WO2018011691A1

WO2018011691A1 - Competitive immunoassay methods

Info

Publication number: WO2018011691A1
Application number: PCT/IB2017/054127
Authority: WO
Inventors: Fabiyola SELVARAJ
Original assignee: Nestec S.A.
Priority date: 2016-07-12
Filing date: 2017-07-07
Publication date: 2018-01-18

Abstract

The invention provides assay methods and kits for detecting, measuring or quantitating the level of 7α-hydroxy-4-cholesten-3-one (7C4) in a biological sample from a subject, such as a human subject. In some embodiments, the human subject has a condition associated with bile acid malabsorption or diarrhea of unknown origin. The invention also provides isolated antibodies or antibody fragment thereof that specifically binds to 7α- hydroxy-4-cholesten-3-one (7C4) and have less than 1% cross-reactivity to one or more members selected from the group consisting of 7-ketocholesterol, 7α-hydroxycholesterol, and trihydroxycholestanoic acid.

Description

COMPETITIVE IMMUNOASSAY METHODS CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The current application claims priority to U.S. Provisional Patent Application No. 62/361,407, filed July 12, 2016, U.S. Provisional Patent Application No. 62/428,930, filed December 1, 2016, and U.S. Provisional Patent Application No. 62/429,025, filed December 1, 2016, the disclosures of which are hereby incorporated by reference in their entireties for all purposes.

BACKGROUND OF THE INVENTION

[0002] Bile acids (BA) are produced in the liver and have major roles in the absorption of lipids in the small intestine. Diarrhea can be produced when excess bile acids (BA) are present in the colon. This condition known as bile acid malabsorption (BAM), has been under recognized since the best diagnostic method, which is known as the ⁷⁵Se-homocholic acid taurine (⁷⁵SeHCAT) test, is not available in many countries.

[0003] Bile acid malabsorption (BAM) is one of the mechanisms underlying the pathophysiology of diarrhea associated with ileal disease, IBS-D and microscopic colitis. In fact, bile acid malabsorption is increasingly being appreciated as a cause of chronic functional diarrhea or IBS-D.

[0004] The ⁷⁵Se-homocholic acid taurine (⁷⁵SeHCAT) test is based on bile acid retention of a radiolabeled homolog of a natural bile acid taurocholate. A seven day retention of less than about 15% of ⁷⁵Se-homocholic acid taurine is abnormal. Other diagnostic tests include a fecal bile acid assay, a ¹⁴C-glycocholate breath test and stool tests. [0005] Failure of bile acid absorption by the distal ileum results in spillover of bile acids into the colon where the excess causes loose, watery stools and diarrhea. However, there is no convenient way to measure a marker indicative of BAM. What is needed in the art are new ways to diagnose BAM and to treat patients with bile acid sequestrates when the condition actually exits. The invention satisfies these and other needs. BRIEF SUMMARY OF THE INVENTION

[0006] In one embodiment, the invention provides an isolated antibody or antibody fragment thereof that specifically binds to 7a-hydroxy-4-cholesten-3-one (7C4) and has less than 1%, e.g., 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, or 0% cross- reactivity to one or more members selected from the group consisting or 7-ketocholesterol, 7a-hydroxycholesterol, and trihydroxycholestanoic acid.

[0007] In certain aspects, the antibody is a polyclonal antibody or a monoclonal antibody.

[0008] In certain aspects, the isolated antibody or antibody fragment thereof is a chimeric or a humanized antibody. [0009] In certain aspects, the antibody fragment is a Fab fragment, a Fab' fragment or F(ab)'2 fragment.

[0010] In certain aspects, the antibody or antibody fragment thereof is produced by the hybridoma cell line deposited under ATCC Accession No. PTA-123441, on AUG 10, 2016 and designated 25G9B1/F2. [0011] In certain aspects, the antibody or antibody fragment thereof is produced by the hybridoma cell line deposited under ATCC Accession No. PTA-123440, on AUG 10, 2016 and designated 23A7D1/F2.

[0012] In certain aspects, the antibody or antibody fragment thereof is produced by the hybridoma cell line deposited under ATCC Accession No. PTA-123439, on AUG 10, 2016 and designated 1G11C12/D2.

[0013] In certain aspects, the antibody or antibody fragment thereof is produced by immunizing an animal with an immunogen comprising a 7C4 derivative conjugated to a carrier protein under conditions such that immune cells of the animal produce an antibody or antibody fragment thereof that specifically binds to 7C4; and isolating the antibody or antibody fragment thereof from the animal, such as a goat, rabbit or mouse.

[0014] In certain aspects, the 7C4 derivative comprises a pegylated derivative of 7C4.

[0015] In certain aspects, the antibody or antibody fragment thereof has a detectable label.

[0016] In certain aspects, the antibody or antibody fragment is immobilized on a solid substrate. [0017] In another embodiment, the invention provides a hybridoma cell line which produces and secretes monoclonal antibodies which selectively bind to 7a-hydroxy-4- cholesten-3-one (7C4) and has been deposited under ATCC Accession No. PTA-123441, on AUG 10, 2016 and designated 25G9B1/F2. [0018] In yet another embodiment, the invention provides a hybridoma cell line which produces and secretes monoclonal antibodies which selectively bind to 7a-hydroxy-4- cholesten-3-one (7C4) and has been deposited under ATCC Accession No. PTA-123440, on AUG 10, 2016 and designated 23A7D1/F2.

[0019] In another embodiment, the invention provides a hybridoma cell line which produces and secretes monoclonal antibodies which selectively bind to 7a-hydroxy-4- cholesten-3-one (7C4) and has been deposited under ATCC Accession No. PTA-123439, on AUG 10, 2016 and designated 1G11C12/D2.

[0020] In still yet another embodiment, the invention provides method for detecting the level of 7a-hydroxy-4-cholesten-3-one (7C4) in a sample from a patient suspected of having bile acid malabsorption using an immunoassay, the method comprising:

(a) contacting an isolated antibody or antibody fragment thereof, a sample obtained from the patient, and immobilized 7C4 under suitable conditions to form a complex comprising the isolated antibody or antibody fragment thereof and 7C4 present in the sample or the immobilized 7C4; (b) detecting the level of antibody or antibody fragment thereof bound to the complex comprising the immobilized 7C4; and

(c) calculating the level of 7C4 in the sample based on the level of antibody or antibody fragment thereof from step (b).

[0021] In certain aspects, the isolated antibody or antibody fragment thereof, the sample, and the immobilized 7C4 are contacted simultaneously.

[0022] In certain aspects, the isolated antibody or antibody fragment thereof, the sample, and the immobilized 7C4 are contacted sequentially.

[0023] In certain aspects, the isolated antibody or antibody fragment thereof is the primary antibody and a secondary antibody is added to generate a signal. [0024] In certain aspects, the immunoassay is a competitive ELISA. [0025] In certain aspects, the antibody or antibody fragment thereof is produced by the hybridoma cell line deposited under ATCC Accession No. PTA-123441, on AUG 10, 2016 and designated 25G9B1/F2.

[0026] In certain aspects, the antibody or antibody fragment thereof is produced by the hybridoma cell line deposited under ATCC Accession No. PTA-123440, on AUG 10, 2016 and designated 23A7D1/F2.

[0027] In certain aspects, the antibody or antibody fragment thereof is produced by the hybridoma cell line deposited under ATCC Accession No. PTA-123439, on AUG 10, 2016 and designated 1G11C12/D2.

[0028] In certain aspects, the sample is a serum, blood or stool sample.

[0029] In still yet another embodiment, method for detecting the level of 7a-hydroxy-4- cholesten-3-one (7C4) in a sample from a patient suspected of having bile acid malabsorption using an immunoassay, the method comprising:

(a) contacting an antibody or antibody fragment thereof, a sample obtained from the patient, and a 7C4-labeled conjugate under suitable conditions to form a complex comprising the isolated antibody or antibody fragment thereof and 7C4 present in the sample or the 7C4-labeled conjugate;

(b) detecting the level of antibody or antibody fragment thereof bound to the complex comprising the 7C4; and

[0030] In certain aspects, the antibody or antibody fragment thereof is immobilized.

[0031] In certain aspects, the 7C4-labeled conjugate is a HRP-7C4-conjugate.

[0032] In certain aspects, the sample and the 7C4-labeled conjugate contact the antibody or antibody fragment simultaneously.

[0033] In certain aspects, the immunoassay is a competitive ELISA.

[0034] In certain aspects, the antibody or antibody fragment thereof is produced by the hybridoma cell line deposited under ATCC Accession No. PTA-123441, on AUG 10, 2016 and designated 25G9B1/F2. [0035] In certain aspects, the antibody or antibody fragment thereof is produced by the hybridoma cell line deposited under ATCC Accession No. PTA-123440, on AUG 10, 2016 and designated 23A7D1/F2.

[0036] In certain aspects, the antibody or antibody fragment thereof is produced by the hybridoma cell line deposited under ATCC Accession No. PTA-123439, on AUG 10, 2016 and designated 1G11C12/D2.

[0037] In certain aspects, the sample is serum, plasma, blood or stool sample.

[0038] In yet another embodiment, the present invention provides a method for diagnosing, and/or monitoring and/or treating bile acid malabsorption, the method comprising: (a) measuring a level of 7C4 in a sample in a patient using an immunoassay with an antibody or an antibody fragment to generate a to concentration;

(b) optionally administering a treatment regimen to the subject to treat the bile acid malabsorption;

(c) measuring a level of 7C4 in a sample in a patient later in time from to using an immunoassay with an antibody or an antibody fragment to generate a ti 7C4 concentration; and

(d) comparing the level of 7C4 at to to the level of 7C4 at ti to ascertain whether the bile acid malabsorption is improving over time.

[0039] These and other aspects, objects and advantages will become more apparent when read with the following detailed description and figures which follow.

BRIEF DESCRIPTION OF THE DRAWINGS

[0040] FIG. 1 illustrates one embodiment of a competitive immunoassay (indirect competitive assay) using monoclonal antibodies of the invention. The higher the amount of 7C4 in the sample the lower the signal produced in the assay of FIG. 1.

[0041] FIGS. 2A-B illustrate one embodiment of a competitive immunoassay (direct competitive assay) using monoclonal antibodies of the invention. FIG. 2A shows the synthesis of a horseradish peroxidase (HRP) 7C4 conjugate. An HRP maleimide is reacted with a 7C4 thiol to yield a HRP-7C4 conjugate. FIG. 2B shows the 7C4 from the sample and the 7C4 from the 7C4-HRP conjugate directly competing to bind to a monoclonal antibody of the invention in a homogeneous assay. The higher the amount of 7C4 in the sample the lower the signal produced in FIG. 2B.

[0042] FIGS. 3A-B illustrate one embodiment of a direct competitive immunoassay using monoclonal antibodies of the invention. FIG. 3 A shows the synthesis of a horseradish peroxidase (HRP) 7C4 conjugate. An HRP maleimide is reacted with a 7C4 thiol to yield a HRP-7C4 conjugate. FIG. 3B shows the 7C4 from the sample and the 7C4 from the 7C4- HRP conjugate directly competing to bind to a monoclonal antibody of the invention in a homogeneous assay. A higher signal produced by the assay of FIG. 3B correlates to a lower level of 7C4 in the sample.

[0043] FIGS. 4A-C illustrate the stabilized synthetic derivatives of 7a-hydroxy-4- cholesten-3-one (7C4) with a carrier protein (A); the generation of polyclonal and

monoclonal antibodies (B); and with a biotinylated conjugate (C).

[0044] FIGS. 5A-C illustrate one embodiment of a standard curve showing increasing concentrations of 7a-hydroxy-4-cholesten-3-one (7C4) in a sample as measured using the indirect competitive assay described herein. FIG. 5A is a graph of the data in FIG. 5B using the monoclonal antibody 23 A7D1/F2. The higher concentration amount of 7a-hydroxy-4- cholesten-3-one (x-axis) in the test sample, will result in a lower signal (Y-axis). FIG. 5B is a tabulation of indirect competitive immunoassay data. FIG. 5C is tabulated control data. [0045] FIGS. 6A-B illustrate one embodiment of control experiments using the 7C4-HRP conjugate versus HRP alone. FIG. 6A shows that using a variety of monoclonal antibodies (x-axis), the 7C4-HRP conjugate binds the antibody and produce a signal whereas negligible signal is produced with HRP alone. FIG. 6B illustrates a dilution series histogram of the 7C4-HRP conjugate. [0046] FIGS. 7A-C illustrate testing of Cohorts 1-5, which Cohorts include 3219 samples having various gastrointestinal disorders along with healthy controls (A). FIG. 7B shows tabulated data from direct competitive ELISAs. FIG. 7C shows a representative standard curve.

[0047] FIGS. 8A-B illustrate the results of Cohort 4 having 300 healthy control samples. FIG. 8A shows the distribution of the levels of 7C4 in the healthy control samples. Using the competitive ELISA, the mean for the 300 samples was 9.8 ng/mL (FIG. 8B). [0048] FIG. 9 illustrates the results of the competitive ELISA of Cohort 1 having 670 samples from various disease indications including Crohn's Disease, Ulcerative Colitis, IBS- D/M, IBS-C, GERD, colitis, celiac, and others.

[0049] FIGS. 10A-E illustrate the results of a competitive ELISA of Cohort 1 having 670 samples. FIG. 10A shows the results for healthy controls; FIG. 10B shows the results for Crohn's Disease (CD) and FIG. IOC shows the results for diarrhea predominant irritable bowel syndrome (IBS-D). FIG. 10D provides another graphical depiction of the results for subjects with Crohn's Disease, IBS-diarrhea, and ulcerative colitis, and for healthy controls. FIG. 10E shows results of oneway analysis of 7C4 level according to disease diagnosis: IBS- diarrhea v. ulcerative colitis; IBS-diarrhea v. healthy controls; Crohn's disease v. ulcerative colitis; Crohn's disease v. healthy control; IBS-diarrhea v. Crohn's disease; and healthy control v. ulcerative colitis.

[0050] FIGS. 11 A-B illustrate the results of the competitive ELISA of Cohort 3 having 483 samples. FIG. 11 A shows the results as a graph and FIG. 1 IB shows the tabulated data. [0051] FIGS. 12A-B illustrate the use of SeHCAT retention results to show severity of BAM. FIG. 12A shows that greater than 15% retention of bile acid is considered normal. FIG. 12B shows that the inventive assay methods described herein correlate with SeHCAT results. The percent retention of bile acid as determined by SeHCAT increases as the level of 7C4 decreases as measured using the competitive assays described herein. [0052] FIG. 13 shows the reactivity of exemplary mouse monoclonal antibodies to 7C4. Antibodies from 3 hybridoma clones (1G11C12/D2, 23A7D1/F2, and 25G9B1/F2) specifically bind to 7C4 and have no cross-reactivity to compounds that are structurally similar to 7C4 such as 7-ketocholesterol, 7a-hydroxycholesterol, and trihydroxycholestanoic acid. In the competitive ELISA, these compounds did not interfere with the binding of the antibody to 7C4.

[0053] FIG. 14 shows one embodiment of the parameters of the 7C4 ELISA assay.

[0054] FIG. 15 shows no significant interference of the 7C4 ELISA assay with common interferrants.

[0055] FIG. 16 shows 7C4 levels in healthy control vs chronic diarrhea population. DETAILED DESCRIPTION OF THE INVENTION

I. Definitions

[0056] The terms "a," "an," or "the" as used herein not only includes aspects with one member, but also includes aspects with more than one member. For example, an

embodiment including "a polyamine compound and an excipient" should be understood to present certain aspects with at least a second polyamine compound, at least a second excipient, or both.

[0057] The term "antigen" refers to any molecule, compound, composition, or particle that can bind specifically to an antibody. An antigen has one or more epitopes that interact with the antibody, although it does not necessarily induce production of that antibody.

[0058] The term "antibody" refers to an immunoglobulin molecule that is immunologically reactive with a particular antigen, and includes both polyclonal and monoclonal antibodies. The term also includes genetically engineered forms such as chimeric antibodies (e.g., humanized murine antibodies) and heteroconjugate antibodies (e.g., bispecific antibodies). The term "antibody" also includes antigen binding forms of antibodies, including fragments with antigen-binding capability e.g., Fab', F(ab')₂, Fab, Fv, scFv and di-scFv (see, e.g., Kuby, Immunology, 3^rd Ed., W.H. Freeman & Co., New York 1998). The term further includes bivalent or bispecific molecules, diabodies, triabodies, and tetrabodies. Bivalent and bispecific molecules are described in, e.g., Zhu et al. (Protein Sci. 1997; 6:781-9, and Hu et al. (Cancer Res. 1996; 56:3055-61). While various antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that fragments can be synthesized de novo either chemically or by utilizing recombinant DNA methodology. Thus, the term antibody, as used herein also includes antibody fragments either produced by the modification of whole antibodies or synthesized using recombinant DNA methodologies

[0059] An antibody can consist of one or more polypeptides substantially encoded by immunoglobulin genes or fragments of immunoglobulin genes. The recognized

immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively. An "antibody" functions as a binding protein and is structurally defined as comprising an amino acid sequence from or derived from the framework region of an immunoglobulin encoding gene of an animal producing antibodies.

[0060] A typical immunoglobulin (antibody) structural unit is known to comprise a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" (about 25 kD) and one "heavy" chain (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms variable light chain (V_L) and variable heavy chain (V_H) refer to these light and heavy chains respectively. [0061] Antibodies can include V_H-V_L dimers, including single chain antibodies (antibodies that exist as a single polypeptide chain), such as single chain Fv antibodies (sFv or scFv) in which a variable heavy and a variable light region are joined together (directly or through a peptide linker) to form a continuous polypeptide. The single chain Fv antibody is a covalently linked V_H-V_L which may be expressed from a nucleic acid including V_H- and V_L- encoding sequences either joined directly or joined by a peptide-encoding linker (e.g.,

Huston, et al. Proc. Nat. Acad. Sci. USA, 85:5879-5883, 1988). While the V_H and V_L are connected to each as a single polypeptide chain, the V_H and V_L domains associate non- covalently. Alternatively, the antibody can be another fragment. Other fragments can also be generated, e.g., using recombinant techniques, as soluble proteins or as fragments obtained from display methods. Antibodies can also include diantibodies and miniantibodies.

Antibodies of the invention also include heavy chain dimers, such as antibodies from camelids. Thus, in some embodiments an antibody is dimeric. In other embodiments, the antibody may be in a monomeric form that has an active isotype. In some embodiments the antibody is in a multivalent form, e.g., a trivalent or tetravalent form, that can cross-link the antigen.

[0062] The term "antibody fragment" or "antigen binding fragment" refers to at least a portion of the variable region of the immunoglobulin molecule, which binds to its target, i.e., the antigen recognition domain or the antigen binding region. Some of the constant region of the immunoglobulin may be included. Examples of antibody fragments include, but are not limited to, linear antibodies, single-chain antibody molecules (scFv), Fv fragments, hypervariable regions ro complementarity determining regions (CDRs), VL (light chain variable region), VH (heavy chain variable region), Fab fragments, F(ab)'₂ fragments, multispecific antibodies formed from antibody fragments, and any combination of those or any other portion of an immunoglobulin peptide capable of binding to target antigen. As appreciated by one of skill in the art, various antibody fragments can be obtained by a variety of methods, for example, digestion of an intact antibody with an enzyme, such as pepsin; or de novo synthesis. Antibody fragments are often synthesized de novo either chemically or by using recombinant DNA methodology.

[0063] The term "polyclonal antibody" refers to an antibody within a collection of antibodies secreted by different B cell lineages that recognize multiple epitopes on the same antigen. [0064] The term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally-occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site or epitope. Furthermore, in contrast to polyclonal antibody preparations which typically include different antibodies directed against different determinants or epitopes, each monoclonal antibody is directed against a single determinant on the antigen. Monoclonal antibodies to be used in accordance with the invention may be made by the hybridoma method first described by Kohler and Milstein, Nature, 256:495 (1975), or may be made by recombinant DNA methods such as described in U.S. Pat. No. 4,816,567. In some cases, monoclonal antibodies may also be isolated from phage libraries generated using the techniques described in McCafferty et al, Nature, 348:552-554 (1990).

[0065] The term "chimeric antibody" refers to an immunoglobulin molecule in which (a) the constant region, or a portion thereof, is altered, replaced or exchanged so that the antigen binding site (variable region) is linked to a constant region of a different or altered class, effector function and/or species, or an entirely different molecule which confers new properties to the chimeric antibody, e.g., an enzyme, toxin, hormone, growth factor, drug, etc.; or (b) the variable region, or a portion thereof, is altered, replaced or exchanged with a variable region, or portion thereof, having a different or altered antigen specificity; or with corresponding sequences from another species or from another antibody class or subclass. [0066] The term "humanized antibody" refers to an antibody in which the antigen binding loops, i.e., complementarity determining regions (CDRs), comprised by the VH and VL regions are grafted to a human framework sequence. Typically, the humanized antibodies have the same binding specificity as the non-humanized antibodies described herein.

Techniques for humanizing antibodies are well known in the art and are described in e.g., Verhoyen et al, Science, 239: 1534 (1988) and Winter and Milstein, Nature, 349: 293 (1991). [0067] The phrase "specifically (or selectively) binds" to an antibody or "specifically (or selectively) immunoreactive with," when referring to an antigen or hapten, refers to a binding reaction that is determinative of the presence of the antigen or hapten, often in a

heterogeneous population of antigens or haptens and other biologies such as a mixture of cells, a cell lysate or a biological sample, e.g., blood, plasma or serum. Thus, under designated immunoassay conditions, the specified antibodies bind to a particular antigen or hapten (at least two times the background and more typically more than 10 to 100 times background. Specific binding to an antibody under such conditions requires an antibody that is selected for its specificity for a particular antigen or hapten. For example, polyclonal antibodies can be selected to obtain only those polyclonal antibodies that are specifically immunoreactive with the selected antigen and not with other proteins. This selection may be achieved by subtracting out antibodies that cross-react with other molecules. A variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein. For example, ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein {see, e.g., Harlow & Lane, Using Antibodies, A Laboratory Manual (1998) for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity).

[0068] Specific binding can be measured, for example, by methods known in the art, e.g., using competition assays with a control molecule that is similar to the target, for example, an excess of non-labeled target. An antibody that specifically binds a target antigen can have a K_d for the antigen of at least about 10^~4 M, alternatively at least about 10^~5 M, alternatively at least about 10^~6 M, alternatively at least about 10^~7 M, alternatively at least about 10^~8 M, alternatively at least about 10^~9 M, alternatively at least about 10^~10 M, alternatively at least about 10^~u M, alternatively at least about 10^~12 M, or greater. In one embodiment, the term "specific binding" refers to binding where an antibody binds to its particular hapten without substantially binding to any other structurally similar haptens or compounds. In such embodiments, the extent of non-specific binding is the amount of binding at or below background and will typically be less than about 10%, preferably less than about 5%, and more preferably less than about 1% as determined by fluorescence activated cell sorting (FACS) analysis, enzyme-linked immunosorbent assay (ELISA) or radioimmunoprecipitation (RIA), for example.

[0069] The term "cross-reactivity" refers to the relative binding of a designated (primary) antigen and a secondary antigen to a purified antibody of interest, wherein the designated or primary antigen is used to produce the antibody of interest. C50_secondaiy is the concentration of the secondary antigen required to cause 50% inhibition of the reaction between the primary antigen and the antibody of interest. Similarly, C50_pri_mar_y is the concentration of the primary antigen required to cause 50% inhibition of the reaction between the primary antigen and the antibody (self-inhibition). Then the relative equilibrium binding constant for the variant antigen, C50_primaiy/C50_secondaiy, measures cross-reactivity (Benjamin and Perdue, Methods, 1996, 9(3):508-515). In other words, the percent cross-reactivity of an antibody produced against compound X with respect to a specific compound is equal to [(a/b)x\00] where a is the amount of compound X required to displace 50% of compound Fbound of the antibody; b is the amount of compound Y required to displace 50% of compound bound to the antibody. The term "cross-reactivity" of an antibody can also refer to the interaction of an antibody to similar or dissimilar epitopes on different antigens. "Cross-reactivity" can be measured using standard assays known to one skilled in the art, such as a competitive ELISA, e.g., a direct competitive ELISA or an indirect competitive ELISA.

[0070] As used herein, the term "isolated" or "purified" antibody refers to an antibody that is substantially or essentially free from components that normally or naturally accompany it. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography.

Contaminant components of its environment are materials that would interfere with uses for the antibody or fragment thereof, and may include enzymes, hormones, and other

proteinaceous or non-proteinaceous solutes. In certain embodiments, the isolated antibody is purified to greater than 95% by weight of polypeptides as determined by the Lowry method, and preferably, more than 99% by weight, or to homogeneity by SDS-page under reducing or non-reducing conditions using Coomassie blue, or silver stain. An isolated antibody includes the antibody in situ within recombinant cells. In some cases, an isolated antibody is prepared by a least one purification step.

[0071] The term "hybridoma cell line" or "hybridoma clone" refers to a hybrid cell line used to produce a monoclonal antibody. In some cases, a hybridoma cell is an antibody- producing cell from a mouse's spleen fused to a myeloma cell, wherein the mouse has been injected with a specific antigen.

[0072] The term "hapten" refers to a small molecule that can elicit an immune response in an animal when the hapten is linked or conjugated to a carrier molecule, e.g., a carrier protein, to form an immunogen or an immunogenic conjugate. The hapten-carrier protein complex is immunogenic (can elicit an immune response) and the hapten alone (unbound hapten) is not immunogenic. Non-limiting examples of a carrier protein include bovine serum albumin (BSA), mouse serum albumin (MSA), rabbit serum albumin (RSA), ovalbumin (OVA), keyhole limpet hemocyanin (KLH), bovine or porcine thyroglobulin, tetanus toxoid, gelatin, or soybean trypsin inhibitor and the like.

[0073] The term "immunogen" refers to a substance, compound, peptide, or composition which stimulates the production of an immune response in an animal.

[0074] As used herein, a "linker" or "spacer" is any molecule capable of binding (e.g., covalently) together a hapten to another molecule or moiety disclosed herein. Linkers include, but are not limited to, straight or branched chain carbon linkers, heterocyclic carbon linkers, peptide linkers, polyether linkers and short hydrophilic molecules. Exemplary linkers can include but are not limited to NH-CH₂-CH₂-O-CH₂-CO- and 5-amino-3-oxopentanoyl. For example, poly(ethylene glycol) linkers are available from Quanta Biodesign, Powell, OH. These linkers optionally have amide linkages, sulfhydryl linkages, or hetero functional linkages.

[0075] The term "label" or a "detectable label" is a composition detectable by

spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means. For example, useful labels include ³²P, fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in an ELISA), biotin, digoxigenin, or peptides and proteins which can be made detectable, e.g., by incorporating a radiolabel into a peptide. The detectable label can be, without limitation, a fluorescent label, a luminescent label, a chemiluminescent label, a bioluminescent label, a radioactive label or an enzymatic label.

[0076] The term "solid substrate" or "solid support" refers to a solid material, membrane, array, chip, bead, and the like. The surface on the solid substrate can be composed of the same material as the substrate. The surface may be composed of any of a wide variety of materials, for example, polymers, plastics, resins, polysaccharides, silica or silica-based materials, carbon, metals, inorganic glasses, membranes, or any of the above-listed substrate materials.

[0077] The term "immunoassay" refers to an assay that detects or measures the presence or concentration (level or amount) of an analyte (small molecule, chemical compound, peptide, polypeptide, biomolecule, antigen, metabolite, etc) by utilizing an antibody, immunoglobulin or a fragment thereof.

[0078] The terms "subject," "patient," and "individual" are used interchangeably and refer to except where indicated, mammals such as humans and non-human primates, as well as rabbits, rats, mice, goats, pigs, and other mammalian species. [0079] The term "sample" includes any biological specimen obtained from an individual. Suitable samples for use include, without limitation, whole blood, plasma, serum, saliva, urine, stool, tears, any other bodily fluid, tissue samples {e.g., biopsy), and cellular extracts thereof {e.g., red blood cellular extract). In a preferred embodiment, the sample is a serum or plasma sample. The use of samples such as serum, saliva, and urine is well known in the art (see, e.g., Hashida et al, J. Clin. Lab. Anal., 11 :267-86 (1997)). One skilled in the art will appreciate that samples such as serum samples can be diluted prior to performing the methods disclosed herein.

[0080] "Acyl" as used herein includes an alkanoyl, aroyl, heterocycloyl, or heteroaroyl group as defined herein. Representative acyl groups include acetyl, benzoyl, nicotinoyl, and the like.

[0081] "Alkanoyl" as used herein includes an alkyl-C(O)- group wherein the alkyl group is as defined herein. Representative alkanoyl groups include acetyl, ethanoyl, and the like.

[0082] "Alkenyl" as used herein includes a straight or branched aliphatic hydrocarbon group of 2 to about 15 carbon atoms that contains at least one carbon-carbon double or triple bond. Preferred alkenyl groups have 2 to about 12 carbon atoms. More preferred alkenyl groups contain 2 to about 6 carbon atoms. In one aspect, hydrocarbon groups that contain a carbon-carbon double bond are preferred. In a second aspect, hydrocarbon groups that contain a carbon-carbon triple bond are preferred {i.e., alkynyl). "Lower alkenyl" as used herein includes alkenyl of 2 to about 6 carbon atoms. Representative alkenyl groups include vinyl, allyl, n-butenyl, 2-butenyl, 3-methylbutenyl, n-pentenyl, heptenyl, octenyl, decenyl, propynyl, 2-butynyl, 3-methylbutynyl, n-pentynyl, heptynyl, and the like. [0083] An alkenyl group can be unsubstituted or optionally substituted. When optionally substituted, one or more hydrogen atoms of the alkenyl group (e.g., from 1 to 4, from 1 to 2, or 1) may be replaced with a moiety independently selected from the group consisting of fluoro, hydroxy, alkoxy, amino, alkylamino, acylamino, thio, and alkylthio. [0084] "Alkenylene" as used herein includes a straight or branched bivalent hydrocarbon chain containing at least one carbon-carbon double or triple bond. Preferred alkenylene groups include from 2 to about 12 carbons in the chain, and more preferred alkenylene groups include from 2 to 6 carbons in the chain. In one aspect, hydrocarbon groups that contain a carbon-carbon double bond are preferred. In a second aspect, hydrocarbon groups that contain a carbon-carbon triple bond are preferred. Representative alkenylene groups include -CH=CH-, -CH₂-CH=CH-, -C(CH₃)=CH-, -CH₂CH=CHCH₂-, ethynylene, propynylene, n- butynylene, and the like.

[0085] "Alkoxy" as used herein includes an alkyl-O- group wherein the alkyl group is as defined herein. Representative alkoxy groups include methoxy, ethoxy, n-propoxy, i- propoxy, n-butoxy, heptoxy, and the like.

[0086] An alkoxy group can be unsubstituted or optionally substituted. When optionally substituted, one or more hydrogen atoms of the alkoxy group (e.g., from 1 to 4, from 1 to 2, or 1) may be replaced with a moiety independently selected from the group consisting of fluoro, hydroxy, alkoxy, amino, alkylamino, acylamino, thio, and alkylthio. [0087] "Alkoxyalkyl" as used herein includes an alkyl-O-alkylene- group wherein alkyl and alkylene are as defined herein. Representative alkoxyalkyl groups include methoxyethyl, ethoxymethyl, n-butoxymethyl and cyclopentylmethyloxy ethyl.

[0088] " Alkoxy carbonyl" as used herein includes an ester group; i.e., an alkyl-O-CO- group wherein alkyl is as defined herein. Representative alkoxycarbonyl groups include methoxycarbonyl, ethoxycarbonyl, t-butyloxycarbonyl, and the like.

[0089] "Alkoxycarbonylalkyl" as used herein includes an alkyl-O-CO-alkylene- group wherein alkyl and alkylene are as defined herein. Representative alkoxycarbonylalkyl include methoxycarbonylmethyl, ethoxycarbonylmethyl, methoxycarbonylethyl, and the like.

[0090] "Alkyl" as used herein includes an aliphatic hydrocarbon group, which may be straight or branched-chain, having about 1 to about 20 carbon atoms in the chain. Preferred alkyl groups have 1 to about 12 carbon atoms in the chain. More preferred alkyl groups have 1 to 6 carbon atoms in the chain. "Branched-chain" as used herein includes that one or more lower alkyl groups such as methyl, ethyl or propyl are attached to a linear alkyl chain.

"Lower alkyl" as used herein includes 1 to about 6 carbon atoms, preferably 5 or 6 carbon atoms in the chain, which may be straight or branched. Representative alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, n-pentyl, and 3-pentyl.

[0091] An alkyl group can be unsubstituted or optionally substituted. When optionally substituted, one or more hydrogen atoms of the alkyl group (e.g., from 1 to 4, from 1 to 2, or 1) may be replaced with a moiety independently selected from the group consisting of fluoro, hydroxy, alkoxy, amino, alkylamino, acylamino, thio, and alkylthio. [0092] "Alkylene" as used herein includes a straight or branched bivalent hydrocarbon chain of 1 to about 6 carbon atoms. Preferred alkylene groups are the lower alkylene groups having 1 to about 4 carbon atoms. Representative alkylene groups include methylene, ethylene, and the like.

[0093] "Alkylthio" as used herein includes an alkyl-S- group wherein the alkyl group is as defined herein. Preferred alkylthio groups are those wherein the alkyl group is lower alkyl. Representative alkylthio groups include methylthio, ethylthio, isopropylthio, heptylthio, and the like.

[0094] "Alkylthioalkyl" as used herein includes an alkylthio-alkylene- group wherein alkylthio and alkylene are defined herein. Representative alkylthioalkyl groups include methylthiomethyl, ethylthiopropyl, isopropylthioethyl, and the like.

[0095] " Amido" as used herein includes a group of formula Y₁Y₂N-C(0)- wherein Y₁ and Y₂ are independently hydrogen, alkyl, or alkenyl; or Y₁ and Y₂, together with the nitrogen through which Yi and Y₂ are linked, join to form a 4- to 7-membered azaheterocyclyl group (e.g., piperidinyl). Representative amido groups include primary amido (H₂N-C(0)-), methylamido, dimethylamido, diethylamido, and the like. Preferably, "amido" is an - C(0) RR' group where R and R' are members independently selected from the group consisting of H and alkyl. More preferably, at least one of R and R' is H.

[0096] "Amidoalkyl" as used herein includes an ami do-alky lene- group wherein amido and alkylene are defined herein. Representative amidoalkyl groups include amidomethyl, ami doethy lene, dimethylamidomethyl, and the like. [0097] "Amino" as used herein includes a group of formula YiY₂N- wherein Y₁ and Y₂ are independently hydrogen, acyl, or alkyl; or Yi and Y₂, together with the nitrogen through which Yi and Y₂ are linked, join to form a 4- to 7-membered azaheterocyclyl group (e.g., piperidinyl). Optionally, when Y₁ and Y₂ are independently hydrogen or alkyl, an additional substituent can be added to the nitrogen, making a quaternary ammonium ion.

Representative amino groups include primary amino (H₂N-), methylamino, dimethylamino, diethylamino, and the like. Preferably, "amino" is an - RR' group where R and R' are members independently selected from the group consisting of H and alkyl. Preferably, at least one of R and R' is H. [0098] "Aminoalkyl" as used herein includes an amino-alkylene- group wherein amino and alkylene are defined herein. Representative aminoalkyl groups include aminomethyl, aminoethyl, dimethylaminomethyl, and the like.

[0099] "Aroyl" as used herein includes an aryl-CO- group wherein aryl is defined herein. Representative aroyl include benzoyl, naphth-l-oyl and naphth-2-oyl. [0100] "Aryl" as used herein includes an aromatic monocyclic or multi cyclic ring system of 6 to about 14 carbon atoms, preferably of 6 to about 10 carbon atoms. Representative aryl groups include phenyl and naphthyl.

[0101] "Aromatic ring" as used herein includes 5-12 membered aromatic monocyclic or fused polycyclic moieties that may include from zero to four heteroatoms selected from the group consisting of oxygen, sulfur, selenium, and nitrogen. Exemplary aromatic rings include benzene, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazine, pyrimidine, naphthalene, benzathiazoline, benzothiophene, benzofurans, indole, benzindole, quinoline, and the like. The aromatic ring group can be substituted at one or more positions with halo, alkyl, alkoxy, alkoxy carbonyl, haloalkyl, cyano, sulfonato, amino sulfonyl, aryl, sulfonyl, aminocarbonyl, carboxy, acylamino, alkyl sulfonyl, amino and substituted or unsubstituted substituents.

[0102] "Biomolecule" as used herein includes a natural or synthetic molecule for use in biological systems. Preferred biomolecules include a protein, a peptide, an enzyme substrate, a hormone, an antibody, an antigen, a hapten, an avidin, a streptavidin, a carbohydrate, a carbohydrate derivative, an oligosaccharide, a polysaccharide, and a nucleic acid. More preferred biomolecules include a protein, a peptide, an avidin, a streptavidin, or biotin. [0103] "Carboxy" and "carboxyl" as used herein include a HOC(O)- group (i.e., a carboxylic acid) or a salt thereof.

[0104] "Carboxyalkyl" as used herein includes a HOC(0)-alkylene- group wherein alkylene is defined herein. Representative carboxyalkyls include carboxymethyl (i.e., HOC(0)CH₂-) and carboxyethyl (i.e. , HOC(0)CH₂CH₂-).

[0105] "Cycloalkyl" as used herein includes a non-aromatic mono- or multicyclic ring system of about 3 to about 10 carbon atoms, preferably of about 5 to about 10 carbon atoms. More preferred cycloalkyl rings contain 5 or 6 ring atoms. A cycloalkyl group optionally comprises at least one sp²-hybridized carbon (e.g., a ring incorporating an endocyclic or exocyclic olefin). Representative monocyclic cycloalkyl groups include cyclopentyl, cyclohexyl, cyclohexenyl, cycloheptyl, and the like. Representative multicyclic cycloalkyl include 1-decalin, norbornyl, adamantyl, and the like.

[0106] "Cycloalkylene" as used herein includes a bivalent cycloalkyl having about 4 to about 8 carbon atoms. Preferred cycloalkylenyl groups include 1,2-, 1,3-, or 1,4- cis- or trans-cyclohexylene.

[0107] "Halo" or "halogen" as used herein includes fluoro, chloro, bromo, or iodo.

[0108] "Heteroatom" as used herein includes an atom other than carbon or hydrogen. Representative heteroatoms include O, S, and N. The nitrogen or sulphur atom of the heteroatom is optionally oxidized to the corresponding N-oxide, S-oxide (sulfoxide), or S,S- dioxide (sulfone). In a preferred aspect, a heteroatom has at least two bonds to alkylene carbon atoms (e.g., -C₁-C9 alkylene-O-Ci-Cg alkylene-). In some embodiments, a heteroatom is further substituted with an acyl, alkyl, aryl, cycloalkyl, heterocyclyl, or heteroaryl group (e. ., -N(Me)-; -N(Ac)-).

[0109] "Hydroxyalkyl" as used herein includes an alkyl group as defined herein substituted with one or more hydroxy groups. Preferred hydroxyalkyls contain lower alkyl.

Representative hydroxyalkyl groups include hydroxymethyl and 2-hydroxy ethyl.

[0110] "Linking group" i.e., L, comprises the atoms joining the metabolite derivative with a biomolecule such as a carrier protein, a biotin or streptavidin. See also R. Haugland, Molecular Probes Handbook of Fluorescent Probes and Research Chemicals, Molecular Probes, Inc. (1992). In one embodiment, L represents the linking group precursor before the attachment reaction with a protein, and R¹¹ represents the resultant attachment between the compound of the invention and the protein or biotin (i.e., R¹¹ is the resultant attachment between the linking group joined to the biomolecule). Preferred reactive functionalities include phosphoramidite groups, an activated ester (e.g., an NHS ester), thiocyanate, isothiocyanate, maleimide and iodoacetamide. L may comprise a terminal amino, carboxylic acid, or sulfhydryl group covalently attached to the ring. In certain instances, the terminal amino, carboxylic acid, or sulfhydryl group is shown and is represented as -L-NH₂, or -L- C(0)OH or -L-SH.

[0111] "Oxo" as used herein includes a group of formula >C=0 (i.e., a carbonyl group - C(O)-) [0112] "Sulfonate)" as used herein includes an -SO₃ ^" group, preferably balanced by a cation such as H⁺, Na⁺, K⁺, and the like.

[0113] "Sulfonatoalkyl" as used herein includes a sulfonato-alkylene- group wherein sulfonato and alkylene are as defined herein. A more preferred embodiment includes alkylene groups having from 2 to 6 carbon atoms, and a most preferred embodiment includes alkylene groups having 2, 3, or 4 carbons. Representative sulfonatoalkyl s include sulfonatomethyl, 3-sulfonatopropyl, 4-sulfonatobutyl, 5-sulfonatopentyl, 6-sulfonatohexyl, and the like.

II. Detailed Description of Embodiments

[0114] The invention provides assay methods and kits for detecting, measuring or quantitating the level of 7a-hydroxy-4-cholesten-3-one (7C4) in a biological sample from a subject, such as a human subject. In some embodiments, the human subject has a condition associated with bile acid malabsorption or diarrhea of unknown origin. The biological sample can be a serum, plasma, blood or stool sample.

[0115] In one embodiment, the invention provides a method for detecting the level of 7a- hydroxy-4-cholesten-3-one (7C4) in a sample from a patient suspected of having bile acid malabsorption (BAM) using an immunoassay, the method comprising:

(a) contacting an isolated antibody or antibody fragment thereof, a sample obtained from the patient, and an immobilized 7C4 under suitable conditions to form a complex comprising the isolated antibody or antibody fragment thereof and 7C4 present in the sample or the immobilized 7C4; (b) detecting the level of antibody or antibody fragment thereof bound to the complex comprising the immobilized 7C4; and

(c) calculating the level of 7C4 in the sample based on the level of antibody or antibody fragment thereof from step (b). [0116] In some aspects, the invention provides a method for assaying, measuring or detecting the presence or level of 7a-hydroxy-4-cholesten-3-one (7C4) in a biological sample such as a fluid or tissue sample from a mammal, e.g., a human. FIG. 1 shows one embodiment of the method 100 of the invention.

[0117] The method 100 includes measuring or quantitating the amount or concentration of 7C4 in a biological sample such as a serum sample 110 obtained from a human subject. The method comprises combining the sample 110 with an antibody 112 that specifically binds to 7C4 under conditions to form a complex 115 between the antibody and 7C4 if present in the sample. The antibody can be any of the anti-7C4 antibodies described herein.

[0118] In some embodiments, the sample and the anti-7C4 antibody are also combined with an immobilized 7a-hydroxy-4-cholesten-3 -one 121 or derivative thereof. The immobilized 7C4 derivative may be biotinylated 7C4 as described herein 121 that has been attached or bound to a streptavidin-coated solid substrate 125 such as a streptavidin-coated multiwell plate. In some embodiments, the sample, the anti-7C4 antibody 112, and immobilized 7C4 derivative are simultaneously contacted or added together. In some cases, the sample and the anti-7C4 antibody 112 are incubated together for a preselected duration, and then incubated with immobilized 7C4 or biotinylated 7C4. In other cases, the

immobilized or biotinylated 7C4 121 derivative is incubated with the anti-7C4 antibody for a preselected duration, and then incubated with the sample. In yet other cases, the sample, the anti-7C4 antibody 112 and immobilized 7C4 are contacted together sequentially in any order. The level of the 7C4 in the sample can be determined by measuring the level of anti-7C4 antibody 112 bound to the immobilized 7C4 derivative 121, and calculating the

corresponding level of 7C4 in the sample.

[0119] The level of anti-7C4 antibody complexed with the immobilized 7C4 derivative can be measured directly using a secondary antibody 151 having a label and the level of 7C4 in the sample 110 is quantitated indirectly. In some cases, there is an inverse proportion of 7C4 in the sample compared to the level of anti-7C4 antibody bound to the immobilized 7C4 derivative. That is, the higher the amount of 7C4 in the sample, the lower the signal 162. In certain instances, the secondary antibody has a label attached thereto (e.g., horseradish peroxidase), which can be detected, whereas the 7C4 and anti-7C4 antibody complex 115 is unlabeled.

[0120] In certain aspects, the step of measuring the level of bound anti-7C4 antibody or the level of 7C4 is performed using an immunoassay. Immunoassays provide reliable and facile ways to monitor metabolites in biological fluids. The invention provides reliable

immunoassays of high specificity and sensitivity for the detection and quantification of 7C4. In some embodiments, the immunoassay is an enzyme linked immunosorbent assay (ELISA), e.g., a competitive ELISA or a proximity immunoassay, e.g., CEER . [0121] In another embodiment, the invention provides a method for detecting the level of 7a-hydroxy-4-cholesten-3-one (7C4) in a sample from a patient suspected of having bile acid malabsorption using an immunoassay employing a HRP-7C4-conjugate. Turning now to FIG. 2A, a horseradish peroxidase (HRP) maleimide 202 is reacted with a 7C4 thiol derivative 209 to yield a HRP-7C4 conjugate 212. FIG. 2B shows the 7C4 from the sample 218 and the 7C4 from the 7C4-HRP conjugate 212 directly competing to bind to a

monoclonal antibody 220 of the invention in a homogeneous assay. A skilled artisan will appreciate that the capture antibody can also be on a solid support. The higher the amount of 7C4 in the sample 218 the lower the signal produced, as the HRP-7C4 conjugate 212 competes for the capture antibody 220. As shown in FIG. 2B, both the 7C4 in the sample 218 and the HRP-7C4 conjugate 212 compete for the capture antibody 240. In certain aspects, the 7C4 in the sample 218 is unlabeled. Therefore, as the HRP-7C4 conjugate 212 is labeled, the more unlabeled 7C4 in the sample, the less signal 250 generated. In certain aspects, the unlabeled 7C4 competes for the labeled HRP-7C4 conjugate.

[0122] FIGS. 3 A-B show an alternative embodiment for detecting the level of 7a-hydroxy- 4-cholesten-3-one (7C4) in a sample from a patient suspected of having bile acid

malabsorption using an immunoassay employing a HRP-7C4-conjugate. FIG. 3 A shows a horseradish peroxidase (HRP) maleimide 302 being reacted with a 7C4 thiol 309 to yield a HRP-7C4 conjugate 312 as above. FIG. 3B shows the 7C4 from the sample 318 and the 7C4 from the 7C4-HRP conjugate 312 directly competing to bind to a monoclonal antibody 320 of the invention in a homogeneous assay. The monoclonal antibody 320 of the present is attached to a substrate using a secondary antibody 329. The secondary antibody 329 can be a generated from any host animal such that it specifically binds to the monoclonal antibody 320. For instance, secondary antibody 329 can be a goat anti-mouse antibody, sheep anti- mouse antibody, rabbit anti-mouse antibody, rat anti-mouse antibody, or other mammalian anti-mouse and the like. The higher the amount of 7C4 in the sample 318, the lower the signal produced, as the FIRP-7C4 conjugate 312 competes for the capture antibody 320. As shown in FIG. 3B, both the 7C4 in the sample 318 and the HRP-7C4 conjugate 312 compete for the capture antibody 320. In certain aspects, the 7C4 in the sample 318 is unlabeled. Therefore, as the HRP-7C4 conjugate 312 is labeled, the more unlabeled 7C4, the less signal 350 generated. In certain aspects, the unlabeled 7C4 competes for the labeled HRP-7C4 conjugate. [0123] In the assay methods herein, the preferred anti-7C4 antibody is the antibody or antibody fragment thereof produced by the hybridoma clone deposited under ATCC

Accession No. PTA-123439, on AUG 10, 2016 and designated 1G11C12/D2; or

alternatively, the antibody or antibody fragment thereof is produced by the hybridoma cell line deposited under ATCC Accession No. PTA-123440, on AUG 10, 2016 and designated 23A7D1/F2; or alternatively, the antibody or antibody fragment thereof is produced by the hybridoma cell line deposited under ATCC Accession No. PTA- 123441, on AUG 10, 2016 and designated 25G9B1/F2.

[0124] In the assay methods of the invention, the sample is a whole blood sample, a plasma sample, a serum sample or stool sample. Such samples can be isolated or obtained from a subject, such as a human subject. In some cases, the subject has been diagnosed as having diarrhea and in certain instances, from unknown etiology. In other cases, it may be suspected that the subject has diarrhea caused from bile acid malabsorption. In other instances, the subject is experiencing or exhibiting one or more symptoms of loose bowels. In some embodiments, the sample used in the assay method is a diluted sample. The sample may be an unprocessed sample. In some instances, the volume of the sample used in the method is less than about 100 μί, e.g., about 99 μί, 90 μί, 85 μί, 80 μί, 75 μί, 70 μί, 65 μί, 60 μί, 55 μΐ,, 50 μί, 45 μί, 40 μί, 35 μί, 30 μΐ,, 25 μί, 20 μΐ,, 15 μί, 10 μί, 5 μί, or less. The sample volume can be less than about 50 μΐ., e.g., about 50 μΐ., 45 μΐ., 40 μΐ., 35 μΐ., 30 μΐ., 25 μί, 20 μί, 15 μί, 10 μί, 5 μί, or less. [0125] In some embodiments, the assay method takes less than 24 hours to perform, e.g., 23 hrs, 22 hrs, 21 hrs, 20 hrs, 19 hrs, 18 hrs, 17 hrs, 16 hrs, 15 hrs, 14 hrs, 13 hrs, 12 hrs, 11 hrs, 10 hrs, 9 hrs, 8 hrs, 7 hrs, 6 hrs, 5 hrs, 4 hrs, 3 hrs, 2 hrs, 1 hr, 30 minutes or less to perform.

[0126] In some embodiments, the antibodies described herein can be conjugated to any detectable label or moiety that can be used to measure the formed antigen-antibody complex. In some cases, the antibody is directly conjugated to a readable signal such as chromophores, colloidal gold, colored latex, fluorophores and the like. In other cases, the antibody is conjugaed to an enzyme, peptide or other biomolecule. In certain instances, a fluorophore is used and a FRET analysis can be performed.

[0127] In one aspect, the invention provides assay methods wherein an antibody-antigen reaction is carried out. In one embodiment of an ELISA, an antigen or 7C4 present in a sample obtained from a subject is allowed to react with an enzyme-labeled, e.g., peroxidase- labeled antibody specific to the metabolite being assayed to form an antigen-antibody complex. The thus formed antigen-antibody complex is then allowed to react with a detection substrate, so that the activity of the enzyme, e.g., peroxidase or phosphatase is measured. In some embodiments, the antibody specific to the metabolite is not enzyme labeled and an enzyme-labeled secondary antibody that recognized the antibody specific to the metabolite is used. A detection substrate can be used to react with the enzyme label of the secondary antibody in order to measure the activity of the enzyme. The enzyme-labeled antibody can be an alkaline phosphatase-, β-galactosidase-, or HRP-labeled antibody. [0128] Any detection substrate recognized by those skilled in the art can be used. For instance, for a chemiluminescent reaction, the substrate can be luminol, Supersignal^® ELISA Pico chemiluminescent substrate (Thermo Fisher), and DynaLight^™ chemiluminescent substrate (Thermo Fisher). For a colorimetric reaction, a substrate such as 4-chloro-l- napthol, p-nitrophenyl phosphate (PNPP), OPD, O PG, or TMB can be used. A substrate such as 4-methylumbelliferyl phosphate disodium salt (MUP), QuantaBlu^™ Fluorogenic substrate (Thermo Fisher), and Amplex^® Red Reagent (Thermo Fisher) can be used for a fluorescent reaction. The presence, concentration and or level of the metabolite can thereby be measured using, for example, a spectrometer or other detection device.

[0129] In another ELISA embodiment, the metabolite or a derivative thereof can be immobilized. An antibody of the invention can be used to bind to the immobilized metabolite to form an antigen-antibody complex. A sample that contains the metabolite can be used to compete for antibody-antigen binding. Thereafter, the conjugate can be detected by another antibody (secondary antibody) with an enzyme label. The enzyme label is then reacted with detection reagents or substrates, and then monitored. In other cases, the antibody of the invention is conjugated to a detectable moiety or label and can be reacted and/or detected without using a secondary antibody. [0130] The assay methods to detect any of the metabolites described herein can comprise any immunoassay known in the art. In some aspects, the assay is performed in a liquid phase. In other embodiments, the assay is performed on a solid phase or solid support, e.g., on a bead or a microplate, for example a 96 well microtiter plate. Non-limiting examples of immunoassays useful in these methods are a radioimmunoassay, a microarray assay, a fluorescence polarization immunoassay, an immunoassay comprising FRET, enzyme linked immunosorbent assay (ELISA) or CEER^™.

[0131] Any ELISA known in the art as useful for hapten detection can be utilized for the instant assays. ELISA for haptens generally utilize a competitive format, i.e., where the hapten (a metabolite) in the sample competes with a labeled hapten (e.g., a biotin-hapten or enzyme-hapten conjugate) for anti-hapten antibody binding sites such that less labeled hapten is bound when there is more hapten in the sample. Thus, in a competitive assay, an increasing amount of hapten in the sample results in less enzyme bound to the solid phase, and consequently less detectable signal. In such competitive assays the sample can be added with the labeled hapten to compete directly for antibody binding sites, or the sample and labeled hapten can be added sequentially such that the labeled hapten simply binds where the sample hapten is not bound. In some embodiments, the ELISA is a direct competitive ELISA, or an indirect competitive ELISA.

[0132] In one embodiment, the antibodies produced herein are bound to a solid phase, either directly or indirectly, the latter being where the solid phase is coated with an anti- antibody (for example goat antibodies that bind to rabbit IgG antibodies (goat anti-rabbit

IgG) and the antibodies are bound to the anti -antibody. The anti-antibodies are also known as secondary antibodies. In these assays, the sample and a labeled hapten are added to the solid phase to compete with antibody binding sites on the coated solid phase. After washing, the signal is generated, which measures the amount of labeled hapten that is bound to the solid phase.

[0133] Provided herein are kits for performing the assay methods described above. In some embodiments, the kit comprises an antibody that specifically binds to 7C4, e.g., an anti- 7C4 monoclonal antibody or polyclonal antibody, and optionally a biotinylated 7C4 derivative. The antibody or antibody fragment thereof against 7C4 may be produced by the hybndoma clone deposited under ATCC Accession No. PTA-123441, on AUG 10, 2016 and designated 25G9B 1/F2; or alternatively, the antibody or antibody fragment thereof is produced by the hybridoma cell line deposited under ATCC Accession No. PTA-123440, on AUG 10, 2016 and designated 23A7D1/F2; or alternatively, the antibody or antibody fragment thereof is produced by the hybridoma cell line deposited under ATCC Accession No. PTA-123439, on AUG 10, 2016 and designated 1G11C12/D2.

[0134] In other embodiments, the kit comprises an antibody that specifically binds to melatonin, e.g., an anti -melatonin monoclonal antibody or polyclonal antibody, and optionally biotinylated melatonin. The antibody or antibody fragment thereof against 7C4 may be produced by the hybridoma clone deposited under ATCC Accession No. PTA- 123439, on AUG 10, 2016 and designated 1G11C12/D2; or alternatively, the antibody or antibody fragment thereof against 7C4 is produced by the hybridoma cell line deposited under ATCC Accession No. PTA-123440, on AUG 10, 2016 and designated 23A7D1/F2; or alternatively, the antibody or antibody fragment thereof against 7C4 is produced by the hybridoma cell line deposited under ATCC Accession No. PTA-123441, on AUG 10, 2016 and designated 25G9B1/F2.

[0135] In certain aspects, the level or concentration of 7C4 can be used to monitor patients or a subject over time. For example, by monitoring the concentration or level of 7C4 longitudinally at various time points, the nature of the bile acid malabsorption can be ascertained, understood and treated. In certain instances, the level or concentration of 7C4 is measured before treatment has started (to). Thereafter, at a time point after treatment, the level or concentration of 7C4 is measured at (ti). At a time point further along the treatment period, the level or concentration of 7C4 is measured again (t₂).

[0136] In one embodiment, the present invention provides a method for diagnosing, and/or monitoring and/or treating bile acid malabsorption, the method comprising:

(a) measuring a level of 7C4 in a sample in a patient using an immunoassay with an antibody or an antibody fragment to generate a to concentration; (b) optionally administering a treatment regimen to the subject to treat the bile acid malabsorption; (c) measuring a level of 7C4 in a sample in a patient later in time from to using an immunoassay with an antibody or an antibody fragment to generate a t₁ 7C4 concentration; and

[0137] In certain aspects, the method is limited to diagnosis. In other aspects, the method includes diagnosing and treating. In still other aspects, the method includes diagnosing, treating and monitoring.

[0138] In certain aspects, the level of serum 7C4 at time to (e.g., fasting serum 7C4) within a mammal is greater than about 20 ng of 7C4 per mL of serum (e.g., greater than about 25 ng/mL, greater than about 26 ng/mL, greater than about 27 ng/mL, greater than about 28 ng/mL, greater than about 29 ng/mL, greater than about 30 ng/mL, greater than about 31 ng/mL, greater than about 32 ng/mL, greater than about 33 ng/mL, greater than about 34 ng/mL, or greater than about 35 ng/mL) which can indicate that the mammal is to be treated with one or more bile acid sequestrants. At time ti after treatment, the level of serum 7C4 is reduced below 20 ng/mL of serum.

[0139] In one embodiment, the present invention provides a method for treating a diarrhea condition, the method comprising

(a) identifying a mammal having a diarrhea condition with a serum level of 7C4 greater than about 20 ng/mL of serum, and

(b) administering a composition comprising a bile acid sequestrant to the identified mammal under conditions wherein the severity of the diarrhea condition is reduced.

[0140] In certain aspects, bile acid sequestrant agents can be used to treat bile acid malabsorption. Cholestyramine and/or colestipol can be used. Colesevelam can also be used and some patients tolerate this more easily. Another treatment option is a farnesoid X receptor agonist obeticholic acid, which has shown clinical and biochemical benefit. A farnesoid X receptor agonist named LJN452 can also be used.

[0141] In certain aspects, the composition containing a bile acid sequestrant can be administered to the subject in any amount, at any frequency, and for any duration effective to achieve a desired outcome (e.g., to treat diarrhea). In some cases, a composition containing a bile acid sequestrant can be administered to a subject to reduce colonic transit by 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70 percent or more. An effective amount of a composition containing a bile acid sequestrant can be any amount that reduces a subject's diarrhea without producing significant toxicity. Typically, an effective amount of a composition containing a bile acid sequestrant can be any amount greater than or equal to about 250 mg of a bile acid sequestrant (e.g., greater than or equal to about 250, 500, 750, 1000, 1250, 1500, 1750, 2000, or more mg of, for example, colesevelam per

administration) provided that that amount does not induce significant toxicity to the mammal upon administration. In some cases, an effective amount of a bile acid sequestrant such as colesevelam can be between 250 mg and 10 g (e.g., between 250 mg and 1250 mg, between 500 mg and 1500 mg, or between 750 mg and 2000 mg). Various factors can influence the actual effective amount used for a particular application. For example, the frequency of administration, duration of treatment, use of multiple treatment agents, route of

administration, and severity of the diarrhea may require an increase or decrease in the actual effective amount administered.

[0142] In certain aspects, the frequency of administration of a composition containing a bile acid sequestrant can be any frequency that reduces a subject's diarrhea without producing significant toxicity. For example, the frequency of administration can be from about three times a day to about twice a week (e.g., once a day). The frequency of administration can remain constant or can be variable during the duration of treatment. For example, a composition containing a bile acid sequestrant can be administered daily, twice a day, five days a week, or three days a week. A composition containing a bile acid sequestrant can be administered for five days, 10 days, three weeks, four weeks, eight weeks, 48 weeks, one year, 18 months, two years, three years, or five years. A course of treatment can include rest periods. For example, a composition containing a bile acid sequestrant can be administered for five days followed by a ten-day rest period, and such a regimen can be repeated multiple times. As with the effective amount, various factors can influence the actual frequency of administration used for a particular application. For example, the effective amount, duration of treatment, use of multiple treatment agents, route of

administration, and severity of the diarrhea may require an increase or decrease in

administration frequency. A. Polyclonal Antibodies

[0143] Polyclonal antibodies provided herein can be of any isotype such as one of the major antibody isotypes: IgA, IgD, IgE, IgG, and IgM. In some embodiments, the antibody can be classified as an IgGi, IgG₂, IgG₃, IgG₄, IgAi or IgA₂ antibody. In some instances, the antibody has a kappa (κ) light chain or a lambda (λ) light chain.

[0144] Polyclonal antibodies are preferably raised in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of an antigen of the invention and an adjuvant. Typically, it is useful to conjugate the antigen of interest to a carrier protein that is immunogenic in the species to be immunized using a bifunctional or derivatizing agent. Non-limiting examples of bifunctional or derivatizing agents include maleimidobenzoyl sulfosuccinimide ester

(conjugation through cysteine residues), N-hydroxysuccinimide (conjugation through lysine residues), glutaraldehyde, succinic anhydride, SOCl₂, and R¹N=C=NR, wherein R and R¹ are different alkyl groups.

[0145] Animals are immunized against the antigens of the invention or an immunogenic conjugate or derivative thereof by combining, e.g., 100 μg (for rabbits) or 5 μg (for mice) of the antigen or conjugate with 3 volumes of Freund's complete adjuvant and injecting the solution intradermally at multiple sites. One month later, the animals are boosted with about 1/5 to 1/10 the original amount of conjugate in Freund's incomplete adjuvant by

subcutaneous injection at multiple sites. Seven to fourteen days later, the animals are bled and the serum is assayed for antibody titer. Animals are typically boosted until the titer plateaus. Preferably, the animal is boosted with the conjugate of the same antigen, but conjugation to a different immunogenic antigen and/or through a different cross-linking reagent may be used. In certain instances, aggregating agents such as alum can be used to enhance the immune response. Detailed descriptions of methods for producing polyclonal antibodies is found in, e.g., Antibodies, A Laboratory Manual, Harlow and Lane, Eds., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1988).

B. Monoclonal Antibodies

[0146] Monoclonal antibodies provided herein can be of any isotype such as one of the major antibody isotypes: IgA, IgD, IgE, IgG, and IgM. In some embodiments, the antibody can be classified as an IgGi, IgG₂, IgG₃, IgG₄, IgAi or IgA₂ antibody. In some instances, the antibody has a kappa (κ) light chain or a lambda (λ) light chain. [0147] Monoclonal antibodies are generally obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally-occurring mutations that may be present in minor amounts. Thus, the modifier "monoclonal" indicates the character of the antibody as not being a mixture of discrete antibodies. For example, monoclonal antibodies can be made using the hybridoma method described by Kohler et al, Nature, 256:495 (1975) or by any recombinant DNA method known in the art {see, e.g., U.S. Patent No. 4,816,567).

[0148] In the hybridoma method, a mouse or other appropriate host animal {e.g., hamster) is immunized as described above to elicit lymphocytes that produce or are capable of producing antibodies which specifically bind to the polypeptide of interest used for immunization. Alternatively, lymphocytes are immunized in vitro. The immunized lymphocytes are then fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form hybridoma cells {see, e.g., Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, pp. 59-103 (1986)). The hybridoma cells thus prepared are seeded and grown in a suitable culture medium that preferably contains one or more substances which inhibit the growth or survival of the unfused, parental myeloma cells. For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT), the culture medium for the hybridoma cells will typically include hypoxanthine, aminopterin, and thymidine (HAT medium), which prevent the growth of HGPRT-deficient cells.

[0149] Preferred myeloma cells are those that fuse efficiently, support stable high-level production of antibody by the selected antibody-producing cells, and/or are sensitive to a medium such as HAT medium. Examples of such preferred myeloma cell lines for the production of human monoclonal antibodies include, but are not limited to, murine myeloma lines such as those derived from MOPC-21 and MPC-11 mouse tumors (available from the Salk Institute Cell Distribution Center; San Diego, CA), SP-2 or X63-Ag8-653 cells

(available from the American Type Culture Collection; Rockville, MD), and human myeloma or mouse-human heteromyeloma cell lines {see, e.g., Kozbor, J. Immunol, 133 :3001 (1984); and Brodeur et al. , Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, pp. 51-63 (1987)).

[0150] The culture medium in which hybridoma cells are growing can be assayed for the production of monoclonal antibodies directed against the polypeptide of interest. Preferably, the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as a radioimmunoassay (RIA) or an enzyme-linked immunoabsorbent assay (ELISA). The binding affinity of monoclonal antibodies can be determined using, e.g., the Scatchard analysis of Munson et al., Anal.

Biochem., 107:220 (1980).

[0151] After hybridoma cells are identified that produce antibodies of the desired specificity, affinity, and/or activity, the clones may be subcloned by limiting dilution procedures and grown by standard methods {see, e.g., Goding, Monoclonal Antibodies:

Principles and Practice, Academic Press, pp. 59-103 (1986)). Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 medium. In addition, the hybridoma cells may be grown in vivo as ascites tumors in an animal. The monoclonal antibodies secreted by the subclones can be separated from the culture medium, ascites fluid, or serum by conventional antibody purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography. [0152] DNA encoding the monoclonal antibodies can be readily isolated and sequenced using conventional procedures {e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). The hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA may be placed into expression vectors, which are then transfected into host cells such as E. coli cells, simian COS cells, Chinese Hamster Ovary (CHO) cells, or myeloma cells that do not otherwise produce antibody, to induce the synthesis of monoclonal antibodies in the recombinant host cells. See, e.g., Skerra et a/., Curr. Opin. Immunol, 5:256-262 (1993); and Pluckthun, Immunol Rev., 130: 151-188 (1992). The DNA can also be modified, for example, by substituting the coding sequence for human heavy chain and light chain constant domains in place of the homologous murine sequences {see, e.g., U.S. Patent No. 4,816,567; and

Morrison et al, Proc. Natl. Acad. Sci. USA, 81 :6851 (1984)), or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non- immunoglobulin polypeptide.

[0153] In a further embodiment, monoclonal antibodies or antibody fragments thereof can be isolated from antibody phage libraries generated using the techniques described in, for example, McCafferty et al, Nature, 348:552-554 (1990); Clackson et al, Nature, 352:624- 628 (1991); and Marks et al, J. Mol. Biol, 222:581-597 (1991). The production of high affinity (nM range) human monoclonal antibodies by chain shuffling is described in Marks et al, BioTechnology, 10:779-783 (1992). The use of combinatorial infection and in vivo recombination as a strategy for constructing very large phage libraries is described in

Waterhouse et al, Nuc. Acids Res., 21 :2265-2266 (1993). Thus, these techniques are viable alternatives to traditional monoclonal antibody hybridoma methods for the generation of monoclonal antibodies.

[0154] The monoclonal antibody against C74 may be produced by the hybridoma clone deposited under ATCC Accession No. PTA- 123441, on AUG 10, 2016 and designated 25G9B1 F2; or alternatively, the antibody or antibody fragment thereof is produced by the hybridoma cell line deposited under ATCC Accession No. PTA-123440, on AUG 10, 2016 and designated 23 A7D1 F2; or the antibody or antibody fragment thereof is produced by the hybridoma cell line deposited under ATCC Accession No. PTA-123439, on AUG 10, 2016 and designated 1G11C12/D2.

C. Antibody Fragments [0155] Various techniques have been developed for the production of antibody fragments. Traditionally, these fragments were derived via proteolytic digestion of intact antibodies {see, e.g., Morimoto et al, J. Biochem. Biophys. Meth., 24: 107-117 (1992); and Brennan et al, Science, 229:81 (1985)). However, these fragments can now be produced directly using recombinant host cells. For example, the antibody fragments can be isolated from the antibody phage libraries discussed above. Alternatively, Fab'-SH fragments can be directly recovered from E. coli cells and chemically coupled to form F(ab')₂ fragments (see, e.g., Carter et al, BioTechnology, 10: 163-167 (1992)). According to another approach, F(ab')₂ fragments can be isolated directly from recombinant host cell culture. Other techniques for the production of antibody fragments will be apparent to those skilled in the art. In other embodiments, the antibody of choice is a single chain Fv fragment (scFv). See, e.g., PCT

Publication No. WO 93/16185; and U.S. Patent Nos. 5,571,894 and 5,587,458. The antibody fragment may also be a linear antibody as described, e.g., in U.S. Patent No. 5,641,870. Such linear antibody fragments may be monospecific or bispecific.

D. Bispecific Antibodies

[0156] Bispecific antibodies are antibodies that have binding specificities for at least two different epitopes. Exemplary bispecific antibodies may bind to two different epitopes of the same polypeptide of interest. Other bispecific antibodies may combine a binding site for the polypeptide of interest with binding site(s) for one or more additional antigens. Bispecific antibodies can be prepared as full-length antibodies or antibody fragments (e.g., F(ab')₂ bispecific antibodies).

[0157] Methods for making bispecific antibodies are known in the art. Traditional production of full-length bispecific antibodies is based on the co-expression of two immunoglobulin heavy chain-light chain pairs, where the two chains have different specificities (see, e.g., Millstein et al., Nature, 305:537-539 (1983)). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. Purification of the correct molecule is usually performed by affinity chromatography. Similar procedures are disclosed in PCT Publication No. WO 93/08829 and Traunecker et al. , EMBO J. , 10:3655-3659 (1991).

[0158] According to a different approach, antibody variable domains with the desired binding specificities (antibody-antigen combining sites) are fused to immunoglobulin constant domain sequences. The fusion preferably is with an immunoglobulin heavy chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It is preferred to have the first heavy chain constant region (CHI) containing the site necessary for light chain binding present in at least one of the fusions. DNA encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transfected into a suitable host organism. This provides for great flexibility in adjusting the mutual proportions of the three polypeptide fragments in embodiments when unequal ratios of the three polypeptide chains used in the construction provide the optimum yields. It is, however, possible to insert the coding sequences for two or all three polypeptide chains into one expression vector when the expression of at least two polypeptide chains in equal ratios results in high yields or when the ratios are of no particular significance.

[0159] In a preferred embodiment of this approach, the bispecific antibodies are composed of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm, and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in the other arm. This asymmetric structure facilitates the separation of the desired bispecific compound from unwanted immunoglobulin chain combinations, as the presence of an immunoglobulin light chain in only one half of the bispecific molecule provides for a facile way of separation. See, e.g., PCT Publication No. WO 94/04690 and Suresh et al., Meth. Enzymol, 121 :210 (1986).

[0160] According to another approach described in U.S. Patent No. 5,731,168, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture. The preferred interface comprises at least a part of the CH3 domain of an antibody constant domain. In this method, one or more small amino acid side-chains from the interface of the first antibody molecule are replaced with larger side chains {e.g., tyrosine or tryptophan). Compensatory "cavities" of identical or similar size to the large side-chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side-chains with smaller ones {e.g., alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers.

[0161] Bispecific antibodies include cross-linked or "heteroconjugate" antibodies. For example, one of the antibodies in the heteroconjugate can be coupled to avidin, the other to biotin. Heteroconjugate antibodies can be made using any convenient cross-linking method. Suitable cross-linking agents and techniques are well-known in the art, and are disclosed in, e.g., U.S. Patent No. 4,676,980.

[0162] Suitable techniques for generating bispecific antibodies from antibody fragments are also known in the art. For example, bispecific antibodies can be prepared using chemical linkage. In certain instances, bispecific antibodies can be generated by a procedure in which intact antibodies are proteolytically cleaved to generate F(ab')₂ fragments {see, e.g., Brennan et al, Science, 229:81 (1985)). These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The Fab' fragments generated are then converted to thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB derivatives is then reconverted to the Fab'-thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount of the other Fab'- TNB derivative to form the bispecific antibody.

[0163] In some embodiments, Fab'-SH fragments can be directly recovered from E. coli and chemically coupled to form bispecific antibodies. For example, a fully humanized bispecific antibody F(ab')₂ molecule can be produced by the methods described in Shalaby et al, J. Exp. Med., 175: 217-225 (1992). Each Fab' fragment was separately secreted from E. coli and subjected to directed chemical coupling in vitro to form the bispecific antibody. [0164] Various techniques for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bispecific antibodies have been produced using leucine zippers. See, e.g., Kostelny et al, J. Immunol, 148: 1547- 1553 (1992). The leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion. The antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers. This method can also be utilized for the production of antibody homodimers. The "diabody" technology described by Hollinger et al, Proc. Natl. Acad. Sci. USA,

90:6444-6448 (1993) has provided an alternative mechanism for making bispecific antibody fragments. The fragments comprise a heavy chain variable domain (VH) connected to a light chain variable domain (VL) by a linker which is too short to allow pairing between the two domains on the same chain. Accordingly, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thereby forming two antigen binding sites. Another strategy for making bispecific antibody fragments by the use of single-chain Fv (sFv) dimers is described in Gruber et al, J.

Immunol, 152:5368 (1994).

E. Antibody Purification

[0165] The antibodies can be purified by methods known to the skilled artisan. Purification methods include, among others, selective precipitation, liquid chromatography, HPLC, electrophoresis, chromatofocusing, gel electrophoresis, dialysis, and various affinity techniques. Selective precipitation may use ammonium sulfate, ethanol (Cohn precipitation), polyethylene glycol, or others available in the art. Liquid chromatography mediums, include, among others, ion exchange medium DEAE, polyaspartate), hydroxylapatite, size exclusion (e.g., those based on crosslinked agarose, acrylamide, dextran, etc.), hydrophobic matrixes (e-g, Blue Sepharose). Affinity techniques typically rely on proteins that interact with the immunoglobulin Fc domain. Protein A from Staphylococcus aureas can be used to purify antibodies that are based on human γΐ, γ2, or γ4 heavy chains (Lindmark et al, J. Immunol. Meth. 62: 1-13 (1983)). Protein G from C and G streptococci is useful for all mouse isotypes and for human γ3 (Guss et al, EMBO J. 5: 15671575 (1986)). Protein L, a

Peptostreptococcus magnus cell-wall protein that binds immunoglobulins (Ig) through k light-chain interactions (BD Bioscience/ClonTech. Palo Alto, CA.), is useful for affinity purification of Ig subclasses IgM, IgA, IgD, IgG, IgE and IgY. Recombinant forms of these proteins are also commercially available. If the antibody contains metal binding residues, such as phage display antibodies constructed to contain histidine tags, metal affinity chromatography may be used.

[0166] When sufficient amounts of specific cell populations are available, antigen affinity matrices may be made with the cells to provide an affinity method for purifying the antibodies. The matrix to which the affinity ligand is attached is most often agarose, but other matrices are available. Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose. Where the antibody comprises a CH3 domain, the Bakerbond ABX^™ resin (J. T. Baker; Phillipsburg, N.J.) can be useful for purification. Other techniques for protein purification such as fractionation on an ion-exchange column, ethanol

precipitation, reverse phase HPLC, chromatography on silica, chromatography on heparin SEPHAROSE™, chromatography on an anion or cation exchange resin (such as a

polyaspartic acid column), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are also available depending on the antibody to be recovered. [0167] When using recombinant techniques, antibodies can be produced inside an isolated host cell, in the periplasmic space of a host cell, or directly secreted from a host cell into the medium. If the antibody is produced intracellularly, the particulate debris is first removed, for example, by centrifugation or ultrafiltration. Carter et al., BioTech., 10: 163-167 (1992) describes a procedure for isolating antibodies which are secreted into the periplasmic space of E. coli. Briefly, cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonylfluoride (PMSF) for about 30 min. Cell debris can be removed by centrifugation. Where the antibody is secreted into the medium, supernatants from such expression systems are generally concentrated using a commercially available protein concentration filter, for example, an Amicon^® or Millipore Pellicon^® ultrafiltration unit. A protease inhibitor such as PMSF may be included in any of the foregoing steps to inhibit proteolysis and antibiotics may be included to prevent the growth of adventitious

contaminants.

[0168] Following any preliminary purification step(s), the mixture comprising the antibody of interest and contaminants may be subjected to low pH hydrophobic interaction

chromatography using an elution buffer at a pH between about 2.5-4.5, preferably performed at low salt concentrations {e.g., from about 0-0.25 M salt). [0169] One of skill in the art will appreciate that any binding molecule having a function similar to an antibody, e.g., a binding molecule or binding partner which is specific for one or more analytes of interest in a sample, can also be used in the methods and compositions of the invention. Examples of suitable antibody -like molecules include, but are not limited to, domain antibodies, unibodies, nanobodies, shark antigen reactive proteins, avimers, adnectins, anticalms, affinity ligands, phylomers, aptamers, affibodies, trinectins, and the like.

F. Methods for Assessing Reactivity of Isolated Antibodies

[0170] The generation and selection of antibodies can be accomplished several ways. The synthesized and purified antigen corresponding to the metabolite of interest is injected, for example, into mice or rabbits or another mammal, to generate polyclonal or monoclonal antibodies. One skilled in the art will recognize that many procedures are available for the production of antibodies, for example, as described in Antibodies, A Laboratory Manual, Harlow and Lane, Eds., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1988). One skilled in the art will also appreciate that binding fragments or Fab fragments which mimic {e.g., retain the functional binding regions of) antibodies can also be prepared from genetic information by various procedures. See, e.g., Antibody Engineering: A Practical Approach, Borrebaeck, Ed., Oxford University Press, Oxford (1995); and Huse et al., J. Immunol, 149:3914-3920 (1992). [0171] The antibodies that are generated by these methods can then be selected by first screening for affinity and specificity with the purified antigen of interest (such as the biotinylated haptens described herein) and, if required, comparing the results to the affinity and specificity of the antibodies with other antigens that are desired to be excluded from binding. The screening procedure can involve immobilization of the purified antigens in separate wells of microtiter plates. The plates can have streptavidin immobilized thereon and the solution containing a potential antibody or group of antibodies is then placed into the respective microtiter wells and incubated for about 30 minutes to 2 hours. The microtiter wells are then washed and a labeled secondary antibody {e.g., an anti-mouse antibody conjugated to alkaline phosphatase if the raised antibodies are mouse antibodies) is added to the wells and incubated for about 30 minutes and then washed. Substrate is added to the wells and a color reaction will appear where antibody to the immobilized antigen, such as the biotinylated antigen, is present. [0172] The antibodies so identified can then be further analyzed for affinity and specificity. In the development of immunoassays for a target metabolite, the purified target metabolite acts as a standard with which to judge the sensitivity and specificity of the immunoassay using the antibodies that have been selected. Because the binding affinity of various antibodies may differ, e.g., certain antibody combinations may interfere with one another sterically, assay performance of an antibody can be a more important measure than absolute affinity and specificity of that antibody.

[0173] Those skilled in the art will recognize that many approaches can be taken in producing antibodies or binding fragments and screening and selecting for affinity and specificity for the various metabolites of interest, but these approaches do not change the scope of the invention.

III. Methods of Use

[0174] The invention provides a method for determining whether a diarrhea is caused by bile acid malabsorption in a subject using the presence or concentrations (amounts or levels) of the 7a-hydroxy-4-cholesten-3-one (7C4) herein. The method may comprise measuring

7C4 in blood, plasma, serum or stools obtained from a patient by the assay methods described herein. The amount of 7C4 in the serum is correlated to the SeHCAT test. For example, retention at 7 days of >15% BA is consistent with a normal result. Mild BAM is considered 10-15%, moderate 5-10% and severe <5% retention (see, FIG. 12A). The invention is based, in part, on the discovery that the level of 7C4 as measured according to the assay methods described herein decreases as % retention of bile acid increases as determined by SeHCAT retention (see, FIG. 12B).

[0175] The invention also provides a method for determining whether a patient is responding to a treatment for diarrhea caused by bile acid malabsorption. The method may comprise measuring 7C4 in blood, plasma, serum or stools of the patient by the assay methods described herein. In some embodiments, the efficacy of a treatment is predicted based on the level of 7C4 in a biological sample from a diarrhea patient before or after administration of the treatment. The method is useful for determining whether patient has had a clinical response to the treatment. [0176] In certain other aspects, the invention provides a method for evaluating a patient previously diagnosed with bile acid malabsorption (BAM) or prognosing a BAM patient. The method comprises measuring 7C4 in blood, plasma, serum or stool of the patient by an assay method described herein. In some embodiments, the method includes measuring the level of 7C4 in a biological sample from a BAM patient at one time point, measuring the level of 7C4 in a second biological sample from the patient at a second time point, and calculating the change or difference between the levels at the two time points. The method can also include using a statistical algorithm to predict the likelihood that the patient has less or more severe BAM compared to before (e.g., the initial diagnosis of BAM).

III. Examples

Example 1 [0177] This example illustrates the synthetic reactions and scheme to prepare stable synthetic derivatives of 7a-hydroxy-4-cholesten-3-one (7C4) for conjugation to a carrier protein and/or biotinylation conjugation.

A. Synthesis of cholest-4,6-diene-3-one

[0178] In a 500 mL round bottom flask Cholest-4-ene-3-one (2.1 15 g, 5.5 mmols) was suspended in 180 mL of tert-butanol-Toluene (2: 1) mixture. Then added /?-chloranil (2.704 g, 1 1.0 mmol) and p-toluene sulfonic acid PTSA (0.209 g, 1.1 mmol) to the reaction mixture. Then continued at 105 °C for 16 hours. Then cooled to room temperature and evaporated volatiles from the reaction. Then diluted with dichloromethane (200 mL) and successively washed with water (2 x 50 mL), 5% sodium hydroxide solution (3 x 50 mL) and brine (2 x 50 mL). Dried the organic layer over sodium sulfate and evaporated to get the crude product, which was then purified on vacuum column chromatography using hexane-EtOAc (0-50 %). Pure product fractions were combined and evaporated to give the desired product as light green solid (1.910 g). The compound was characterized by TLC comparing with the authentic sample purchased from Steraloids and by 6,7-alpha epoxide formation given below. B. Synthesis of cholest-6,7-alpha epoxy-4-ene-3-one

[0179] In a 250 mL round bottom flask, cholest-4,6-dien-3-one (1.91 g, 5.0 mmol) was dissolved in chloroform (40 mL). Then added w-chloro perbenzoic acid (77%) (w-CPBA) (1.29 g, 7.5 mmol) to the reaction flask. Closed the flask with septum and continued the reaction at room temperature for 5 hours. Tested the reaction mixture on TLC (20% EtOAc in hexanes) only 50% completion was observed. Added additional m-CPBA (0.43 g) to the reaction mixture to complete the conversion. Then diluted the reaction mixture with dichloromethane (50 mL) and washed with aq.NaHC0₃ (1.0 M) solution. Then dried the organic layer over sodium sulfate and evaporated. The resulted crude product was purified on vacuum column chromatography using hexane-EtOAc (0-100%). Pure product fractions were combined and evaporated to give the desired product as tan solid (0.92 g).

[0180] 1H MR (500 MHz, CMoroform-d) δ 6.11 (s, IB), 3.45 (d, 1 = 3.6 Hz, 1H), 3.34 (d, J = 3.5 Hz, 1 H), 2.75 - 2.31 (m, 2H), 2.02 (dt J = 13.0, 3.3 Hz, 1 H), 1.99 - 1.79 (m, 4H), 1.71 (td, J = 13.7, 5.3 Hz, 1H), 1.59 - 1.22 (m, I IH), 1.23 - 1.11 (m, 5H), 1.09 (s, 4H), 1.01 (q, J = 9.2 Hz, I I ! ).. 0.92 (d, J = 6.4 Hz, 3H), 0.87 (dd, J = 6.7, 2,4 Hz, (:»! ! }, 0.73 (s, 3H ) MS: 399.6 [M+l] calculated for C₂₇H₄₂O₂

C. Synthesis of 7-alpha-hydroxy cholest-4-ene-3-one

[0181] Formic acid (38.6 μΕ) and triethylamine (75 μΕ) were added to a suspension of Pd₂(dba)₃ (15 mg) and triphenylphosphene (5.0 mg) in degassed THF (1.0 mL) under inert atmosphere (under Argon). After 10 minutes, a solution of epoxide (150 mg, 0.377 mmol) was added using syringe. Then continued the reaction under argon at room temperature for 16 hours. Tested the reaction on TLC (30 % EtOAc-hexanes), a single product spot at R_f = 0.3 was observed. Then filtered the reaction mixture over celite layer and washed the layer using dichloromethane (10 mL). Then washed the organic layer using NaHC0₃ (1.0 M) (10 mL) solution (pH ~ 7). Then separated organic layer dried over sodium sulfate and evaporated. Purified the resulting residue on vacuum column chromatography using hexane-EtOAc (0- 30%) as gradient eluent. Pure product fractions were combined and evaporated to give the desired product as white solid (65 mg).

[0182] ⁵H NMR (500 MHz, Chloroform-d) δ 5.80 (s, 1H), 3.97 (s, 1H), 2.62 (ddd, J= 14.9, 3.3, 2.1 Hz, 1H), 2.52 - 2.28 (m, 3H), 2.03 (tq, J= 10.0, 3.3 Hz, 2H), 1.90 (dtd, J= 13.2, 9.4, 6.1 Hz, 1H),1.83 - 1.63 (m, 3H), 1.62 - 1.41 (m, 9H), 1.34 (dddd, J= 20.6, 13.4, 7.7, 2.7 Hz, 6H), 1.19 (s, 3H), 1.18 - 1.09 (m, 6H), 1.06 - 0.97 (m, 1H), 0.92 (d, J= 6.5 Hz, 3H), 0.87 (d, J= 2.2 Hz, 3H), 0.86 (d, J= 2.2 Hz, 3H), 0.72 (s, 3H). MS: 423.5 [M+Na] calculated for

D. Synthesis of 3-keto-D-4-cholic -XYL-PEG-disulfide and 3-keto-D-4-cholic-PEG- biotin (side-chain linked cholestenone)

[0183] Procedure: To a solution of 7-a,12-a dihydroxy cholin-4-ene-3-one carboxylic acid methyl ester(400mg, 0.96mmol) in 13 ml methanol was added 0.5M LiOH solution in water(6.1ml, 3.05mmol) slowly over a period of 3 hours. After completion, the reaction was stirred at RT for 2 hours. TLC (95:5 EtO Ac/5% methanol) indicates the formation of desired products. The PH of the reaction solution was adjusted to slightly acidic (pH=5 ) with 1M acetic acid solution at 0°C. White precipitates were collected by filtration, then the solid was dried under high vacuum. After drying, 340mg product was obtained. The crude product was used in the next step 13-120 without further purification.

[0184] To a solution of 7-alpha, 12-alpha dihydroxy cholin-4-ene-3-one carboxylic acid (340mg, 0.84 mmol, from reaction 13-117) in anhydrous DMF(2.5ml), DCC (0.21g, l .Ommol) and DMAP (catalytic amount, 5mg) were added. The reaction mixture was stirred at RT for 10 min and tetrafluorophenol (0.21g, 1.26 mmol) was added and continued stirring another 5 h. TLC indicates the starting material acid is gone. The resulting mixture was filtered and the filtrate was concentrated. The crude product was loaded onto a 12g ISCO silica gel column, and then eluted with 10-80% EtOAc/Hexane. The desired product was collected, after removing solvent under rotovap and drying under high vacuum, 0.265g product was obtained. Yield for two steps 13-117/13-120: 50%

[0185] 1H MR (500 MHz, Chloroform-i ) δ 7.06 - 6.92 (m, 1H), 5.82 (d, J= 1.9 Hz, 1H), 4.01 (d, J= 38.5 Hz, 2H), 2.74 (ddd, J= 13.8, 8.9, 4.7 Hz, 1H), 2.67 - 2.55 (m, 2H), 2.51 - 2.30 (m, 3H), 2.06 - 1.09 (m, 18H), 1.06 (d, J= 6.1 Hz, 3H), 0.77 (d, J= 0.7 Hz, 3H). Mass: calculated (M+23): 575.3, Observed:575.6

E. Synthesis of 3-keto-D-4-cholic-PEG-biotin-final step

[0186] In a 4.0 mL brown glass vial, 7-alpha, 12-alpha dihydroxy cholin-4-ene-3-one carboxylic acid (23 mg, 0.057 mmol) and Biotin-PEG7- H2 (34 mg, 0.057 mmol) were dissolved in DMF (0.3 mL). Then disopropyl ethylamine (0.015 g, 0.11 mmol) and PyBOP (30 mg, 0.057 mmmol) were added and the reaction mixture was stirred for 18 hours. Solvent DMF was evaporated and the crude product (81 mg) was purified using dry column on silica (5-15 % methanol in EtOAc) and 21 mg of final product 3-keto-D-4-cholic-PEG-biotin (R_f = 0.3) was obtained. MS: 982.1(M+1)⁺. 1H MR (499 MHz, Chloroform-i ) δ 6.85 (s, 1H), 6.72 (s, 1H), 5.82 (d, J= 1.3 Hz, 1H), 5.70 (s, 1H), 4.93 (s, 1H), 4.56 - 4.43 (m, 1H), 4.35 - 4.25 (m, 1H), 4.06 - 3.91 (m, 2H), 3.72 - 3.53 (m, 29H), 3.45 (d, J= 5.8 Hz, 5H), 3.22 - 3.09 (m, 1H), 2.92 (dd, J= 12.9, 5.0 Hz, 1H), 2.73 (d, J= 12.9 Hz, 1H), 2.59 (d, J= 14.9 Hz, 1H), 2.50 - 2.31 (m, 3H), 2.32 - 1.23 (m, 50H), 1.19 (s, 3H), 1.02 (d, J= 6.3 Hz, 3H), 0.75 (s, 3H).

[0187] In a 10 mL round bottom flask, SS-PEG- HS ester (90 mg, 0.081 mmol) and N- Boc-xylyl diamine (42 mg, 0.178 mmol) suspended in DMF (1 mL), Et₃N (0.018 g, 0.178 mmol) was added. The reaction mixture was stirred at room temperature with occasional heating by heat gun (to make everything homogenous solution) for 2 hours under argon atmosphere. Oxalic acid (8 equiv) was added to the mixture and DMF was evaporated. The crude reaction mixture was purified via dry column using 5-10% MeOH in DCM. Solvent was evaporated and then the Boc was removed by dissolving in MeOH (2 ml) and treating with 2N HCl in ether (2ml) for 16 hours. The resulted reaction mixture was evaporated to obtain crude product (0.11 g) and used in the next step without any purification. F. Synthesis of 3-keto-D-4-cholic -XYL-PEG-disulfide-final step

[0188] To a solution of PEG disulfide amine derivative (33 mg) in 1 ml anhydrous DMF at 0°C was added triethyl amine(0.1 ml), then 2,3,5,6-tetrafluorophenyl 4- ((7R, 1 OR, 12S, 13R)-7, 12-dihydroxy- 10, 13-dimethyl-3 -oxo-

2,3,6,7,8,9, 10,11, 12,13, 14,15, 16,17-tetradecahydro-lH-cyclopenta[a]phenanthren-17- yl)pentanoate (50 mg) in 0.5ml anhydrous DMF was added dropwise. The reaction mixture was stirred at room temperature for 16 hours. Solvent DMF was evaporated and the crude product was purified using dry column on silica (5-10 % methanol in DCM) and 35 mg of final product 3-keto-D-4-cholic -XYL-PEG-di sulfide (R_f 0.4) was obtained. MS: 1948.4(M+Na)⁺. 1H MR (499 MHz, Methanol-^) δ 7.30 - 7.22 (m, 4H), 6.25 (dd, J= 9.7, 2.0 Hz, 1H), 6.18 (dd, J= 9.9, 2.5 Hz, 1H), 4.47 - 4.24 (m, 4H), 4.08 - 3.93 (m, 2H), 3.87 (s, 1H), 3.74 (dt, J= 10.6, 6.2 Hz, 4H), 3.63 - 3.53 (m, 27H), 3.15 - 2.96 (m, 1H), 2.95 - 2.81 (m, 3H), 2.72 - 2.43 (m, 4H), 2.42 - 2.12 (m, 5H), 2.10 - 1.53 (m, 8H), 1.45 (dd, J= 9.2, 5.5 Hz, 1H), 1.18 (d, J= 38.7 Hz, 5H), 1.05 (dd, J= 6.5, 3.8 Hz, 4H), 0.86 - 0.70 (m, 5H).

Example 2

[0189] This example illustrates generating antibodies against derivatives of 7a-hydroxy-4- cholesten-3-one (7C4). [0190] Monoclonal antibodies against 7a-hydroxy-4-cholesten-3-one (7C4) derivative described herein were produced. For example, as is shown in FIG. 4A, the 7C4 derivative is linked to a carrier protein via amine or thiol activation. The immunogen was injected into mice to generate monoclonal antibodies or rabbits to produce polyclonal antibodies (FIG 4B). Standard methods known to those skilled in the art were used for antibody generation, for example, techniques as described in ANTIBODIES, A LABORATORY MANUAL, Harlow and Lane, Eds., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1988).

[0191] The 7C4 derivative was also linked to biotin instead of a carrier protein (FIG. 4C). Such a biotinylated hapten was used to test the validity and specificity of the antibodies generated using the following assay. The biotinylated hapten of interest (2 μg/ml) was coated onto a streptavidin plate for 1 hour at room temperature. The antigen was coated on the plate for about 2 hours at about 4° C. To evaluate the specificity or sensitivity of mouse monoclonal antibodies generated against the derivatized antigen, the antibodies were added to the wells and incubated for about 1 hour at room temperature. The plate was washed several times with wash buffer, e.g., PBS and the like. A goat anti -mouse antibody -HRP conjugate was added and incubated for about 1 hour at room temperature. The plate was washed several times with wash buffer. A color substrate was added for the colorimetric reaction. A stop solution was added prior to reading the plate at about 405 nm.

[0192] Assays (direct competitive assays) of antibody specificity showed that the monoclonal antibodies against 7C4 were specific and do not bind to structurally similar compounds such as 7-ketocholesterol, 7a-hydroxy cholesterol, and trihydroxycholestanoic acid (FIG. 13). It was also determined that the monoclonal antibodies against 7C4 also do not bind other compounds present in serum, plasma, blood, or stool samples, such as 5- hydroxy tryptophan, 5HIAA, tryptophan, kynurenine and melatonine. In fact, these other compounds show 0 to < 0.5% cross-reactivity to the monoclonal antibodies.

Example 3

[0193] This example shows that the antibodies generated using the methods disclosed herein can be used in a competitive ELISA assay to accurately and effectively detect, measure and quantitate 7C4 in samples, e.g., patient serum. Advantageously, the antibodies exhibited no cross-reactivity (or substantially no cross-reactivity, <1%) to other probable antigens. The competitive ELISA provides an accurate, quantitative measure of 7C4 concentration or level.

[0194] The competitive ELISA is based on novel antibodies raised to the synthetically made 7C4 analogs (haptens), which serve as the immunogenic conjugate (e.g., antigens). The analogs were specifically designed with a linker to project the small molecule and elicit an immune response specific to the hapten.

Biotinylated hapten

[0195] A biotinylated hapten was generated for 7C4 or a derivative thereof. Instead of conjugating the linker arm to a carrier protein, the linker was conjugated to biotin (FIG. 4C). For example, a biotinylated derivative of 7C4 was chemically synthesized to contain a linker arm, wherein 7C4 is at one end and biotin is at the other end of the linker (FIG. 4C).

Competitive ELISA

[0196] FIG. 1 provides an exemplary embodiment of an indirect competitive ELISA that was be used to detect 7C4 in a patient's sample. The assay plate was made by coating a streptavidin plate with the biotinylated hapten of interest {e.g., biotinylated 7C4). Patient sample or a dilution of the sample was admixed with the antibody against 7C4 {e.g., the anti- 7C4 antibody), and then transferred to the plate. The plate was incubated for 1 hour at room temperature. The incubation condition was selected to provide sufficient time for the antibody to bind to the biotinylated 7C4 or to the 7C4 in the serum. The plate was washed several times with wash buffer, e.g., PBS buffer. A secondary antibody, such as a goat anti- rabbit antibody-URP conjugate or a goat anti-mouse antibody-URP conjugate was added and the plate was incubated at room temperature for 1 hour. The plate was washed several times with wash buffer. A substrate solution was added for a detection reaction, e.g., color reaction, fluorescent reaction, chemiluminescent reaction, or luminescent reaction. The stop solution as added to arrest the substrate reaction. Then, the plate was read at an appropriate wavelength in a spectrophotometer to monitor the detection reaction. Based on the measured concentration of antibody bound to the biotinylated hapten, the concentration of the metabolite of interest is calculated. In this type of assay, there is an inverse relationship between the amount of the metabolite in the sample and the measured level of bound antibody.

[0197] Using the assay format shown in FIG. 1, a standard curve showing increasing concentrations of 7a-hydroxy-4-cholesten-3-one (7C4) in a sample was generated. FIG. 5A is a graph of the data tabulated in FIG. 5B. The higher the concentration of 7C4 in the sample, the less signal that is generated. FIG. 5C shows a tabulation of control data.

[0198] FIGS. 2A-B and FIGS. 3A-B represent alternative assay formats to the format depicted in FIG. 1. FIG. 2B shows an exemplary embodiment of a direct competitive assay with a capture antibody that specifically binds to 7C4 coated onto the assay plate. FIG. 3B shows another exemplary embodiment of a direct competitive assay. In this assay, a secondary antibody such as a goat anti-mouse antibody is coated onto the assay plate, and specifically binds to the monoclonal antibody described herein. Results from the assay format depicted in FIG. 2B are shown in FIG. 6 A-B. FIGS. 6 A-B illustrate one embodiment of control experiments using the 7C4-HRP conjugate versus HRP alone. FIG. 6A shows that using a variety of monoclonal antibodies (x-axis), the 7C4-HRP conjugate binds the antibody and produce a signal whereas negligible signal is produced with HRP alone. FIG. 6B illustrates a dilution series histogram of the 7C4-HRP conjugate.

Example 4 [0199] This example investigates the amount or concentration of 7C4 in 5 different previously validated cohorts.

[0200] 3219 sample were analyzed and the amount of 7C4 was measured and quantitated using methods herein. As shown in FIG. 7 A, Cohort-4 contained 300 healthy control samples. Cohort- 1 contained 670 samples. Cohort-2 contained 470 samples. Cohort-3 contained 483 samples. Cohort-5 contained 1296 samples. Each of the 5 cohorts contained validated samples with the indication as listed. [0201] FIG. 7B shows a sampling of the standard curves, a total of 81 standard curves were generated. An example of a standard curve is shown in FIG. 7C.

[0202] FIG. 8 A illustrates the results of Cohort-4, which cohort contained 300 healthy control samples. Using a direct competitive ELISA, the mean for the 300 samples was 9.8 ng/mL of 7C4 (FIG. 8B). 7C4 is reported as a serum concentration (ng/mL).

[0203] FIG. 9 illustrates the results of the competitive ELISA of Cohort- 1, which cohort has 670 samples. The distribution of 7C4 for the various indications is shown in FIG. 9.

[0204] FIGS. lOA-C illustrate the results of a competitive ELISA of Cohort- 1 having 670 samples. FIG. 10A shows the results for healthy controls; FIG. 10B shows the results for Crohn' s Disease (CD) and FIG. IOC shows the results for diarrhea predominant irritable bowel syndrome (IBS-D). The table provides a summary of the results. FIG. 10D shows the level of 7C4 for various disease indications including CD, IBS-D, UC, and healthy controls. FIG. 10E shows oneway analysis of 7C4 levels by diagnosis. The data shows that the difference in 7C4 level between IBS-D and UC subjects, IBS-D and healthy control subjects, CD and UC subjects, and CD and healthy control subjects is statistically significant.

[0205] Cohort-3 contained 483 samples. The distribution of 7C4 levels in this cohort is shown in the graph of FIG. 1 1. The data is also presented in the table of FIG. 1 IB.

Example 5 [0206] This example compares the ⁷⁵Selenium HomotauroCholic Acid Test (SeHCAT) and the current methods to measure 7 a-hydroxy-4-cholesten-3-one (7C4).

[0207] In the SeHCAT test, a patient ingests a capsule of ⁷⁵selenium homotaurocholic acid (gamma radiolabeled BA). The ⁷⁵selenium is appropriately distributed in the gut after approximately 1 hour, at which time a baseline scan is obtained and represents 100% retention. A follow-up scan on Day 7 is performed. The amount of radioactivity from ⁷⁵selenium on subsequent scans is divided by the baseline scan on Day 1, indicating the percentage of ⁷⁵selenium homotaurocholate remaining in the body and, indirectly, how much was lost in the stool. A single subsequent scan on Day 7 has a sensitivity of 89% and specificity of 100% using whole body retention value. In healthy subjects, 83% of ⁷⁵ selenium homotaurocholic BA is passed into the colon by Day 7. Retention at 7 days of >15% BA is consistent with a normal result. Mild BAM is considered 10-15%), moderate 5-10% and severe <5%> retention (see, FIG. 12A). [0208] FIG. 12B shows that the inventive assay methods described herein correlate with SeHCAT results. As the percent retention of bile acid increases (normal), the amount of serum 7C4 decreases. In other words, bile acid malabsorption is indicated by high 7C4 levels and less retention of bile acid. It should be noted that the levels of 7C4 presented in FIG. 12B can not be directly compared to the levels presented in the other figures, for example, FIG. 8A. It is recognized by those skilled in the art that 7C4 can undergo degradation due to repeated freezing and thawing.

Example 6 [0209] This example evaluates the specificity and sensitivity of the monoclonal antibodies generated herein.

[0210] A direct competitive ELISA assay was used evaluate the specificity and sensitivity of the monoclonal antibodies generated as described herein. The results shows that antibodies from hybridoma clones 1G11C12/D2, 23A7D1/F2, and 25G9B1/F2 specifically bind to 7C4 and have no cross-reactivity to 7-ketocholesterol, 7a-hydroxy cholesterol, and trihydroxycholestanoic acid. These antibodies also do not bind to other metabolites found in serum such as serotonin (5-HT), 5-hydroxyindole-3-acetic acid (5-HIAA), tryptophan (Trp), kynurenic acid (KA), and melatonin (MT). These compounds did not interfere with the binding of the antibody to 7C4. Example 7A

[0211] This example evaluates the analytical and clinical validation of ELISA (enzyme- linked immunosorbent assay) for 7a-Hydroxy-4-cholesten-3-one (7C4), a biomarker for bile acid diarrhea (BAD). Purpose

[0212] 7a-Hydroxy-4-cholesten-3-one (7C4) is an intermediate in bile acid synthesis and a surrogate for hepatic bile acid (BA) synthesis rate. The cause of diarrhea in about 50% of Crohn's disease and about 25% in irritable bowel syndrome - diarrhea (IBS-D) is attributed to bile acid malabsorption. Bile acid diarrhea can be diagnosed by whole body retention of selenium labeled (⁷⁵Se) homotaurocholic acid (⁷⁵SeHCAT), however this technology is not widely available. 7C4 has also been measured by UPLC-MS using a complex extraction procedure. To address these ease of use issues, we have developed and validated a simple competitive ELISA assay to detect 7C4 levels in serum.

Methods

[0213] Monoclonal antibodies (mAb) against synthetic 7C4 were developed and used to coat microtiter plate wells in a competitive ELISA format to measure serum 7C4 levels. Patient serum is mixed with 7C4 conjugated to HRP and then competes to bind to mAb coated on microtiter plate. Method was validated analytically per CLSI guidance doc EP17-A to determine analytical sensitivity, LLOD, LLOQ, reproducibility (intra and inter assay variability), interference and cross reactivity. 7C4 marker was tested in a chronic diarrhea cohort for its prevalence. FIG. 14 shows one embodiment of the parameters of the 7C4 ELISA assay.

Results

[0214] Assay sensitivity for 7C4 was 2.45 ng/mL using calibration curve generated with synthetic quantified 7C4 diluted into normal healthy serum (NHS). With a lower limit of quantitation (LLOQ) of 4.45 ng/ml the assay yields a dynamic range of 4.45 - 101.8 ng/mL. Inter- and intra-assay precision were 11% and 10% CV respectively and accuracy of between two 7C4 spiked serum samples was within 25%. There was no significant interference from lipemic, hemolytic, human anti-mouse antibody (HAMA) or rheumatoid factor in serum. FIG. 15 shows no significant interference of the 7C4 ELISA assay with common

interferrants. 7C4 mAb showed no cross reactivity with 7-ketocholesterol, 7a- hydroxy cholesterol and Trihydroxycholestanoicacid. 31.4% positivity for 7C4 marker was found in a chronic diarrhea cohort using a cutoff of 12ng/mL based on the cut-off study performed internally comparing healthy population (n=224) with chronic diarrhea population (n=306). Conclusions

[0215] A sensitive and specific easy to use competitive ELISA assay has been developed to measure 7C4 in serum. The development of unique monoclonal antibodies measure only 7C4 owing to the complexity of the presence of other similar metabolites in serum. This ELISA assay demonstrates high accuracy and precision with tolerance to known interfering agents. With the observed prevalence of 7C4 being 31.4% in chronic diarrhea cohort, development of 7C4 assay will be useful for diagnosing one of the underlying causes of diarrhea. Example 7B Purpose

[0216] 7a-Hydroxy-4-cholesten-3-one (7C4) is an intermediate in bile acid synthesis and a surrogate for hepatic bile acid (BA) synthesis rate. The cause of diarrhea in about 50% of Crohn's disease and about 25% in irritable bowel syndrome - diarrhea (IBS-D) is attributed to bile acid malabsorption. Bile acid diarrhea can be diagnosed by whole body retention of selenium labeled (⁷⁵Se) homotaurocholic acid (⁷⁵SeHCAT), however this technology is not widely available. 7C4 has also been measured by HPLC-MS using a complex extraction procedure. To address these ease of use issues, we have developed and validated a simple competitive ELISA assay to detect 7C4 levels in serum.

Methods

[0217] Monoclonal antibodies (mAb) against synthetic 7C4 were developed and used to coat microtiter plate wells in a competitive ELISA format to measure serum 7C4 levels. Patient serum is mixed with 7C4 conjugated to HRP and incubated in mAb coated microtiter plate. This 7C4 ELISA was validated analytically per CLSI guidance EP17-A to determine analytical sensitivity, LLOD, LLOQ, reproducibility (intra and inter assay variability), interference and cross reactivity. 7C4 was determined in chronic diarrhea patients (n=306) recruited from 11 sites in the US and in a corresponding healthy cohort (n=224).

Results

[0218] Assay sensitivity for 7C4 was 2.45 ng/mL using calibration curve generated with synthetic quantified 7C4 diluted into normal healthy serum (containing low level of 7C4). The lower limit of quantitation (LLOQ) was 4.45 ng/ml. The assay yields a dynamic range of 4.45 - 101.8 ng/mL. Inter- and intra-assay precision were 11% and 10% CV, respectively. There was no significant interference from lipemic, hemolytic, human anti-mouse antibody (HAMA) or rheumatoid factor in serum. 7C4 ELISA showed no cross reactivity with 7- ketocholesterol, 7a-hydroxy cholesterol and trihydroxycholestanoicacid. 31.4% positivity for 7C4 marker was found in a chronic diarrhea cohort using a clinically-determined cutoff of 12 ng/mL, upper 90^th percentile of a healthy population. Significant differentiation of 7C4 was demonstrated between chronic diarrhea patients and healthy controls (p < 0.0001, see Fig 16). Average 7C4 level in chronic diarrhea was 11.2 ng/mL ±10 vs 7 ng/mL measured in healthy controls. Conclusions

[0219] A simple competitive ELISA assay has been developed to measure 7C4 in serum which demonstrated high analytical sensitivity and >2 log dynamic range. The critical element of the ELISA is use development of a unique monoclonal antibody which detects only 7C4 with little or no reactivity to similar metabolites in serum. This 7C4 ELISA assay demonstrates good reproducibility and tolerance to known interfering agents. The test demonstrates that chronic diarrhea patients have significantly higher levels of 7C4 than healthy controls.

[0220] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

Claims

WHAT IS CLAIMED IS: 1. An isolated antibody or antibody fragment thereof that specifically binds to 7a-hydroxy-4-cholesten-3-one (7C4) and has less than 1% cross-reactivity to one or more members selected from the group consisting of 7-ketocholesterol, 7a- hydroxycholesterol, and trihydroxycholestanoic acid.

2. The isolated antibody or antibody fragment thereof of claim 1, wherein the antibody is a polyclonal antibody or a monoclonal antibody.

3. The isolated antibody or antibody fragment thereof of claim 1, wherein the antibody is a chimeric or a humanized antibody.

4. The isolated antibody or antibody fragment thereof of claim 1, wherein the antibody fragment is a Fab fragment, a Fab' fragment or F(ab)'2 fragment.

5. The isolated antibody or antibody fragment thereof of claim 1, wherein the antibody or antibody fragment thereof is produced by the hybridoma cell line deposited under ATCC Accession No. PTA-123441, on AUG 10, 2016 and designated 25G9B1/F2.

6. The isolated antibody or antibody fragment thereof of claim 1, wherein the antibody or antibody fragment thereof is produced by the hybridoma cell line deposited under ATCC Accession No. PTA-123440, on AUG 10, 2016 and designated 23A7D1/F2.

7. The isolated antibody or antibody fragment thereof of claim 1, wherein the antibody or antibody fragment thereof is produced by the hybridoma cell line deposited under ATCC Accession No. PTA-123439, on AUG 10, 2016 and designated 1G11C12/D2.

8. The isolated antibody or antibody fragment thereof of claim 1, wherein the antibody or antibody fragment thereof is produced by immunizing an animal with an immunogen comprising a 7C4 derivative conjugated to a carrier protein under conditions such that immune cells of the animal produce an antibody or antibody fragment thereof that specifically binds to 7C4; and isolating the antibody or antibody fragment thereof from the animal.

9. The isolated antibody or antibody fragment thereof of claim 8, wherein the animal is a goat, rabbit or mouse.

10. The isolated antibody or antibody fragment thereof of claim 8, wherein the 7C4 derivative comprises a pegylated derivative of 7C4.

11. The isolated antibody or antibody fragment thereof of any one of claims 1 to 10, which has a detectable label.

12. The isolated antibody or antibody fragment thereof of any one of claims 1 to 11, which is immobilized on a solid substrate.

13. A hybridoma cell line which produces and secretes monoclonal antibodies which selectively bind to 7a-hydroxy-4-cholesten-3-one (7C4) and has been deposited under ATCC Accession No. PTA-123441, on AUG 10, 2016 and designated 25G9B1/F2.

14. A hybridoma cell line which produces and secretes monoclonal antibodies which selectively bind to 7a-hydroxy-4-cholesten-3-one (7C4) and has been deposited the antibody or antibody fragment thereof is produced by the hybridoma cell line deposited under ATCC Accession No. PTA-123440, on AUG 10, 2016 and designated 23A7D1/F2.

15. A hybridoma cell line which produces and secretes monoclonal antibodies which selectively bind to 7a-hydroxy-4-cholesten-3-one (7C4) and has been deposited under ATCC Accession No. PTA-123439, on AUG 10, 2016 and designated 1G11C12/D2.

16. A method for detecting the level of 7a-hydroxy-4-cholesten-3-one (7C4) in a sample from a patient suspected of having bile acid malabsorption using an immunoassay, the method comprising:

(a) contacting the isolated antibody or antibody fragment thereof of claim 1, a sample obtained from the patient, and immobilized 7C4 under suitable conditions to form a complex comprising the isolated antibody or antibody fragment thereof and 7C4 present in the sample or the immobilized 7C4;

(b) detecting the level of antibody or antibody fragment thereof bound to the complex comprising the immobilized 7C4; and (c) calculating the level of 7C4 in the sample based on the level of antibody or antibody fragment thereof from step (b).

17. The method of claim 16, wherein the isolated antibody or antibody fragment thereof, the sample, and the immobilized 7C4 are contacted simultaneously.

18. The method of claim 16, wherein the isolated antibody or antibody fragment thereof, the sample, and the immobilized 7C4 are contacted sequentially.

19. The method of claim 16, wherein the isolated antibody or antibody fragment thereof is the primary antibody and a secondary antibody is added to generate a signal.

20. The method of claim 16, wherein the immunoassay is a competitive ELISA.

21. The method of claim 16, wherein the antibody or antibody fragment thereof is produced by the hybridoma cell line deposited under ATCC Accession No. PTA- 123441, on AUG 10, 2016 and designated 25G9B 1/F2.

22. The method of claim 16, wherein the antibody or antibody fragment thereof is produced by the hybridoma cell line deposited under ATCC Accession No. PTA- 123440, on AUG 10, 2016 and designated 23A7D1/F2.

23. The method of claim 16, wherein the antibody or antibody fragment thereof is produced by the hybridoma cell line deposited under ATCC Accession No. PTA- 123439, on AUG 10, 2016 and designated 1G11C12/D2.

24. The method of claim 16, wherein the sample is a member selected from the group consisting of serum, plasma, blood, and stool.

25. A method for detecting the level of 7a-hydroxy-4-cholesten-3-one (7C4) in a sample from a patient suspected of having bile acid malabsorption using an immunoassay, the method comprising:

(a) contacting the antibody or antibody fragment thereof of claim 1, a sample obtained from the patient, and a 7C4-labeled conjugate under suitable conditions to form a complex comprising the isolated antibody or antibody fragment thereof and 7C4 present in the sample or the 7C4-labeled conjugate;

26. The method of claim 25, wherein the antibody or antibody fragment thereof is immobilized.

27. The method of claim 25, wherein the 7C4-labeled conjugate is a HRP- 7C4-conjugate.

28. The method of claim 25, wherein the sample and the 7C4-labeled conjugate contact the antibody or antibody fragment simultaneously.

29. The method of claim 25, wherein the immunoassay is a competitive ELISA.

30. The method of claim 25, wherein the antibody or antibody fragment thereof is produced by the hybridoma cell line deposited under ATCC Accession No. PTA- 123441, on AUG 10, 2016 and designated 25G9B 1/F2.

31. The method of claim 25, wherein the antibody or antibody fragment thereof is produced by the hybridoma cell line deposited under ATCC Accession No. PTA- 123440, on AUG 10, 2016 and designated 23A7D1/F2.

32. The method of claim 25, wherein the antibody or antibody fragment thereof is produced by the hybridoma cell line deposited under ATCC Accession No. PTA- 123439, on AUG 10, 2016 and designated 1G11C12/D2.

33. The method of claim 25, wherein the sample is a member selected from the group consisting of serum, plasma, blood, and stool.

34. A method for diagnosing, and/or monitoring and/or treating bile acid malabsorption, the method comprising: (a) measuring a level of 7C4 in a sample in a patient using an immunoassay with an antibody or an antibody fragment to generate a tO concentration;

(c) measuring a level of 7C4 in a sample in a patient later in time from tO using an immunoassay with an antibody or an antibody fragment to generate a t₁ 7C4 concentration; and

(d) comparing the level of 7C4 at to to the level of 7C4 at tl to ascertain whether the bile acid malabsorption is improving over time.

35. The method of claim 34, wherein the treatment regimen includes a bile acid sequestrant agent.

36. The method of claim 35, wherein the bile acid sequestrant agent is a member selected from cholestyramine, colestipol, colesevelam, and an a farnesoid X receptor agonist.