a doxy cy cline inducible expression vector encoding a CEMIP- V5/6xHis tagged construct. Cells are grown in the presence [Dox (+)] or absence [Dox (-)] of doxycycline and media was collected and analyzed for presence of secreted CEMIP protein by immunoprecipitation / western blot against the V5-tag. Lanes labeled Empty Pool 1 show analysis of control media collected from HeLa cells transfected with an empty expression vector. Also indicated is the volume of cell culture media analyzed in each assay. Molecular weights of 97 and 191 are indicated by bars and an arrow indicate the presence of CEMIP. Thus, secreted CEMIP is detectable by immunoprecipitation / western blot methods.

Example 2: Detection of CEMIP protein in the blood of mice bearing xenografts of CEMIP secreting clone A11-4

[00100] FIG 2 shows detection of CEMIP protein present in the blood of mice bearing xenografts of CEMIP secreting clone A11-4. Shown is the detection of V5 -epitope-tagged CEMIP protein in plasma collected from 6 athymic mice bearing xenografts of HeLa cell clone Al l-4 cells, and maintained on drinking water supplemented with doxycycline (lanes labeled Clone A11- 4, 1-6). Also shown is an assay of plasma harvested from 8 control mice bearing xenografts from HeLa cells transfected with an empty expression vector (lanes labeled Empty Pool 1, 1-8). Control Media I.P. shows detection of the ~150kDa CEMIP protein secreted into the cell culture media of doxycycline treated A11-4 cells. Marker denotes molecular weight marker of size indicated.

[00101] In this experiment athymic female nude mice, 4-6 weeks of age, were injected subcutaneously on each flank with 5 x 10 ⁶ T-REx™-HeLa cells expressing an inducible V5-tagged CEMIP or a control empty vector (clone Al l-4). Three weeks after injection, the regular water was replaced with doxycycline-containing water (750 pg/ml) and the water was changed twice weekly. Mice were sacrificed 7-9 weeks after injection and exsanguinated blood was collected into tubes containing 100 mM EDTA in order to prevent clotting. The tubes were spun for 10 min at 5,000 rpm (2,040 x g) at 4°C and the plasma was transferred to a new tube.

[00102] To enable immunodetection of CEMIP against the background of murine antibody present in mouse plasma, we developed a“direct” immunoassay in which V5-tagged CEMIP was immunoprecipitated by anti-V5 antibodies conjugated to beads, and then detected by western blot assay using anti-V5 antibodies directly conjugated to horseradish peroxidase. For detection of V5 tagged CEMIP in mouse plasma, 30 pL of agarose-conjugated anti-V5 antibody beads (Invitrogen, Carlsbad, CA) was added to the plasma and rocked overnight at 4°C. The next day the beads were washed 3 times with RIPA buffer containing protease inhibitors, and the entire sample was loaded onto a 4-12% Bis-Tris, SDS-PAGE gel for protein separation. Western blotting was performed using for a primary antibody a horseradish peroxidase-conjugated anti-V5 antibody (Invitrogen, Carlsbad, CA). The direct CEMIP-V5 assay was validated by showing it detected ~150kDa CEMIP protein secreted into the cell culture medium of doxy cy cline treated A11-4 cells but not in cell culture medium of cells maintained without doxy cy cline treatment.

[00103] These observations indicate the utility of developing a method for detection of native human CEMIP protein or its fragments in blood of human individuals.

Example 3 : Development of monoclonal antibodies against native human CEMIP

[00104] FIG 3 A shows testing of anti-CEMIP antibodies for Western blot detection of purified human V5/6xHis-tagged CEMIP (purified from media of Al l-4 cells). In this experiment antibodies tested are IP5, IP6, IP14, and PW-3. Positive detection is demonstrated for PW-3 and IP-14. Fink et al. described purification of CEMIP protein from Al l-4 cells and Western detection of purified CEMIP using monoclonal antibody PW-3 (Fink et al. 2015 Oncotarget).

[00105] FIG 3B shows testing of anti-CEMIP antibodies for detection of native CEMIP protein by immune-precipitation from cell culture media of the colorectal cancer cell line FET. Microarray analysis identifies FET cells as expressing high levels of CEMIP, and identifies colorectal cancer cell line RKO as not expressing CEMIP. CEMIP was immunoprecipitated using the monoclonal antibodies IP5, IP6, and IP14, then detected using PW-3 on subsequent Western blot. IP5, IP6 and IP14 each is active in immunoprecipitating native CEMIP.

[00106] FIG 4 shows mapping of regions within the CEMIP protein that are recognized by anti- CEMIP antibodies IP5, IP6, and IP14. Tagged fragments of CEMIP were expressed as recombinant proteins and then tested for recognition in immunoprecipitation and/or western blot assays. IP5 and IP6 were determined to recognized epitopes that map to between CEMIP amino acids 340-543 (SEQ ID NO: 2), and IP14 was determined to recognize an epitope mapping between CEMIP amino acids 29-151 (SEQ ID NO: 3),

[00107] To enable detecting CEMIP protein, or protein fragments, in human blood, anti-CEMIP monoclonal antibodies were tested in pairs for compatibility in CEMIP Elisa assays. The table shown in FIG 5 summarizes results of testing a panel of CEMIP monoclonal antibodies in capture and detection antibody pairs to detect recombinant CEMIP- V5 tagged protein at 10 ng/ml, with the OD of the resulting ELISA signal indicated on an increasing scale of (-), (+), (++), and (+++). This initial testing of different pairs of monoclonal antibodies for compatibility in CEMIP Elisa assays demonstrated that anti-CEMIP monoclonal antibodies IP5 and WB1 were active in certain combinations as a CEMIP capture antibody, that is that IP5 and WB 1 functioned well in capturing CEMIP protein onto antibody coated plates. The study additionally showed that antibodies IP6 and IP14 both functioned well in detecting CEMIP captured by either IP5 or WB1. Specifically, when IP5 was used as a CEMIP capture antibody, positive ELISA signals for detecting CEMIP were obtained with IP6 (++ reactivity) and IP14 (with + reactivity). Other active antibody combinations were also identified.

[00108] In the following examples, the detection antibody was biotinylated using Thermo Scientific EZ-Link™ Sulfo-NHS-Biotinylation Kit, catalogue #21425. In these examples, the CEMIP ELISA method is summarized as follows:

Coat plates with capture antibody;

Block; wash;

Antigen reaction: Add plasma sample and incubate; wash;

Detection Antibody: Add detection antibody and incubate; wash;

Detection: Add means to detect detection antibody.

[00109] More specifically, the CEMIP ELISA method may be described as:

[00110] 1. Coat a self-contained reaction container, such as an ELISA plate with 100 pL of capture antibody at a concentration of 2 pg/mL in a capture buffer. Shaking the ELISA plate is optional. Preferably, the ELISA plate is in contact with the capture antibody overnight, but preferably for at least 12 hours. Non-adhered antibody is removed by inverting and tapping plate. Tapping plate on papers towels is typically sufficient for non-adhered antibody removal, though other means of removing remaining liquid from the wells of the ELISA plate are encompassed by the invention. A standard 96 well ELISA plate is suggest, though the ELISA methods are usable in any desired format. The term well in this context is used to refer to an individual self-contained sample. Covering the plate with Parafilm ^® during incubation is an option. Incubation may preferably occur at 4°C. Preferably, antibody, IP5, IP13, or WB1 are selected as the coating antibody.

[00111] 2. Block with 200 pL of, for example, StartingBlock™ for 2 hours at RT on orbital shaker (120 rpm) followed by washing. StartingBlock™ Blocking Buffer is a single purified protein for fast blocking of western blots and ELISA assays available from ThermoFisher Scientific. It provides broad compatibility with a wide range of antibodies, antibody combinations, and other protein probing and assay systems. Any typical ELISA plate washing protocol may be applied to the plate. For example, the plate may be washed at least about three times with 300 pL new buffer

[00112] 3. Sample preparation, while plate is incubating with Block solution, Samples, such as plasma may be diluted 1 :5 into StartingBlock™ and kept on ice for up to lhr. Specifically, if a 48 pL of plasma is added to 192 pL of StartingBlock™ to create a 240 pL ELISA, then 2 duplicate 100 pL aliquots are available for testing.

[00113] 4. Antigen reaction: 100 pL/well of the diluted plasma sample is added to a well of the plate and then incubated 3-4 hours at RT (room temperature) on orbital shaker (120 rpm). This step is followed by washing at least about four times as directed above.

[00114] 5. Addition of Detection Antibody: 100 pL/well of a Biotinylated Antibody IP-14 Bio or IP-6 Bio at 0.4 pg/ml in StartingBlock™ buffer is added and the plate incubated for at least 12 hours to overnight at 4°C optionally without shaking. This step is followed by washing at least about four times as directed above.

[00115] 6. Detection of the detection antibody: Add 100 pL/well of Streptavidin-HRP to the well at a 1 : 1000 dilution in blocking buffer and incubate about 1 hour at RT on orbital shaker (120 rpm). This step is followed by washing at least about five times as directed above. Add 100 pL of TMB substrate to each well, incubate for about 15-30 minutes at RT, stop the reaction by adding 100 pL of 2M sulfuric acid to each well; and read at 450 nm using plate reader within about lhr of stopping reaction

[00116] The following buffers and reagents can be used for Sandwich ELISA for CEMIP. Capture/coating Buffer (pH 9.6) comprising 1.59 g sodium carbonate (Na2CCb) = 15 mM, 2.93 g sodium bicarbonate (NaHCCh) = 35 mM, 0.20 g sodium azide (NaN3). Dissolve in 900 ml water, adjust pH to 9.6 with HC1 and make up to 1 L. As an alternative, Carbonate-Bicarbonate Buffer can be purchased from Sigma: C3041-50CAP and prepared by adding one capsule to 100 ml water = 0.05M. Buffer can use for up to 1 week. Store in refrigerator. PBS (pH 7.4 no Mg ²⁺ or Ca ²⁺ ) is prepared by diluting lOxPBS (from Gibco) into distilled water

[00117] New Buffer comprises: lxPBS with 0.05% Tween-20, and a FINAL cone of 0.5M NaCl (pH 5.0). Prepare by adding 100 ml of lOxPBS to 750 ml of distilled water; adding 5.0 ml of a 10% Tween-20 (BioRad #1706531); adding 70 ml of 5M NaCl (note the 1 x PBS already makes the NaCl cone. 0.15M) and pH the buffer by adding the appropriate amount of acid to bring to pH 5.0 and bringing the final volume to 1 L with water

[00118] Blocking buffer may be StartingBlock™ T20 from ThermoFisher (Pierce: cat# 37539) TMB substrate may be 1-Step Ultra TMB-ELISA from Pierce: cat # 34028. ELISA plates may be Corning Inc., Costar 3590, 96-well EIA/RIA plates flat bottom from Fisher: cat # 07-200-35 or 96-well deep well (300 pi) plates may be ThermoScientific ABgene SuperPlate 96-well PCR Plate (AB-2400) . Streptavidin-HRP may be BD Biosciences, cat #: 554066. Anti-V5 Biotinylated (Used only in studies employing detection of a V5-tag) may be Serotec, cat #: MCA1360B. 5M NaCl (500ml) may be prepared by addition of 146.1 g of NaCl into 400 ml of water. Adjust the volume to 500 ml with H2O. Sterilize by autoclaving. Store the NaCl solution at room temperature. 4N Sulfuric Acid (for making 2N Sulfuric Acid to stop TMB reaction) may be purchased from Fisher Scientific, cat# SA818-1, and prepared by diluting 1 :2 with water.

[00119] FIG 6 shows specific results demonstrating successful ELISA detection of CEMIP using several of these specific antibody combinations. In this experiment 10 ng/ml of V5-tagged recombinant CEMIP protein is incubated on an ELISA plate coated with either no capture antibody, a negative control anti -HA antibody, a positive control anti-V5 antibody, or the IP5 anti- CEMIP monoclonal antibody. The plate is then interrogated with either a negative control no detection antibody, a positive control biotinylated anti-V5 antibody, or biotinylated detection antibody anti-CEMIP antibody IP6 or IP14. The results show that antibodies IP6 and IP14 successfully detect CEMIP protein when captured onto the ELISA plate either by the positive control anti-V5 antibody or by capture anti-CEMIP monoclonal antibody IP5. Biotinylated anti- V5 antibody also detects CEMIP when captured onto the plate by antibody IP5 and also to some extent when captured by anti-V5 antibody. This may indicate that CEMIP oligomerizes.

[00120] Table 1 summarizes studies mapping regions within the CEMIP protein that are recognized by anti-CEMIP antibodies that were active as capture or detection antibodies in CEMIP ELISA assays. Tagged fragments of CEMIP were expressed as recombinant proteins and then tested for recognition in immunoprecipitation and/or western blot assays. Sequences of regions to which antibody epitopes were mapped are provided for CEMIP amino acids 340-543 of SEQ ID NO: l, amino acids 29-151 (SEQ ID NO:3), and amino acids 205-235 (SEQ ID NO:4).

[00121] Table 1 : Mapping regions within the CEMIP protein

Example 4: CEMIP ELISA of single spun patient Serum and Plasma

[00122] FIG 7 shows the initial testing of human serum (Set A) using the CEMIP ELISA, using capture with IP5 and detection with IP6 (IP5/IP6) or using capture with IP5 and detection with IP14 (IP5/IP14). Serum was tested from normal subjects (n=20) and from colorectal cancer patients (n= 13). The median CEMIP level for all samples is 0.32 (IP5/IP6) or 0.30 (IP5/IP14) ng/100 pL serum. Data is presented as a scatter plot (Bars = Mean with STD) and as a box plot: (Whiskers = Min to Max value, box =25-75% range, line = median, + = mean). Interestingly, in these serum samples CEMIP values from colon cancer patients were actually a bit lower than in normal individuals, although the differences were not statistically significant. The ELISA was quantitated by performing parallel ELISA assays on a standard curve of dilutions of purified CEMIP protein. As a negative control we demonstrated that pre-incubating the plasma samples for 1 hour on ice with the corresponding CEMIP capture antibody abolished all of the ELISA signal (i.e. reduced the Elisa signal to the 0 value on the standard curve). (This negative control was performed by adding the capture antibody at 10 pg/ml into the preparation of plasma sample diluted in StartingBlock™). FIG 7 shows no significant difference in serum CEMIP levels between normal subjects and CRC patients.

[00123] FIG 8 shows the initial testing of human plasma (Set B) from blood drawn into an EDTA containing vacutainer tube with plasma separated off using a conventional method for plasma preparation in which blood was centrifuged at room temperature for 10 minutes at 3000 rpm (1650 x g) in a Centra GP8R centrifuge. The ELISA used capture with IP5 and detection with IP6 (IP5/IP6) or used capture with IP5 and detection with IP14 (IP5/IP14). Plasma was tested from normal subjects (n=20) and from colorectal cancer patients (n= 16). Median CEMIP level for all samples is 2.95 (IP5/IP6) or 2.47 (IP5/IP14) ng/100 pL PLASMA. Thus, plasma CEMIP levels were ~8-9 fold higher than in serum. Data is presented as a scatter plot (Bars = Mean with STD). Thus, the testing of plasma and not serum shows a trend towards increased CEMIP levels in CRC patient plasma. The ELISA was quantitated by performing parallel ELISA assays on a standard curve of dilutions of purified CEMIP protein. As a negative control we demonstrated that preincubating the plasma samples with the corresponding CEMIP capture antibody abolished all of the ELISA signal (i.e. reduced it to the 0 value on the standard curve). In these plasma samples, mean and median CEMIP levels were higher in colorectal cancer patients than in normal controls, with the difference approaching statistical significance (p=0.078 for IP5/IP6 combination and p=0.055 for the IP5/IP14 combination). However, there was still considerable overlap between the plasma CEMIP values measured in normal and in colon cancer patients.

Example 5: CEMIP ELISA of DOUBLE SPUN Patient PLASMA and SERUM (SET C)

[00124] It might be possible that plasma CEMIP values might be contaminated by cellular CEMIP derived from formed elements of the blood. A cell free plasma sample was obtained by performing two serial centrifugations. In the first centrifugation EDTA anti coagulated blood sample was centrifuged at room temperature for 10 minutes at 3000 rpm (1650 x g) in a Centra GP8R centrifuge. The plasma fraction was collected and transferred into 1.5 ml Eppendorf ^® microfuge tubes and then centrifuged a second time for 10 minutes at 13,500 rpm (19,357 x g) in a tabletop Eppendorf 5417R centrifuge at 4 °C. The cell free plasma supernate was then harvested, with any cellular elements remaining from the plasma having been trapped into the pellet.

[00125] FIG 9A-C shows analysis of cell free plasma prepared from EDTA anti coagulated blood samples and assayed using the IP5/IP6 CEMIP ELISA. Samples were tested from 39 normal individuals and 129 colorectal cancer individuals (19 stage 1, 28 stage 2, 19 stage 3, 63 stage 4). Scatter plots (FIG 9A) show individual sample values and bars show group mean and standard deviation. In this example, compared to samples from normal individuals, there was a statistically significant increase in mean CEMIP in each of stage I, II, III and IV colorectal cancer samples (FIG 9B-C). However, considerable overlap was still observed between the normal range and the range of samples from cancer cases.

[00126] FIG 10 shows assay of serum samples that were available from a subset of the cases of FIG 9 tested using the IP5/IP6 CEMIP ELISA. Samples were tested from 6 normal individuals and 21 colorectal cancer individuals (4 stage 1, 6 stage 2, 5 stage 3, 6 stage 4). Scatter plots (FIG 10 A) show individual sample values and bars show group mean and standard deviation. Again, CEMIP values in serum were much lower than in plasma, and no significant difference in serum CEMIP levels between normal subjects and CRC patients (FIG lOB-C).

[00127] FIG 11 shows direct comparison of CEMIP values in individuals from which both serum and matched cell free plasma were tested. FIG 11 A shows raw CEMIP values (symbols). Bars show group mean and standard deviation. FIG 1 IB graphs the ratio of CEMIP levels in plasma/serum for each individual. Median plasma levels are >3-fold higher than matched serum samples.

Example 6: No Difference in CEMIP Plasma Levels Based On Ethnicity or Gender [00128] FIG 12 shows that in the experiment of FIG 9, CEMIP levels in cell free plasma of normal and cancer patients do not significantly differ between Caucasians and African Americans (using the IP5/IP6 ELISA). A summary of the results is found in FIG 12B.

[00129] FIG 13 shows that in the experiment of FIG 9, CEMIP levels in cell free plasma of normal and cancer patients do not significantly differ between males and females (using the

IP5/IP6 ELISA).

Example 7: Sensitivity to Detect Early Stage CRC Combining Markers

[00130] Tables 2 and 3 shows results of analysis of cell free plasma prepared from EDTA anticoagulated blood samples (using the same protocol as in FIG 9) for a panel including CEMIP protein plus two methylated DNAs, methylated vimentin gene DNA (VIM) and methylated DNA at the UnUpl46 locus. 20 normal and 78 colorectal cancer samples were analyzed, including 9 stage I, 18 stage II, 9 stage 3 and 42 stage IV. When markers were analyzed at 100% specificity (Table 2), addition of CEMIP increased sensitivity for the panel to detect stage I and stage III colorectal cancers. CEMIP levels were assayed using the IP5/IP6 ELISA. Data was also analyzed based on 87% specificity (Table 3).

[00131] Table 2: Analysis based on 100% specificity

[00132] Table 3: Analysis based on 87% specificity

[00133] FIG 14 shows values of CEMIP assayed by IP5/IP6 ELISA in cell free plasma prepared from EDTA anti coagulated blood from 25 normal and 116 colorectal cancer cases also tested for methylated DNA markers VIM and UnUpl46. In FIG 14A, the plasma CEMIP levels as shown in a scatter plot. The samples included 18 stage 1, 26 stage II, 18 stage 3 and 54 stage IV. Compared to samples from normal individuals, there was a statistically significant increase in mean CEMIP in each of stage I, II, III and IV colorectal cancer samples tested. FIGs 14B-C summarize the statistical analysis of the scatter plot shown in FIG 14A

[00134] FIG 15 shows the distribution of values for methylated VIM DNA in the same sample set as FIG 15. Statistical analysis of the scatter plot of FIG 15A is shown in FIG 15B.

[00135] FIG 16 shows the distribution of values for methylated UnUpl46 DNA in same samples also assayed for CEMIP and methylated VIM DNA. Statistical analysis of the scatter plot of FIG 16A is shown in FIG 16B.

Example 8: Blood draw methodology

[00136] As CEMIP levels were highly different between serum and plasma, the type of anticoagulant into which blood samples were drawn was evaluated. [00137] FIG 19 shows measurement of CEMIP in cell free plasma prepared from 10 individuals each of whom had blood serially drawn into a vacutainer with K2EDTA anticoagulant (BD 367899), a vacutainer with Sodium Polyanetholesulfonate (abbreviation SPS) (BD#364960), and a vacutainer with 3.2% buffered sodium citrate (BD 369714). CEMIP was measured using the IP5/IP6 ELISA assay. Symbols indicate individual sample results with bars denoting group mean and standard deviation. CEMIP values were substantially higher in cell free plasma prepared from EDTA anti coagulated samples, suggesting the possibility that EDTA activates release of CEMIP from formed elements of the blood, and that drawing blood into 3.2% sodium citrate may protect from this effect. CEMIP levels were also low in cell free plasma prepared from blood drawn into SPS.

[00138] In this and in successive data (FIGs 17-29) the method of preparing cell free plasma was modified to minimize platelet activation as follows. In the first centrifugation, the anticoagulated blood sample was transferred to a 15 ml falcon tube and centrifuged for 10 min at 133 x g (800 rpm in a Benchtop Sorvall RTH-750 rotor). The plasma fraction was collected and transferred into 1.5 ml Eppendorf ^® microfuge tubes and then centrifuged a second time for 20 minutes at 2200 rpm (455 x g) in a tabletop Eppendorf 5424 with FA-45-24-11 rotor, at room temperature). The cell free plasma supernatant was then harvested, with any cellular elements remaining from the plasma having been trapped into the pellet.

[00139] FIG 18 displays CEMIP levels in cell free plasma from the cohort presented in FIG 17 alongside with the data from the separately studied cohort of individuals presented in FIG 9. Baseline CEMIP levels lower in blood collected in SPS and NaCitrate vacutainer tubes. CEMIP values in cell free plasma prepared from blood samples drawn into K2EDTA were lower when prepared by the method of FIG 17, suggesting that the lower g-forces of this method may reduce background release of CEMIP from formed elements of the blood into the K2EDTA anticoagulated plasma.

[00140] FIG 19 shows further exploration of effects on blood CEMIP determinations of drawing blood into different citrate based anticoagulants. The measurement of CEMIP by the IP5/IP6 ELISA in cell free plasma prepared from 7 individuals each of whom has blood sequentially drawn into a vacutainer with K2EDTA anticoagulant (BD 367899), a vacutainer with 3.2% buffered sodium citrate (BD 369714), and a vacutainer with anticoagulant citrate dextrose solution, solution A (ACD-A) (BD 364606). Symbols indicate individual sample results with bars denoting group mean and standard deviation. CEMIP values were substantially higher in cell free plasma prepared from EDTA anti coagulated samples that in cell free plasma prepared from 3.2 % sodium citrate or from ACD-A anti coagulated samples. However, CEMIP values were significantly lower in the cell free plasma prepared from ACD-A anti coagulated samples than from 3.2% sodium citrate anti coagulated samples.

[00141] It may be that the background levels of CEMIP in blood are due to release of CEMIP from platelets. We note that in the preparation of cell free plasma from blood drawn into 3.2% buffered sodium citrate, that the pH of plasma harvested from the first centrifugation step was in this and all other studies further acidified to 6.5 using 5 pL of 1M citric acid added to 1.5 ml of plasma, after which the sample was then processed by a second centrifugation for 20 minutes at 455 x g. No pH adjustments were made to plasma prepared from blood anticoagulated with EDTA or with ACD-A prior to these samples being processed by a second centrifugation step.

[00142] FIG 20 shows an expansion of the study cohort of FIG 19 to now compare measurement of CEMIP by IP5/IP6 ELISA in cell free plasma prepared from 13 individuals each of whom has blood serially drawn into drawn into a vacutainer with K2EDTA anticoagulant (BD 367899), and a vacutainer with 3.2% buffered sodium citrate (BD 369714), and a vacutainer with anticoagulant citrate dextrose solution, solution A (ACD-A) (BD 364606). Symbols indicate individual sample results with bars denoting group mean and standard deviation. CEMIP values remain substantially higher in cell free plasma prepared from EDTA anti coagulated samples that in cell free plasma prepared from 3.2 % sodium citrate or from ACD-A anti coagulated samples. However again, CEMIP values were significantly lower in the cell free plasma prepared from ACD-A anti coagulated samples than from 3.2% sodium citrate anticoagulated samples.

[00143] FIG 21 shows the raw data from the three studies summarized in FIGs 17, 19 and 20. Red symbols indicate individuals who donated repeat blood samples to multiple of the different studies (but who are shown only once in the data of FIG 20). FIG 20 indicates that CEMIP levels are highly reproducible in repeat samples from the same individuals.

[00144] FIG 22 shows assay of CEMIP levels by IP5/IP6 ELISA in cell free plasma prepared from a fresh cohort of 7 individuals from each of whom blood was serially drawn into drawn into a vacutainer with K2EDTA anticoagulant (BD 367899), and a vacutainer with 3.2% buffered sodium citrate (BD 369714), and a vacutainer with anticoagulant citrate dextrose solution, solution A (ACD-A) (BD 364606). In FIG 22A, symbols indicate individual sample results with bars denoting group mean and standard deviation. Statistical analysis of the scatter plot is presented in FIG 22B. Again CEMIP values are highest in cell free plasma prepared from EDTA anticoagulated blood samples and lowest in cell free plasma prepared from ACD-A anti coagulated blood samples. In addition, FIG 22A shows assay of CEMIP in cell free plasma prepared from EDTA anti coagulated blood, after this cell free plasma has then been transferred into a vacutainer containing 3.2% sodium citrate (denoted EDTA/NaCit), or into a vacutainer containing ACD-A (denoted EDTA/ ACD-A). Mixing EDTA plasma with ACD or NaCitrate additive DOES NOT deplete CEMIP protein signal. Values of CEMIP shown are corrected for the added dilution of the 0.5 ml sodium citrate or 1.5 ml ACD-A into the vacutainers into which the cell free plasma has been transferred. The data show that adding already prepared cell free plasma into sodium citrate or into ACD-A solution into does not lower measured CEMIP values. Thus, the lower values of CEMIP measured in cell free plasma prepared from blood samples anti coagulated with 3.2% sodium citrate or with ACD-A is not due to an effect of these anticoagulants on reducing CEMIP values in the plasma, but is likely due to an effect of these citrate buffers on preventing release of CEMIP from formed elements of the blood.

[00145] FIG 23 shows a repeat of the study of FIG 22. However, whereas in the data of FIG 24 samples were diluted into StartingBlock™ in Eppendorf ^® tubes in preparation for ELISA assay, in the data of FIG 23A samples were diluted into StartingBlock™ on 96-well ELISA plates. Statistical analysis of the scatter plot data is presented in FIG 23B. CEMIP values measure somewhat higher in the samples that were diluted in the 96-well ELISA plates, suggesting the potential for CEMIP to be lost on the walls of Eppendorf ^® tubes when diluted into StartingBlock™.

[00146] FIG 24 shows a head to head comparison of measuring CEMIP in cell free plasma versus in plasma in a cohort of individuals from whom has blood serially been drawn into a vacutainer with Sodium Polyanethole Sulfonate (abbreviation SPS) (BD#364960), a vacutainer with 3.2% buffered sodium citrate (BD 369714), and a vacutainer with anticoagulant citrate dextrose solution, solution A (ACD-A) (BD 364606). Each sample was drawn in duplicate, with one sample processed for preparation of plasma, using a single low speed centrifugation (10 min at 133xg; denoted lx) and a duplicate sample processed for preparation of cell free plasma by two successive centrifugation steps (10 min at 133 x g followed by re-centrifugation of the decanted plasma for 20 minutes at 455 x g; denoted 2x). CEMIP values were then determined in the multiple blood fractions from each individual using the IP5/IP6 ELISA assay (FIG 24A) and a statistical analysis of the data is shown in FIG 24B. For samples collected into ACD-A buffer, CEMIP levels were significantly lower in the cell free plasma than in the simple plasma preparation. CEMIP levels in cell free plasma prepared from ACD-A anticoagulated blood were also lower than levels in plasma or cell free plasma prepared from blood anticoagulated with 3.2% citrate. Cell free plasma from ACD-A anti coagulated blood may have the least CEMIP background derived from formed elements of the blood, and may thus give the most accurate measurement of circulating CEMIP blood levels in the individual. Symbols indicate the individual sample values, and bars indicate group mean and standard deviation.

[00147] FIG 25 shows assay of CEMIP levels by IP5/IP6 ELISA in cell free plasma prepared from a fresh cohort of 7 individuals each of whom has blood serially drawn into drawn into a vacutainer with K2EDTA anticoagulant (BD 367899), and a vacutainer with SPS (BD 364960). Symbols indicate individual sample results with bars denoting group mean and standard deviation. Again, CEMIP values are highest in cell free plasma prepared from EDTA anti coagulated blood samples and lowest in cell free plasma prepared from SPS anticoagulated blood samples. In addition, FIG 25 shows assay of CEMIP in cell free plasma prepared from EDTA anticoagulated blood, after this cell free plasma is then transferred into a vacutainer containing SPS (denoted EDTA/SPS). Values of CEMIP are also shown after correction for the added dilution of the 1.7 ml of SPS buffer in the vacutainers into which the cell free plasma was transferred (EDTA/SPS x DF). Symbols indicate the individual sample values, and bars indicate group mean and standard deviation. The data show that adding SPS solution into already prepared cell free plasma markedly lowers CEMIP values. Thus, the lower values of CEMIP measured in cell free plasma prepared from blood samples anti coagulated with SPS appears to be due to an effect of SPS on reducing CEMIP values in the plasma, perhaps by aggregating or destabilizing the CEMIP protein. Thus, unlike findings observed with citrate or ACD-A anti coagulated blood samples, (which did not affect CEMIP levels when added into cell free plasma), these data show that low values of CEMIP assayed in SPS anticoagulated blood samples maybe an artifact due to an interaction of SPS with CEMIP protein.

[00148] FIG 26 shows analysis of the stability of CEMIP protein determinations in 6 different blood samples collected in ACD-A anticoagulant and held at room temperature for various times before preparation of cell free plasma. CEMIP values were measured using the IP5/IP6 ELISA. CEMIP values steadily decline upon aging of the blood samples from 1 to 3 to 6 to 24 hours, with decline of mean CEMIP by 35% at 24 hours. FIG 26 A shows mean CEMIP values (and standard error) for the group of blood samples tested at each time point. FIG 26 indicates the relative stability of CEMIP values in assays of blood held at room temperature for up to 3 hours, with perhaps 13.5% decline at 6 hours, and 28.8% decline at 24 hours.

[00149] FIG 26B represents the data of FIG 26 showing decline in blood CEMIP for each sample relative to the samples’ value at 1 hour. This date demonstrated high stability of CEMIP values in assays of blood held at room temperature for up to 3 hours, with 9% decline at 6 hours, and 28% decline at 24 hours.

[00150] FIG 27A shows analysis of the stability of CEMIP protein when purified CEMIP is spiked into ACD-A anti coagulated blood samples from 7 different individuals, and then held at room temperature for various times prior to preparation of cell free plasma. CEMIP was spiked into the anti coagulated blood samples at a level of 60 ng/ml (6 ng/100 pL as shown on the Y-axis of the graphs). CEMIP values were measured using the IP5/IP6 ELISA. CEMIP values were not significantly different between 1 and 3 hours of sample incubation, but declined upon aging of the blood samples of from 6 to 24 hours prior to the preparation of cell free plasma, with decline of mean CEMIP by 22.6% at 24 hours. FIG 27A shows mean CEMIP values (and standard error) for the group of blood samples tested at each point during the time course.

[00151] FIG 27B represents the data of FIG 27A showing decline in CEMIP following spike- in of CEMIP into each blood sample expressed relative to the samples’ value at 1 hour. FIG 27B shows the decline in mean relative CEMIP values (and standard error) for the group of blood samples tested at each time point. In this analysis the level of the spiked in CEMIP proteins declines by approximately 8.1% at 6 hours and 22.6% at 24 hours.

[00152] FIG 28 shows the design of a study comparing different parameters for measuring CEMIP in blood, including comparing preparation of cell free plasma from blood collected into a tube with ACD-A anticoagulant buffer by using either: one low speed centrifugation at 800 rpm in a Benchtop Sorvall RTH-750 rotor (133 x g) for 10 minutes (designated 1L); two successive centrifugations, the first at 800 rpm in a Benchtop Sorvall RTH-750 rotor rpm (133 x g) for 10 minutes, followed by centrifuging the supernatant again at 2200 rpm in an Eppendorf centrifuge (5424) with a FA-45-23-11 rotor (455 x g) for 10 minutes (designated 2L); one high speed centrifugation at 2823 rpm in a Benchtop Sorvall RTH-750 rotor (1650 x g) for 10 minutes (designated 1H); or two successive centrifugations, the first at 2823 rpm in a Benchtop Sorvall RTH-750 rotor (1650 x g) for 10 minutes, followed by centrifuging the supernatant again at 14,350 rpm in an Eppendorf centrifuge (5424) with a FA-45-23-11 rotor (19,350 x g) for 10 minutes (designated 2H).

[00153] FIG 29 shows results of measuring CEMIP in blood fractions defined by the experimental design of FIG 28. Background CEMIP levels are highest in 1L type samples. Background is reduced in 2L type samples. Background is also reduced in 1H or 2H type samples that undergo a first high speed centrifugation at 1650 x g for 10 minutes. In one scenario, samples that undergo a first centrifugation at 133 x g for 10 minutes, a second centrifugation step is required to reduce CEMIP background; however, samples with low CEMIP background may alternatively be prepared using a single centrifugation step at higher speed such as that provides 1650 x g. FIG 29 also indicates that any appropriate type of 1.5 mL tube is usable in these assays. FIG 29 also indicates use of ACD-A has slightly less background that sodium citrate under conditions where sample is to be spun twice at low speeds. FIG 29 also indicates that at a low first speed, two spins are recommended whereas at high speed only one spin is necessary.

Example 9: CEMIP ELISA Studies: Testing alternate Spin Conditions and Temperature Effects

[00154] Experiments were conducted to compare the results from studying blood from healthy human volunteers, collected into ACD-A blood drawing tubes, by measuring CEMIP in cell free plasma prepared using three alternative protocols, which are described below. The first step was to obtain three ACD-A tubes from each patient.

Protocol 1: Preparation of cell free plasma using one high speed centrifugation

[00155] Sample sits up to 3 h at RT (room temperature)

[00156] First spin at RT at 1650 x g for 10 min in 15 ml conical (or vacutainer)

[00157] Transfer plasma (upper layer) to 1.5 ml microcentrifuge tubes

[00158] Second spin: at RT at 19357 x g for 10 min

[00159] Transfer plasma supernatant to 2 ml Coming tubes

Protocol 2: Preparation of cell free plasma using two centrifugations at room temperature [00160] Sample sits up to 4 h at RT

[00161] First spin at RT at 1,300 x g for 10 min in vacutainer

[00162] Transfer plasma (upper layer) to 15 ml conical tube

[00163] Second spin: RT at 1,300 x g for 10 min

[00164] Transfer plasma supernatant to 1.5 ml Eppendorf tubes

Protocol 3: Preparation of cell free plasma using two centrifugations at 4 °C

[00165] Sample sits up to 4 h at 4°C

[00166] First spin at 4 °C at 1,300 x g for 10 min in vacutainer

[00167] Transfer plasma (upper layer) to 15 ml conical tube

[00168] Second spin: 4 °C at 1,300 x g for 10 min

[00169] Transfer plasma supernatant to 1.5 ml Eppendorf tubes

[00170] Figures 30, 31, and 32 show the results of these experiments, with Fig. 30 showing each individual data point graphed as a dot plot along with the bars that denote the means and standard errors of the mean (SEM) for each test protocol set. Figure 31 shows the data as a bar graph of the mean and standard error of the mean for each test protocol, and with Fig. 32 showing the data as a bar graph of the mean and standard error of the mean for each test protocol with the mean of test protocol number 1 normalized to a value of 1.0.

[00171] Results of these studies show that preparing cell free plasma with two successive centrifuge steps, the first at 1300 x g for ten minutes and the second at 1300 x g for 10 minutes, yields equivalent results as when preparing cell free plasma using two successive centrifuge steps with the first at 1650 x g for 10 minutes and the second at 19,350 x g for 10 minutes. Results of these studies also show that background is lowest when all sample material is maintained at room temperature from the time that blood is drawn from the patient through the time of centrifugation to prepare cell free plasma, up until cell free plasma is frozen away.

[00172] A property of ACD-A buffer is that it prevents blood coagulation and platelet activation. This suggests that the method for preparing blood for assay of CEMIP may be further enhanced by additional steps to reduce or prevent platelet activation. These include any, either singly or in combination, of: drawing blood from the patient through a 21 gauge or larger needle; discarding the first 2 or more milliliters of blood before collecting the sample for CEMIP analysis; mixing the blood with the anticoagulant buffer by gentle inversion 3 to 5 or more times immediately after drawing the sample; maintaining the sample at room temperature (not 4 degrees) from the time of blood draw from the patient through the time of preparing all plasma fractions and up to the time of freezing the final plasma fraction; addition to the blood drawing tube or to any or each step of plasma preparation of 1 uM PGE1 (Prostaglandin El), or of 0.1 - 10 mM or more PGE1, or of a biologically equivalent PGE1 analog or mimetic (such as misoprostol).

Conclusions

[00173] The aim was to develop a blood test for accurately detecting cancer biomarker proteins (e.g., human CEMIP protein) in a human blood fraction that would have utility in detecting individuals with cancer. To this end, monoclonal antibodies against CEMIP were developed and used these to develop western blot assays, immunoprecipitation assays, and ELISA assays for CEMIP detection. The initial studies comparing CEMIP protein levels in serum or in plasma of humans with and without colon cancer showed high background levels of CEMIP protein in the serum and plasma of control normal individuals, with substantial overlap in the levels of CEMIP protein detected in blood fractions of normal individuals and of individuals with colon cancer. These observations led the inventors to hypothesize that CEMIP protein detected in the blood of normal individuals might be generated artifactually during the process of drawing blood or in preparing blood fractions of plasma and serum through the release of CEMIP from formed elements of the blood. To overcome this obstacle, a method of handling and processing blood samples was developed that successfully minimizes the level of CEMIP generated and detected in blood or blood fractions from normal individuals, without interfering with the ability to detect CEMIP as assayed in simulated colon cancer samples that were constructed by spiking recombinant CEMIP into blood samples from normal individuals. This new method provides the first ability to accurately measure CEMIP protein in blood, without interference from CEMIP artifactually released during blood processing. This invention should have utility in discriminating individuals with CEMIP associated diseases such as colon cancer from normal healthy persons.

[00174] The complete disclosure of all patents, patent applications, and publications, and electronically available material cited herein are incorporated by reference. The foregoing detailed description and examples have been given for clarity of understanding only. No unnecessary limitations are to be understood there from. The invention is not limited to the exact details shown and described, for variations obvious to one skilled in the art will be included within the invention defined by the claims.