WO2013132338A2

WO2013132338A2 - Competitive immunoassay for calprotectin

Info

Publication number: WO2013132338A2
Application number: PCT/IB2013/000733
Authority: WO
Inventors: Magne FAGERHOL
Original assignee: Calpro As
Priority date: 2012-03-06
Filing date: 2013-03-05
Publication date: 2013-09-12
Also published as: WO2013132338A3

Abstract

A competitive immunoassay for the S100A9 subunit of calprotectin is described; the immunoassay can be performed on a solid phase platform or a lateral flow platform. Also described are suitable assay devices for carrying out such competitive immunoassays, including lateral flow competitive immunoassays, and methods of use of such immunoassays in the diagnosis of inflammatory bowel disease.

Description

COMPETITIVE IMMUNOASSAY FOR CALPROTECTIN by

Magne K. Fagerhol

CROSS-REFERENCES

[0001] This application claims the benefit of United States Provisional

Application Serial No. 61/607,257 by M.K. Fagerhol, filed March 6, 2012, and entitled "Competitive Immunoassay for Calprotectin," the contents of which are incorporated herein in their entirety by this reference.

FIELD OF THE INVENTION

[0002] This invention is directed to an improved competitive immunoassay format for protein or polypeptide analytes, particularly the protein calprotectin.

BACKGROUND OF THE INVENTION

[0003] The protein calprotectin is used as a marker for a number of inflammatory conditions, including, but not limited to, inflammatory bowel disease (Crohn's disease or ulcerative colitis), cystic fibrosis, psoriasis, rheumatoid arthritis, and chronic bronchitis. Calprotectin is also associated with a number of other inflammatory disease processes, such as allograft rejection, coronary heart disease, atheromatosis, sepsis,

preeclampsia, cirrhosis, periodontitis, obesity, type 2 diabetes, acne vulgaris, asthma, autoimmune disorders, chronic prostatitis, glomerulonephritis, pelvic inflammatory disease, sarcoidosis, and vasculitis. Additionally, inflammatory processes associated with calprotectin contribute to the etiology of cancer, including pancreatic cancer, ovarian cancer, and uterine cancer, and are especially prominent in inflammatory breast cancer. Calprotectin is especially useful as a marker for the diagnosis of inflammatory bowel disease, for screening for inflammatory bowel disease, and for determining the prognosis of inflammatory bowel disease in patients already diagnosed with that condition.

[0004] Calprotectin belongs to the S100 family of proteins. The name derives from the fact that they are resistant to precipitation by ammonium sulfate, so that they are soluble even in 100 per cent saturated (thus S100) ammonium sulfate solution. It is believed that they have evolved by a large number of point mutations, but many amino acid sequence homologies remain. For this reason, some antibodies can bind to epitopes that are common for many or at least several S100 proteins. A common feature of these proteins is that they can bind calcium and zinc and thereby become resistant to enzymatic degradation; this is especially true for calprotectin. Calprotectin is a heterotrimer consisting of two subunits called S100A9 (A9) and one called S100A8 (A8). In the presence of calcium calprotectin will form dimers, while S100A12 (hereafter called A12) will form oligomers, mostly dimers, tetramers, and hexamers. In

calprotectin, each of these subunits (the S100A8 subunit and the S100A9 subunit), can bind two calcium ions, for a total of six calcium ions bound per calprotectin molecule.

[0005] Both calprotectin and A12 are abundant in neutrophil granulocytes and monocytes and are released from these cells during inflammation or cell damage or death. They are therefore found in increased concentration in blood, other bodily fluids, secretions, and excretions during inflammation, for which they may be useful markers.

[0006] Inflammatory bowel disease (ulcerative colitis and Crohn's disease) is an extremely severe condition of the digestive tract. Although generally not life- threatening, it is a condition that severely impacts the quality of life and can substantially disrupt a patient's lifestyle, livelihood and relationships. Moreover, patients with inflammatory bowel disease have an increased risk of colorectal cancer, and there is recent research that suggests that patients with inflammatory bowel disease have an increased risk of endothelial dysfunction and coronary artery disease.

[0007] Inflammatory bowel disease is a chronic disease for which there is no certain cure, although the symptoms can be treated by a number of drugs as well as measures such as dietary changes. Chronic inflammatory bowel disease is characterized by unpredictable flare-ups of symptoms. Such flare-ups of symptoms can severely impair the quality of life of patients with inflammatory bowel disease. Most patients have recurrent periods of active disease, with significant symptoms, or even continuous active inflammation. For patients who do respond to treatment, the duration of the response varies considerably. The course of the disease, the variability of the response to existing treatments, and the unpredictability of flare-ups all make accurate monitoring of the course of the disease a necessity. This is especially significant during periods of low disease activity, when it is important to be able to detect subclinical intestinal inflammation and to be able to predict the possible occurrence of relapses.

[0008] A number of markers have been proposed for use for monitoring inflammatory bowel disease. These markers, in addition to calprotectin, include immunoassays for

cerevisiae antibody (ASCA) (either IgG or IgA), anti OmpC (Escherichia coli outer membrane porin) antibody, and anti-CBirl (flagellin protein) antibody. There are also additional assays for IBD-specific pANCA (perinuclear antineutrophilic cytoplasmic antibody): autoantibody ELISA, IFA perinuclear staining, and DNase sensitivity. Some of these tests are described in T.D. Jaskowski, "Analysis of Serum Antibodies in Patients Suspected of Having Inflammatory Bowel Disease," Clin. Vacc. Immunol. 13: 655-660 (2006), incorporated herein by this reference.

However, these assays have had little acceptance in the medical community. Another alternative immunoassay technique proposed involves diagnosis based on the level of anti-glycan antibodies. Specifically, the antibodies detected are one or more of anti-GIc (β) antibody, anti-GIc (β1 -4) Glc (β) antibody, anti-GIc (β1 -3) Glc (β) antibody, anti- GlcNAc 6-sulfate antibody, anti-dextran antibody, anti-xylan antibody, anti-GlcNAc (β 1 - 4) GlcNac (β) antibody, anti-Gal-3-sulfate (β) antibody, anti-GlcNAc (β 1 -3) GalNAc (β) antibody, anti-GlcNAc (β 1 -3) Gal (β 1 -4) Glc (β) antibody, or anti-Gal (a 1 -3) Gal (β 1 -4) GlcNAc (β) antibody. These assays also have seen little use. Another marker that has been proposed for the diagnosis, detection, or monitoring of inflammatory bowel disease is lactoferrin. However, lactoferrin is not nearly as specific a marker for inflammatory bowel disease as is calprotectin. Additional potential immunoassays employing fecal biomarkers are described in A.D. Sutherland et al., "Review of Fecal Biomarkers in Inflammatory Bowel Disease," DOI: 10.1007/s10350-008-930-8 (2008), incorporated herein by this reference. The markers considered, in addition to

calprotectin and lactoferrin, included lysozyme, polymorphonuclear neutrophil elastase, human neutrophil lipocalin, myeloperoxidase, eosinophil-derived proteins, α-ι-antitrypsin, and nitric oxide. These markers were considered not particularly useful or significant for diagnosis.

[0009] Other non-immunological diagnostic and assay methods are known.

These methods are described, for example, in M.R. Konikoff & L.A. Denson, "Role of Fecal Calprotectin as a Biomarker of Intestinal Inflammation in Inflammatory Bowel Disease," Inflamm. Bowel Pis. 12: 524-533 (2006), incorporated herein by this

reference; in this publication, endoscopy with biopsies is described as the "gold standard" for assessing intestinal inflammation. This cannot be done routinely because it is far too invasive. This publication also mentions other imaging methods such as CT and MRI exams, barium enemas, and enteroclysis, but these methods also cannot be performed routinely. Also known is the labeling of leukocytes with the radioactive isotopes ¹¹¹ In and ⁹⁹Tc with subsequent fecal monitoring of these radioactive isotopes. Despite the high sensitivity and specificity of such radioactive techniques, they have substantial limitations; they are cumbersome, costly, difficult to perform, and expose the patient, particularly the liver and spleen, to radiation. Therefore, the use of such radioactive techniques is not recommended for children, adolescents, or fertile women.

[0010] An important prerequisite for quantitative immunoassays is that the analyte calibrators should have the same molecular configuration as that in the test sample. Calprotectin, a major protein in the cytosol of neutrophil granulocytes, is a heterotrimer of one S100A8 and two S100A9 subunits. The structure of this protein in leukocyte extracts has been determined by electrophoresis, isoelectric focussing, ion exchange chromatography, gel filtration, circular dichroism, equilibrium dialysis and analytical ultracentrifugation (Fagerhol et al., 1980, Dale et al., 1983; Naess-Andresen et al.; 1995; Berntzen & Fagerhol, 1990; Berntzen et al., 1988; and reviewed in the book chapter by Fagerhol et al., 1990). In the presence of calcium in relatively low

concentrations, for instance 2 mM, a dimeric form consisting of two trimers is generated. By immunization of animals, for instance rabbits, antibodies against several epitopes of S100A8 and S100A9 will be generated. Even if the test antibodies react only with one epitope on each of the two subunits, the signal in immunoassays, for instance ELISA, will be stronger (theoretically doubled) if calprotectin is present as a double heterotrimer compared to the single heterotrimer in the absence of calcium. When an assay for calprotectin in stool samples was developed (R0seth et al., 1992), it was found that using a simple extraction buffer, for instance phosphate buffered saline, pH 7.4, only about 15% of the total calprotectin was found in the extract; the sample had to be re- extracted several times to obtain a yield that was close to 100% yield. When extracts were run on gel filtration (gel permeation chromatography), about 90 % of the anti- calprotectin reactivity was found in the 100 to 1000 kDa region suggesting that in stool extracts, calprotectin is present mostly in high molecular size complexes. By use of an improved stool extraction buffer (H. ΤΘΠ et al., "Improved Assay for Fecal Calprotectin," Clin. Chem. Acta 292: 41 -54 (2000), incorporated herein by this reference), the yield of calprotectin increased significantly to 41-100%, with a mean of about 50%. By gel filtration, using a buffer with calcium, of an extract with the higher yield, calprotectin reactivity was found only in the 60 to 80 kDa fractions corresponding to the size of a double heterotrimer; see Figure 1 of ΤΘΠ et al., supra. In samples with lower yields, reactivity was also found in much higher molecular size fraction. Clearly, there are big differences between the native calprotectin purified from leukocytes and that in stool extracts and even between extracts from different individuals. We have also found another important difference, namely that calprotectin in stool samples does not react with a specific monoclonal antibody against the S100A8 subunit, in contrast to strong reactions against native as well as recombinant calprotectin. This should theoretically give falsely low values when stool extracts are run in immunoassays, for instance ELISA, using antibodies reacting both with S100A8 and S100A9 and native calprotectin in the calibrators. This has been supported by extensive experiments where antibodies, both monoclonal antibodies and polyclonal antibodies, have been tried. An additional source of error in estimates is that some epitopes on calprotectin may be altered or hidden in the large complexes referred to above. [0011] Another source of error in measuring analytes such as calprotectin in immunoassays such as sandwich immunoassays is the high dose hook effect. The high dose hook effect refers to measured levels of antigen displaying a significantly lower absorbance than the actual level present in a sample. This appears when a

simultaneous ELISA assay is saturated by a very high concentration of sample antigen binding to all available sites on both the solid phase antibody as well as the detection antibody and thereby preventing the formation of the "sandwich" that is subject to detection in the immunoassay. The "sandwich" consists of antigen bound to two antibody molecules; typically, one of these antibody molecules is unlabeled and bound to a solid support, while the other antibody molecule is labeled with a detectable label. The antigen-saturated detection antibodies in solution will be washed off giving a falsely low signal. A "hook" is observed in the curve when data is plotted as a signal versus antigen concentration. The possible existence of a high dose hook effect is very significant with respect to the use of calprotectin concentrations for the diagnosis or monitoring of inflammatory bowel disease, as, in the event a calprotectin concentration in a stool sample is high enough to trigger the high dose hook effect, it will be incorrectly measured as a low concentration of calprotectin. This, in turn, will cause a failure to properly diagnosis the existence of inflammatory bowel disease, as the concentration apparently measured would typically be below the cutoff value for the existence of inflammatory bowel disease. Thus, active cases of inflammatory bowel diseases would be missed. The clinical consequences of this error are significant, because such patients would then be assumed not to have inflammatory bowel disease, but rather a less serious condition such as irritable bowel syndrome. They would then be given incorrect treatment and their inflammatory bowel disease would go untreated.

[0012] Therefore, the most practical and specific biomarker for the diagnosis, detection, or monitoring of inflammatory bowel disease is calprotectin. Although a number of assays for calprotectin are known, there is a necessity for an improved assay for calprotectin that is specific for calprotectin and has a wide dynamic range, such that it is not susceptible to interference that can occur. Additionally, there is a necessity for an improved assay for calprotectin that is highly specific for calprotectin and can distinguish calprotectin from other closely related S100 proteins. Moreover, there is a necessity for an improved calprotectin assay that avoids a high dose hook effect.

SUMMARY OF THE INVENTION

[0013] It has been found that an improved immunoassay for calprotectin capable of accurately detecting wide ranges of calprotectin in stool samples without interference and without the existence of a high dose hook effect can be achieved by use of a competitive immunoassay using immobilized S100A9 subunit and labeled anti-S100A9 antibody.

[0014] In such a competitive immunoassay, the S100A9 subunit of calprotectin in the sample competes with the immobilized S100A9 subunit for binding of the labeled anti-S100A9 antibody. As only labeled anti-S100A9 antibody bound to the immobilized S100A9 subunit is eventually detected in such a competitive immunoassay, the higher the concentration of calprotectin in the sample, the less labeled antibody is bound to the solid support. A standard curve then can be constructed using appropriate calibrators; the standard curve will reflect the properties of a competitive immunoassay in which a lower signal detected indicates a higher concentration of analyte such as S100A9.

[0015] Competitive immunoassays according to the present invention can be performed on either a solid phase platform, in which the reagents required are successively added to a solid phase without migration of macromolecular components participating in the competitive immunoassay reaction, or a lateral flow platform, in which a sample and a labeled anti-S100A9 antibody, and possibly other components, migrate through a solid support for lateral flow with a detection zone including immobilized S100A9 polypeptide.

[0016] Accordingly, one aspect of the present invention is a method for determining the concentration of calprotectin in a sample comprising the steps of:

(1 ) providing an antigen selected from the group consisting of purified S100A9 polypeptide and purified calprotectin;

(2) coating the antigen onto the solid phase; (3) reacting the solid phase onto which the antigen has been coated with: (i) a sample that may contain calprotectin; and (ii) a labeled antibody specifically binding the antigen;

(4) washing the solid phase subsequent to the reaction of any calprotectin in the sample and the labeled antibody specifically binding the antigen to remove unbound labeled antibody specifically binding the antigen from the solid phase; and

(4) determining the quantity of labeled antibody specifically binding the antigen bound to the solid phase to determine the concentration of calprotectin in the sample;

wherein the quantity of the antigen coated onto the solid phase and the quantity of labeled antibody specifically binding the antigen are chosen such that the antigen coated onto the solid phase and any calprotectin in the sample compete for the labeled antibody specifically binding the antigen such that the quantity of labeled antibody specifically binding the antigen bound to the solid support is related inversely to the concentration of calprotectin in the sample.

[0017] When the antibody is anti-S100A9 antibody, the anti-S100A9 antibody can be a monoclonal antibody or a polyclonal antibody. The anti-S100A9 antibody can be produced using a recombinant S100A9 polypeptide as immunogen. The

recombinant S100A9 polypeptide can have the sequence

MTCKMSQLERNIETIINTFHQYSVKLGHPDTLNQGEFKELVRKDLQNFLKKENKNEKVI EHIMEDLDTNADKQLSFEEFIMLMARLTWASHEKMHEGDEGPGHHHKPGLGEGTP

(SEQ ID NO: 1 ).

[0018] The recombinant S100A9 polypeptide of SEQ ID NO: 1 can be produced by expression of a DNA molecule with the sequence

GGTACCATATGACCTGCAAAATGAGCCAGCTGGAACGTAACATTGAAACCATCATC

AACACCTTTCATCAGTATAGCGTGAAACTGGGCCATCCGGATACCCTGAACCAGG

GCGAATTTATGATCGAACACATCATGGAAGATCTGGATACCAACGCGGATAAACAG

CTGTCTTTCGAAGAAGAACTGGTGCGTAAAGATCTGCAGAACTTCCTGAAAAAAGA

AAACAAAAACGAAAAAGAATTTATTATGCTGATGGCGCGTCTGACCTGGGCGAGCC ATGAAAAAATGCATGAAGGCGATGAAGGCCCGGGTCATCATCATAAACCGGGCCT GGGCGAAGGCACCCCGTGATAACTCG (SEQ ID NO: 3).

[0019] The S100A9 polypeptide coated onto the solid phase can be a purified and isolated naturally occurring S100A9 polypeptide. Alternatively, the S100A9 polypeptide coated onto the solid phase is a recombinant S100A9 polypeptide, produced as described above.

[0020] When calprotectin is used as the immobilized component, the

calprotectin can be purified naturally occurring calprotectin or can be produced by recombinant DNA technology that generates S100A8 polypeptide and S100A9 polypeptide; the S100A8 polypeptide and S100 A9 polypeptide then associate to form calprotectin. When recombinant DNA technology is used, the production of S100A9 polypeptide is as described above. For S100A8 polypeptide, the amino acid sequence of human S100A8 is known and is

MLTELEKALNSIIDVYHKYSLIKGNFHAVYRDDLKKLLETECPQYIRKKGADVWFKELDI NTDGAVNFQEFLILVIKMGVAAHKKSHEESHKE (SEQ ID NO: 4). The sequence of human DNA encoding S100A8 is

ATGTCTCTTGTCAGCTGTCTTTCAGAAGACCTGGTGGGGCAAGTCCGTGGGCATC ATGTTGACCGAGCTGGAGAAAGCCTTGAACTCTATCATCGACGTCTACCACAAGTA CTCCCTGATAAAGGGGAATTTCCATGCCGTCTACAGGGATGACCTGAAGAAATTGC TAGAGACCGAGTGTCCTCAGTATATCAGGAAAAAGGGTGCAGACGTCTGGTTCAA AG AGTTG GATATCAACACTG ATG GTGCAGTTAACTTCCAG G AGTTCCTCATTCTG G TGATAAAGATGGGCGTGGCAGCCCACAAAAAAAGCCATGAAGAAAGCCACAAAGA GTAGCTGAGTTACTGGGCCCAGAGGCTGGGCCCCTGGACATGTACCTGCAGAATA ATAAAGTCATCAATACCTCAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 5). The sequence of SEQ ID NO: 5 is derived from the mRNA and therefore excludes introns possibly present in the genomic sequence. A suitable DNA sequence for expression of S100A8 in Escherichia coli is

GGTACCATATGCTGACCGAACTGGAAAAAGCGCTGAACAGCATCATTGATGTGTAC

CACAAATATAGCCTGATCAAAGGTAACTTTCATGCGGTGTATCGTGATGATCTGAA

AAAACTGCTGGAAACCGAATGCCCGCAGTATATTCGTAAAAAAGGCGCGGATGTG TGGTTTAAAGAACTGGATATCAACACCGATGGCGCGGTGAACTTTCAGGAATTTCT GATCCTGGTTATCAAAATGGGCGTGGCGGCGCATAAAAAAAGCCATGAAGAAAGC CACAAAGAATGATAACTCGAGCTC (SEQ ID NO: 6). Anti-calprotectin antibodies can be polyclonal or monoclonal antibodies.

[0021] Typically, the label of the labeled antibody is an enzyme label, such as a label selected from the group consisting of alkaline phosphatase, horseradish peroxidase, glucose 6-phosphate dehydrogenase, and β-galactosidase. The enzyme typically produces a product that is detected and/or quantitated photometrically.

Alternatively, the enzyme can produce a product that is detected and/or quantitated by a technique selected from the group consisting of detection and/or quantitation of fluorescence, detection and/or quantitation of chemiluminescence, and detection and/or quantitation of bioluminescence. Alternatively, the label of the labeled antibody can be a label selected from the group consisting of a radioactive label, a fluorescent label, a chemiluminescent label, an electrochemiluminescent label, a bioluminescent label, a colloidal metal label, a colloidal metal oxide label, a dye label, a colored latex particle label, a colored polystyrene or polypropylene label, and a liposome label.

[0022] The step of coating the solid support with the S100A9 polypeptide or calprotectin is typically performed in Tris-buffered saline with from about 0.2 mM to 2 mM calcium present. Typically, the incubation of the S100A9 polypeptide or

calprotectin with the solid support occurs at a temperature of from about 0° C to about 10° C. Typically, the incubation of the S100A9 polypeptide or calprotectin with the solid support occurs for an incubation period of from about 6 hours to several weeks.

Typically, subsequent to the step of binding the S100A9 polypeptide or calprotectin to the solid support, the solid support is washed with T s-buffered saline with from about 0.2 mM to 2 mM calcium present. Typically, subsequent to the washing step, a conditioning step is performed on the solid support, wherein the conditioning step comprises incubation of the solid support with a solution of sucrose and bovine serum albumin containing a phosphate buffer. Typically, subsequent to the conditioning step, the solid support is washed with a washing buffer comprising Tris, sodium chloride, magnesium chloride, and a compatible biocide. [0023] Typically, in the performance of the assay, the size of the sample is from about 50 μΙ_ to about 150 μΙ_. Typically, the sample is a fecal sample or a

gastrointestinal (Gl) tract sample, although other samples, such as whole blood, serum, plasma, urine, or crevicular fluid can be used. Typically, in the performance of the assay, subsequent to the addition of the sample and the labeled antibody to the solid support, the solid support is incubated at a temperature of from about 20-25° C for from about 10 minutes to about 20 minutes.

[0024] Typically, the method further comprises the steps of constructing a standard curve and determining the concentration of calprotectin by comparison with the standard curve. The standard curve can be constructed using a plurality of concentrations of purified calprotectin. Alternatively, the standard curve can be constructed using a plurality of concentrations of purified S100A9 polypeptide.

[0025] Accordingly, a method for diagnosing or detecting the presence of active inflammatory bowel disease comprises the steps of:

(2) coating the antigen onto the solid phase;

(3) reacting the solid phase onto which the antigen has been coated with: (i) a sample that may contain calprotectin; and (ii) a labeled antibody specifically binding the antigen;

(4) washing the solid phase subsequent to the reaction of any calprotectin in the sample and the labeled antibody specifically binding the antigen to remove unbound labeled antibody specifically binding the antigen from the solid phase;

(5) determining the quantity of labeled antibody specifically binding the antigen bound to the solid phase to determine the concentration of calprotectin in the sample; and

(6) determining the presence or absence of active inflammatory bowel disease in the patient according to whether the calprotectin concentration in the sample is at least 50 mg/kg, in which case active inflammatory bowel disease is present in the patient, or is less than 50 mg/kg, in which case active inflammatory bowel disease is absent in the patient.

[0026] Similarly, a method for determining whether a patient with inflammatory bowel disease who has been in remission is at risk of suffering a relapse comprising the steps of:

(2) coating the antigen onto the solid phase;

(6) determining the risk of relapse of inflammatory bowel disease in the patient according to whether the calprotectin concentration in the sample is at least 50 mg/kg, in which case a risk of relapse of inflammatory bowel disease exists, or is less than 50 mg/kg, in which case a risk of relapse of inflammatory bowel disease does not exist.

[0027] Yet another method employing a solid phase competitive immunoassay for S100A9 polypeptide is a method of performing an immunoassay for S100A9 and for a related antigen to determine whether inflammatory bowel disease exists in a subject, the method comprising the steps of:

(1 ) providing a stool sample from a subject;

(2) performing a competitive immunoassay for the antigen selected from the group consisting of calprotectin and S100A9 using: (i) an immobilized antigen selected from the group consisting of immobilized calprotectin and immobilized S100A9 polypeptide; and (ii) a labeled antibody that is anti-calprotectin antibody when the innnnobilized antigen is calprotectin and is labeled anti-S100A9 antibody when the innnnobilized antigen is S100A9 polypeptide, the competitive immunoassay employing a solid phase platform;

(3) performing an immunoassay for a related antigen;

(4) determining the concentration of the antigen selected from the group consisting of calprotectin and S100A9 in the stool sample from the results of the competitive immunoassay for the antigen selected from the group consisting of calprotectin and S100A9 employing the solid phase platform;

(5) determining the concentration of the related antigen in the stool sample from the results of the immunoassay for the related antigen; and

(6) comparing the concentration of the antigen selected from the group consisting of calprotectin and S100A9 and the concentration of the related antigen in the stool sample with the cutoff values for the antigen selected from the group consisting of calprotectin and S100A9 and for the related antigen to determine whether or not inflammatory bowel disease exists in the subject.

[0028] The related antigen can be S100A12 polypeptide or lactoferrin. The immunoassay for the related antigen can be performed in a competitive immunoassay or a sandwich immunoassay.

[0029] As indicated above, competitive immunoassays for S100A9 polypeptide or for calprotectin can be performed on a lateral flow platform. In general, a device for performing such competitive immunoassays for S100A9 polypeptide or for calprotectin on a lateral flow platform comprises:

lateral flow competitive immunoassay for S100A9 or for calprotectin comprising:

(1 ) a solid support providing lateral flow having a first end and a second end;

(2) a detection zone having an immobilized protein or polypeptide selected from the group consisting of immobilized calprotectin and immobilized S100A9 polypeptide, the detection zone located on the solid support; (3) a conjugate zone having a mobilizable labeled antibody selected from the group consisting of anti-calprotectin antibody and anti-S100A9 antibody located on the solid support; and

(4) a sample pad for application of a sample in operable contact with the first end of the solid support;

wherein a sample applied to the sample pad migrates to the conjugate zone to solubilize and mobilize the mobilizable labeled antibody and the sample and the mobilized labeled antibody migrate to the detection zone, such that any calprotectin or S100A9 polypeptide in the sample competes for binding of the labeled antibody with the immobilized calprotectin or immobilized S100A9 polypeptide bound at the detection zone to perform a competitive immunoassay.

[0030] The S100A9 polypeptide or the calprotectin can be covalently or non- covalently immobilized at the detection zone of the device.

[0031] In such devices, the solid support can be constructed of a material selected from the group consisting of nitrocellulose, glass fiber, paper, nylon, polyvinylidene fluoride, and a synthetic nanoporous polymer. Typically, the solid support is nitrocellulose. In such devices, the labeled anti-S100A9 antibody or the labeled anti-calprotectin antibody can be present on the device before the

commencement of the immunoassay. In another alternative, the labeled anti-S100A9 antibody or the labeled anti-calprotectin antibody is added to the device at the commencement of the immunoassay. The device can further comprise a control zone. Additionally, the device can further comprise a casing having at least one aperture therein.

[0032] In one alternative in which the labeled anti-S100A9 antibody or labeled anti-calprotectin antibody is present on the device before the commencement of the assay, the device comprises:

(1 ) a solid support providing lateral flow having a first end and a second end; (2) a detection zone having an immobilized protein or polypeptide selected from the group consisting of immobilized calprotectin and immobilized S100A9 polypeptide, the detection zone located on the solid support;

(3) a conjugate zone having a mobilizable labeled antibody selected from the group consisting of anti-calprotectin antibody and anti-S100A9 antibody located on the solid support; and

[0033] In another alternative in which the labeled anti-S100A9 antibody or labeled anti-calprotectin antibody is present on the device before the commencement of the assay, the device comprises:

(2) a detection zone having an immobilized protein or polypeptide selected from the group consisting of immobilized calprotectin and immobilized S100A9 polypeptide, the detection zone located on the solid support; and

(3) a conjugate pad having a mobilizable labeled antibody selected from the group consisting of anti-calprotectin antibody and anti-S100A9 antibody in operable contact with the first end of the solid support;

wherein a sample applied to the conjugate pad solubilizes and mobilizes the labeled antibody in the conjugate pad and the sample and the mobilized labeled antibody migrate to the detection zone, such that any calprotectin or S100A9 polypeptide in the sample competes for binding of the labeled antibody with the immobilized calprotectin or immobilized S100A9 polypeptide bound at the detection zone to perform a competitive immunoassay.

[0034] In yet another alternative in which the labeled anti-S100A9 antibody or anti-calprotectin antibody is present on the device before the commencement of the assay, the device comprises:

(2) a detection zone having an immobilized protein or polypeptide selected from the group consisting of immobilized calprotectin and immobilized S100A9 polypeptide, the detection zone located on the solid support;

(3) a conjugate pad having a mobilizable labeled antibody selected from the group consisting of anti-calprotectin antibody and anti-S100A9 antibody in operable contact with the first end of the solid support; and

(4) a sample pad for application of the sample in operable contact with the conjugate pad;

wherein a sample applied to the sample pad migrates to the conjugate pad to solubilize and mobilize the labeled antibody and the sample and the mobilized labeled antibody migrate to the detection zone, such that any calprotectin or S100A9 polypeptide in the sample competes for binding of the labeled antibody with the immobilized calprotectin or immobilized S100A9 polypeptide bound at the detection zone to perform a competitive immunoassay.

[0035] In yet another alternative in which the labeled anti-S100A9 antibody or anti-calprotectin antibody is present on the device before the commencement of the assay, the device comprises:

(1 ) a first substantially planar part, the first substantially planar part comprising a solid support that provides lateral flow, the solid support that provides lateral flow comprising a detection zone having an immobilized protein or polypeptide selected from the group consisting of immobilized calprotectin and immobilized S100A9 polypeptide; (2) a second substantially planar part, the second substantially planar part comprising a conjugate pad having a mobilizable labeled antibody selected from the group consisting of anti-calprotectin antibody and anti-S100A9 antibody; and

(3) a hinge connecting the first substantially planar part with the second substantially planar part;

wherein, after a sample is applied to the conjugate pad to solubilize and mobilize the labeled antibody, the hinge connecting the first substantially planar part with the second substantially planar part is closed so that the conjugate pad is placed into operable contact with the solid support such that any calprotectin or S100A9 polypeptide in the sample competes for binding of the labeled antibody with the immobilized calprotectin or immobilized S100A9 polypeptide bound at the detection zone to perform a competitive immunoassay.

[0036] In yet another alternative in which the labeled anti-S100A9 antibody or anti-calprotectin antibody is present on the device before the commencement of the assay, the device comprises:

(1 ) a first substantially planar part, the first substantially planar part comprising a solid support that provides lateral flow, the solid support that provides lateral flow comprising a detection zone having an immobilized protein or polypeptide selected from the group consisting of immobilized calprotectin and immobilized S100A9 polypeptide;

(2) a second substantially planar part, the second substantially planar part comprising a sample pad for application of a sample and a conjugate pad having a mobilizable labeled antibody selected from the group consisting of anti-calprotectin antibody and anti-S100A9 antibody in operable contact with the sample pad; and

wherein, after a sample is applied to the sample pad and the sample migrates to the conjugate pad to solubilize and mobilize the labeled antibody, the hinge connecting the first substantially planar part with the second substantially planar part is closed so that the conjugate pad is placed into operable contact with the solid support such that any calprotectin or S100A9 polypeptide in the sample competes for binding of the labeled antibody with the immobilized calprotectin or immobilized S100A9 polypeptide bound at the detection zone to perform a competitive immunoassay.

[0037] In another alternative as described above, mobile labeled anti-S100A9 antibody or mobile anti-calprotectin antibody is applied to the device at the

commencement of the assay. One such device comprises:

(1 ) a solid support providing lateral flow having a first end and a second end; and

wherein, after a sample and a mobile labeled antibody selected from the group consisting of anti-calprotectin antibody and anti-S100A9 antibody are applied to the solid support, the sample and the mobile labeled antibody migrate to the detection zone, such that any calprotectin or S100A9 polypeptide in the sample competes for binding of the labeled antibody with the immobilized calprotectin or immobilized S100A9

polypeptide bound at the detection zone to perform a competitive immunoassay.

[0038] Another such device comprises:

(2) a second substantially planar part, the second substantially planar part comprising a sample pad for application of a sample and a conjugate application pad for application of a mobile labeled antibody selected from the group consisting of anti-calprotectin antibody and anti-S100A9 antibody in operable contact with the sample pad; and

(3) a hinge connecting the first substantially planar part with the second substantially planar part; wherein, after a sample is applied to the sample pad and a mobile labeled antibody is applied to the conjugate application pad, the hinge connecting the first substantially planar part with the second substantially planar part is closed so that the sample pad and the conjugate application pad are placed into operable contact with the solid support and the sample and the mobile labeled antibody migrate to the detection zone, such that any calprotectin or S100A9 polypeptide in the sample competes for binding of the labeled antibody with the immobilized calprotectin or immobilized S100A9 polypeptide bound at the detection zone to perform a competitive immunoassay.

[0039] Yet another such device comprises:

(2) a second substantially planar part, the second substantially planar part comprising a sample pad for application of a sample and a mobile labeled antibody selected from the group consisting of anti-calprotectin antibody and anti-S100A9 antibody to the sample pad;

wherein, after a sample and a mobile labeled antibody are both applied to the sample pad, the hinge connecting the first substantially planar part with the second substantially planar part is closed so that the sample pad is placed into operable contact with the solid support and the sample and the mobile labeled antibody migrate to the detection zone, such that any calprotectin or S100A9 polypeptide in the sample competes for binding of the labeled antibody with the immobilized calprotectin or immobilized S100A9 polypeptide bound at the detection zone to perform a competitive immunoassay.

[0040] Labels suitable for use in these lateral flow devices are described above.

[0041] These devices can be used for assays and diagnostic methods, such as an assay of calprotectin concentration, a method of detecting or diagnosing the presence of active inflammatory bowel disease, a method of determining whether a patient with inflammatory bowel disease who has been in remission is at risk of suffering a relapse, or a method of performing an immunoassay for S100A9 polypeptide or calprotectin and for a related antigen to determine whether inflammatory bowel disease exists in a subject, as described above for the solid phase assay alternative.

[0042] Another aspect of the present invention is a kit for immunoassay of S100A9 polypeptide and diagnosis, screening, or monitoring of inflammatory bowel disease comprising, separately packaged:

(1 ) a solid support for performing a lateral flow competitive immunoassay for detection of S100A9 polypeptide; and

(2) a labeled anti-S100A9 antibody to be applied to the solid support.

[0043] The kit can include a single solid support and a quantity of labeled anti- Si 00A9 antibody sufficient for a single assay. Alternatively, the kit can include a plurality of solid supports and a quantity of labeled anti-S100A9 antibody sufficient for a plurality of assays such that the quantity of labeled anti-S100A9 antibody is sufficient for use with each of the solid supports included in the kit. If the label of the labeled anti- Si 00A9 antibody is an enzyme label, the kit can further comprise, separately packaged, a substrate for the enzyme label. As another alternative, the kit can further comprise, separately packaged, a set of S100A9 polypeptide standards of differing concentrations so that a standard curve for S100A9 polypeptide can be established.

[0044] Similarly, yet another aspect of the invention is a kit for immunoassay of calprotectin and diagnosis, screening, or monitoring of inflammatory bowel disease comprises, separately packaged:

(a) a solid support for performing a lateral flow competitive immunoassay for detection of calprotectin; and

(b) a labeled anti-calprotectin antibody to be applied to the solid support.

BRIEF DESCRIPTION OF THE DRAWINGS [0045] These and other features, aspects, and advantages of the present invention will become better understood with reference to the following description, appended claims, and accompanying drawings where:

[0046] Figure 1 A depicts a top view of a first alternative for an assay device that can perform a competitive immunoassay in which the labeled anti-S100A9 antibody is present in the device before the commencement of the assay.

[0047] Figure 1 B depicts a side view of the first alternative for an assay device that can perform a competitive immunoassay of Figure 1 A showing an optional backing and casing.

[0048] Figure 2A depicts a top view of a second alternative for an assay device that can perform a competitive immunoassay in which the labeled anti-S100A9 antibody is present in the device before the commencement of the assay.

[0049] Figure 2B depicts a side view of the second alternative for an assay device that can perform a competitive immunoassay of Figure 2A showing an optional backing and casing.

[0050] Figure 3A depicts a third alternative for an assay device that can perform a competitive immunoassay in which the labeled anti-S100A9 antibody is present in the device before the commencement of the assay.

[0051] Figure 3B depicts a side view of the third alternative for an assay device that can perform a competitive immunoassay of Figure 3A showing an optional backing and casing.

[0052] Figure 4 depicts a fourth alternative for an assay device that can perform a competitive immunoassay in which the labeled anti-S100A9 antibody is present in the device before the commencement of the assay.

[0053] Figure 5 depicts a fifth alternative for an assay device that can perform a competitive immunoassay in which the labeled anti-S100A9 antibody is present in the device before the commencement of the assay.

[0054] Figure 6A depicts a top view of a first alternative for an assay device that can perform a competitive immunoassay in which the labeled anti-S100A9 antibody is added to the device at the commencement of the assay. [0055] Figure 6B depicts a side view of the first alternative for an assay device that can perform a competitive immunoassay of Figure 6A showing an optional backing and casing.

[0056] Figure 7A depicts a top view of a second alternative for an assay device that can perform a competitive immunoassay in which the labeled anti-S100A9 antibody is added to the device at the commencement of the assay.

[0057] Figure 7B depicts a side view of the second alternative for an assay device that can perform a competitive immunoassay of Figure 7A showing an optional backing and casing.

[0058] Figure 8 depicts a third alternative for an assay device that can perform a competitive immunoassay in which the labeled anti-S100A9 antibody is added to the device at the commencement of the assay.

[0059] Figure 9 depicts a standard curve for S100A9 polypeptide obtained by a competitive immunoassay procedure employing a solid phase method.

[0060] Figure 10 depicts the comparison between results obtained by the solid phase competitive immunoassay procedure of Example 1 and the original sandwich immunoassay for calprotectin, indicating that a satisfactory correlation between the results obtained by the solid phase competitive immunoassay procedure and the sandwich immunoassay.

[0061] Figure 1 1 is a photograph of the apparatus used to scan and read the results for the lateral flow competitive immunoassay of Example 3.

[0062] Figure 12 depicts the results with recombinant S100A9 polypeptide in the competitive lateral flow immunoassay of Example 3, showing the stained results with samples at 16 ng/mL, 64 ng/mL, 256 ng/mL, and 1024 ng/mL.

[0063] Figure 13 depicts a standard curve based on the results of Figure 12.

DETAILED DESCRIPTION OF THE INVENTION

[0064] Unexpectedly, it has been found that an improved immunoassay for calprotectin that is effective in determining the concentration of calprotectin in stool samples, such as would be used in clinical practice, and that is not subject to the high dose hook effect that can affect sandwich immunoassays, employs the principle of a competitive immunoassay using either: (i) immobilized S100A9 polypeptide and labeled anti-S100A9 antibody or (ii) immobilized calprotectin and labeled anti-calprotectin antibody.

[0065] In such a competitive immunoassay, the S100A9 subunit of calprotectin or the calprotectin in the sample competes with the immobilized S100A9 polypeptide or with the calprotectin for binding of the labeled anti-S100A9 antibody or labeled anti- calprotectin antibody. As only labeled anti-S100A9 antibody bound to the immobilized S100A9 polypeptide or labeled anti-calprotectin antibody bound to the immobilized calprotectin is eventually detected in such a competitive immunoassay, the higher the concentration of calprotectin in the sample, the less labeled antibody is bound to the solid support. A standard curve then can be constructed using appropriate calibrators; the standard curve will reflect the properties of a competitive immunoassay in which a lower signal detected indicates a higher concentration of analyte such as S100A9 polypeptide or calprotectin.

[0066] The competitive immunoassay for calprotectin uses immobilized S100A9 polypeptide or calprotectin on a solid support. When immobilized S100A9 polypeptide is used, although the S100A9 polypeptide can be isolated and purified naturally- occurring S100A9 polypeptide, it is generally preferred to use recombinant S100A9 polypeptide as described below. Similarly, when immobilized calprotectin is used, the calprotectin can be either purified naturally occurring calprotectin or recombinantly produced calprotectin as described below.

[0067] In the competitive immunoassay for calprotectin, the antibody that specifically binds the immobilized S100A9 polypeptide or calprotectin can be a polyclonal antibody or a monoclonal antibody. However, as described below, it is typically preferred to use a monoclonal antibody that specifically binds the S100A9 polypeptide or the calprotectin.

[0068] As used herein, unless further defined or limited, the term "antibody" encompasses both polyclonal and monoclonal antibodies, as well as genetically engineered antibodies such as chimeric or humanized antibodies of the appropriate binding specificity. As used herein, unless further defined, the term "antibody" also encompasses antibody fragments such as sFv, Fv, Fab, Fab' and F(ab)'2 fragments. Because, in a typical competitive immunoassay, there is no requirement for an antibody molecule to bind to more than one antigen (i.e., S100A9 polypeptide or calprotectin), suitable antibody fragments that can bind only one antigen molecule can be used in assays according to the present invention.

[0069] As described below, various formats can be employed in the

performance of the assay. In one format, a platform similar to that typically employed for ELISA assays is used, designated herein as a "solid phase platform," with the S100A9 polypeptide or calprotectin being bound to the solid support and then sample and labeled antibody added to the solid support after a washing step. The immobilized S100A9 polypeptide or calprotectin and any free S100A9 polypeptide or calprotectin in the sample are then allowed to react and compete with the limited quantity of labeled antibody added to the solid support at this stage. After any S100A9 polypeptide or calprotectin in the sample has been allowed to react and compete for binding of the labeled anti-S100A9 antibody or labeled anti-calprotectin antibody with the immobilized S100A9 polypeptide or immobilized calprotectin bound to the solid support, a washing step is then performed to remove unbound antibody. Labeled antibody bound to the solid support is then detected. A number of alternatives for this assay format are described below.

[0070] In another format, a lateral flow platform is employed as is generally known in the art; this alternative platform is designated herein as a "lateral flow platform." In the lateral flow platform format, immobilized S100A9 polypeptide or immobilized calprotectin is bound at a defined detection zone in one portion of a test strip that is permeable to sample and to mobile labeled antibody. The sample and the mobile labeled antibody are then applied to the test strip and allowed to migrate through the test strip so that any S100A9 polypeptide or calprotectin present in the sample competes for the binding of the antibody with the S100A9 polypeptide or the calprotectin immobilized at the detection zone of the test strip. Various alternatives for applying the sample and the mobile labeled antibody are described below. The quantity of labeled antibody bound to the detection zone is then determined to provide an indication of the quantity of calprotectin in the sample. A number of alternatives for this assay format are described below.

[0071] Although isolated and purified S100A9 polypeptide can be used as the immobilized S100A9 polypeptide, it is generally preferred to use recombinant S100A9 polypeptide. A suitable recombinant A9 protein has the sequence

(SEQ ID NO: 1 ). The genomic DNA sequence encoding S100A9 is

AAACACTCTGTGTGGCTCCTCGGCTTTGACAGAGTGCAAGACGATGACTTGCAAAA TGTCGCAGCTGGAACGCAACATAGAGACCATCATCAACACCTTCCACCAATACTCT GTGAAGCTGGGGCACCCAGACACCCTGAACCAGGGGGAATTCAAAGAGCTGGTG CGAAAAGATCTGCAAAATTTTCTCAAGAAGGAGAATAAGAATGAAAAGGTCATAGA ACACATCATGGAGGACCTGGACACAAATGCAGACAAGCAGCTGAGCTTCGAGGAG TTCATCATGCTGATGGCGAGGCTAACCTGGGCCTCCCACGAGAAGATGCACGAGG GTGACGAGGGCCCTGGCCACCACCATAAGCCAGGCCTCGGGGAGGGCACCCCCT AAGACCACAGTGGCCAAGATCACAGTGGCCACGGCCACGGCCACAGTCATGGTG GCCACGGCCACAGCCACTAATCAGGAGGCCAGGCCACCCTGCCTCTACCCAACC AGGGCCCCGGGGCCTGTTATGTCAAACTGTCTTGGCTGTGGGGCTAGGGGCTGG GGCCAAATAAAGTCTCTTCCTCCAAGTCAAAAAAAAAA (SEQ ID NO: 2). The sequence of SEQ ID NO: 2 is derived from mRNA and therefore excludes introns possibly present in the genomic sequence. A DNA sequence encoding A9 that is suitable for expression in Escherichia coli is

GGTACCATATGACCTGCAAAATGAGCCAGCTGGAACGTAACATTGAAACCATCATC

AACACCTTTCATCAGTATAGCGTGAAACTGGGCCATCCGGATACCCTGAACCAGG

GCGAATTTATGATCGAACACATCATGGAAGATCTGGATACCAACGCGGATAAACAG

CTGTCTTTCGAAGAAGAACTGGTGCGTAAAGATCTGCAGAACTTCCTGAAAAAAGA

AAACAAAAACGAAAAAGAATTTATTATGCTGATGGCGCGTCTGACCTGGGCGAGCC

ATGAAAAAATGCATGAAGGCGATGAAGGCCCGGGTCATCATCATAAACCGGGCCT GGGCGAAGGCACCCCGTGATAACTCG (SEQ ID NO: 3); this sequence is optimized for expression in E. coli.

[0072] The antibody to S100A9 can be either a polyclonal antibody or a monoclonal antibody, although it is generally preferred to use a monoclonal antibody. Whether a polyclonal antibody or a monoclonal antibody is used, it is generally preferred to use purified S100A9 polypeptide as immunogen as described above.

[0073] Methods for expression of recombinant S100A9 polypeptide and purification of the expressed polypeptide for use as immunogen and for binding to the solid support are well known in the art. SEQ ID NO: 3 is only one example of a DNA sequence that can be used to express the amino acid sequence of SEQ ID NO: 1 .

Other DNA sequences can readily be constructed by one of ordinary skill in the art employing the known degeneracy of DNA codons in the genetic code. In general, any DNA sequence encoding the amino acid sequence of SEQ ID NO: 1 can be used, although, in some cases, particular DNA sequences are preferred because of codon utilization biases in some organisms used for expression, such as Escherichia coli or because of the presence of negative c/s-acting sites such as internal RBS sites, TATA boxes, or Chi recombination sites, which may negatively influence expression. The DNA sequence of SEQ ID NO: 3 is optimized for expression in E. coli, with a CAI (codon adaptation index) of 0.97, which indicates that the sequence is extremely well adapted for expression in E. coli. Other DNA sequences can be selected by one of ordinary skill in the art for expression in other organisms as described below.

[0074] Expression techniques are well known in the art. A variety of host- expression vector systems may be utilized to express the S100A9 protein or other proteins intended to be used as immunogens. These include but are not limited to microorganisms such as bacteria transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing a suitable coding sequence; yeast transformed with recombinant yeast expression vectors containing the zinc finger- nucleotide binding coding sequence; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing an appropriate coding sequence; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing an appropriate coding sequence; or animal cell systems infected with recombinant virus expression vectors (e.g., retroviruses, adenovirus, vaccinia virus) containing an appropriate coding sequence, or transformed animal cell systems engineered for stable expression. In such cases where glycosylation may be important, expression systems that provide for translational and post-translational modifications may be used; e.g., mammalian, insect, yeast or plant expression systems.

[0075] Depending on the host/vector system utilized, any of a number of suitable transcription and translation elements, including constitutive and inducible promoters, transcription enhancer elements, transcription terminators, etc. may be used in the expression vector (see e.g., Bitter et al., Methods in Enzymology, 153:516-544, 1987). For example, when cloning in bacterial systems, inducible promoters such as pL of bacteriophage λ, plac, ptrp, ptac (ptrp-lac hybrid promoter) and the like may be used. When cloning in mammalian cell systems, promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the retrovirus long terminal repeat; the adenovirus late promoter; the vaccinia virus 7.5K promoter) may be used. Promoters produced by recombinant DNA or synthetic techniques may also be used to provide for transcription of the inserted coding sequence.

[0076] In bacterial systems a number of expression vectors may be

advantageously selected. For example, when large quantities are to be produced, vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable. Those which are engineered to contain a cleavage site to aid in recovering the protein are preferred. Such vectors include but are not limited to the Escherichia coli expression vector pUR278 (Ruther et al., EM BO J., 2:1791 ,1983), in which the coding sequence may be ligated into the vector in frame with the lac Z coding region so that a hybrid A9 protein-lac Z protein is produced; pIN vectors (Inouye & Inouye, Nucleic Acids Res. 13:3101 -3109, 1985; Van Heeke & Schuster, J. Biol. Chem. 264:5503-5509, 1989); and the like. [0077] In yeast, a number of vectors containing constitutive or inducible promoters may be used. For a review, see Current Protocols in Molecular Biology, Vol. 2, 1988, ed. Ausubel et al., Greene Publish. Assoc. & Wiley Interscience, Ch. 13; Grant, et al., 1987, Expression and Secretion Vectors for Yeast, in Methods in Enzymology, Eds. Wu & Grossman, 31987, Acad. Press, N.Y., Vol. 153, pp. 516-544; Glover, 1986, DNA Cloning, Vol. II, IRL Press, Wash., D.C., Ch. 3; and Bitter, 1987, Heterologous Gene Expression in Yeast, Methods in Enzymology, Eds. Berger & Kimmel, Acad.

Press, N.Y., Vol. 152, pp. 673-684; and The Molecular Biology of the Yeast

Saccharomyces, 1982, Eds. Strathern et al., Cold Spring Harbor Press, Vols. I and II. A constitutive yeast promoter such as ADH or LEU2 or an inducible promoter such as GAL may be used (Cloning in Yeast, Ch. 3, R. Rothstein In: DNA Cloning Vol. 1 1 , A Practical Approach, Ed. D M Glover, 1986, IRL Press, Wash., D.C.). Alternatively, vectors may be used which promote integration of foreign DNA sequences into the yeast chromosome.

[0078] In cases where plant expression vectors are used, the expression of a coding sequence may be driven by any of a number of promoters. For example, viral promoters such as the 35S RNA and 19S RNA promoters of CaMV (Brisson et al., Nature, 310:51 1 -514, 1984), or the coat protein promoter to TMV (Takamatsu et al., EMBO J., 6:307-31 1 , 1987) may be used; alternatively, plant promoters such as the small subunit of RUBISCO (Coruzzi et al., EMBO J. 3:1671 -1680, 1984; Broglie et al., Science 224:838-843, 1984); or heat shock promoters, e.g., soybean hsp17.5-E or hsp17.3-B (Gurley, et al. Mol. Cell. Biol., 6:559-565, 1986) may be used. These constructs can be introduced into plant cells using Ti plasmids, Ri plasmids, plant virus vectors, direct DNA transformation, microinjection, electroporation, or other techniques known in the art. For reviews of such techniques see, for example, Weissbach & Weissbach, Methods for Plant Molecular Biology, Academic Press, NY, Section VIII, pp. 421 -463, 1988; and Grierson & Corey, Plant Molecular Biology, 2d Ed., Blackie,

London, Ch. 7-9, 1988.

[0079] An alternative expression system that can be used to express a protein of the invention is an insect system. In one such system, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells. The polypeptide coding sequence may be cloned into non-essential regions (in Spodoptera frugiperda, for example, the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter). Successful insertion of the polypeptide coding sequence will result in inactivation of the polyhedrin gene and production of non-occluded recombinant virus (i.e., virus lacking the proteinaceous coat coded for by the polyhedrin gene). These recombinant viruses are then used to infect cells in which the inserted gene is expressed. (E.g., see Smith et al., J. Biol. 46:584, 1983; Smith, U.S. Pat. No.

4,215,051 ).

[0080] Eukaryotic systems, and preferably mammalian expression systems, allow for proper post-translational modifications of expressed mammalian proteins to occur. Therefore, eukaryotic cells, such as mammalian cells that possess the cellular machinery for proper processing of the primary transcript, glycosylation,

phosphorylation, and, advantageously secretion of the gene product are suitable host cells for the expression of a polypeptide such as an S100A8, S100A9, or S100A12 polypeptide. Such host cell lines may include but are not limited to CHO, VERO, BHK, HeLa, COS, MDCK, 293, and WI38. However, as detailed above, expression can also be obtained in E. coli bacterial cells and such expression may be preferable; codon optimization for expression in E. coli is described above.

[0081] Mammalian cell systems that utilize recombinant viruses or viral elements to direct expression may be engineered. For example, when using adenovirus expression vectors, the coding sequence of a polypeptide such as an S100A8, S100A9, or S100A12 polypeptide may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene may then be inserted into the adenovirus genome by in vitro or in vivo

recombination. Insertion in a non-essential region of the viral genome (e.g., region E1 or E3) will result in a recombinant virus that is viable and capable of expressing the desired polypeptide in infected hosts (e.g., see Logan & Shenk, Proc. Natl. Acad. Sci. USA 81 :3655-3659, 1984). Alternatively, the vaccinia virus 7.5K promoter may be used, (e.g., see, Mackett et al., Proc. Natl. Acad. Sci. USA, 79:7415-7419, 1982;

Mackett et al., J. Virol. 49:857-864, 1984; Panicali et al., Proc. Natl. Acad. Sci. USA, 79:4927-4931 , 1982). Of particular interest are vectors based on bovine papilloma virus which have the ability to replicate as extrachromosomal elements (Sarver et al., Mol. Cell. Biol. 1 :486, 1981 ). Shortly after entry of this DNA into mouse cells, the plasmid replicates to about 100 to 200 copies per cell. Transcription of the inserted cDNA does not require integration of the plasmid into the host's chromosome, thereby yielding a high level of expression. These vectors can be used for stable expression by including a selectable marker in the plasmid, such as the neo gene. Alternatively, the retroviral genome can be modified for use as a vector capable of introducing and directing the expression of the desired polypeptide in host cells (Cone & Mulligan, Proc. Natl. Acad. Sci. USA 81 :6349-6353, 1984). High level expression may also be achieved using inducible promoters, including, but not limited to, the metallothionein IIA promoter and heat shock promoters.

[0082] For long-term, high-yield production of recombinant proteins, stable expression is preferred. Rather than using expression vectors which contain viral origins of replication, host cells can be transformed with a cDNA controlled by

appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines. For example, following the introduction of foreign DNA, engineered cells may be allowed to grow for 1 -2 days in enriched media, and then are switched to a selective medium. A number of selection systems may be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler et al., Cell 1 1 :223, 1977), hypoxanthine-guanine

phosphoribosyltransferase (Szybalska & Szybalski, Proc. Natl. Acad. Sci. USA,

48:2026, 1962), and adenine phosphoribosyltransferase (Lowy et al., Cell, 22:817, 1980) genes, which can be employed in tk^", hgprt^" or aprf cells respectively. Also, antimetabolite resistance-conferring genes can be used as the basis of selection; for example, the genes for dhfr, which confer resistance to methotrexate (Wigler et al., Natl. Acad. Sci. USA, 77:3567, 1980; O'Hare et al., Proc. Natl. Acad. Sci. USA, 78:1527, 1981 ); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA, 78:2072, 1981 ; neo, which confers resistance to the aminoglycoside G418 (Colberre-Garapin et al., J. Mol. Biol., 150:1 , 1981 ); and hygro, which confers resistance to hygromycin (Santerre et al., Gene, 30:147, 1984). Recently, additional selectable genes have been described, namely trpB, which allows cells to utilize indole in place of tryptophan; hisD, which allows cells to utilize histinol in place of histidine (Hartman & Mulligan, Proc. Natl. Acad. Sci. USA, 85:804, 1988); and ODC (ornithine decarboxylase) which confers resistance to the ornithine decarboxylase inhibitor, 2- (difluoromethyl)-DL-ornithine, DFMO (McConlogue L, In: Current Communications in Molecular Biology, Cold Spring Harbor Laboratory ed., 1987).

[0083] Isolation and purification of microbially expressed protein, or fragments thereof provided by the invention, may be carried out by conventional means including preparative chromatography and immunological separations involving monoclonal or polyclonal antibodies. A particularly suitable anti-S100A9 monoclonal antibody is that designated as Calpro Mab CAL1 -4H1/2/2.

[0084] Once suitably purified preparations of S100A9 polypeptides, are available, antibodies, including both polyclonal and monoclonal antibodies, can be prepared by standard techniques, such as those disclosed in E. Harlow & D. Lane, "Antibodies: A Laboratory Manual" (Cold Spring Harbor Laboratory, 1988), incorporated herein by this reference. Polyclonal antibodies can be produced by immunization of suitable antibody-producing animals such as rabbits, rats, mice, hamsters, guinea pigs, sheep, or goats with the polypeptides. Typically, immunization is performed with the use of an adjuvant such as Freund's adjuvant, aluminum hydroxide adjuvant, Lipid A, muramyl dipeptide, SAF, or RAS. Once polyclonal antibodies have been prepared and antibody-secreting lymphocytes are available, these cells can be fused with appropriate myeloma fusion partners and hybridomas can be selected that grow in culture and produce monoclonal antibodies of the appropriate specificity according to the protocol originally described by Kohler and Milstein. The steps required for fusion, selection, and screening are well known in the art and are described, for example, in E. Harlow & D. Lane, "Antibodies: A Laboratory Manual" (Cold Spring Harbor Laboratory, 1988), incorporated herein by this reference.

[0085] When calprotectin is used as the immobilized component, the

GGTACCATATGCTGACCGAACTGGAAAAAGCGCTGAACAGCATCATTGATGTGTAC

CACAAATATAGCCTGATCAAAGGTAACTTTCATGCGGTGTATCGTGATGATCTGAA

AAAACTGCTGGAAACCGAATGCCCGCAGTATATTCGTAAAAAAGGCGCGGATGTG

TGGTTTAAAGAACTGGATATCAACACCGATGGCGCGGTGAACTTTCAGGAATTTCT

GATCCTGGTTATCAAAATGGGCGTGGCGGCGCATAAAAAAAGCCATGAAGAAAGC

CACAAAGAATGATAACTCGAGCTC (SEQ ID NO: 6). This sequence has been optimized for expression in E. coli and has a codon adaptation index of 0.97. SEQ ID NO: 6 is only one example of a DNA sequence that can be used to express the amino acid sequence of SEQ ID NO: 4. Other DNA sequences can readily be constructed by one of ordinary skill in the art employing the known degeneracy of DNA codons in the genetic code. In general, any DNA sequence encoding the amino acid sequence of SEQ ID NO: 4 can be used, although, in some cases, particular DNA sequences are preferred because of codon utilization biases in some organisms used for expression, such as Escherichia coli or because of the presence of negative c/s-acting sites such as internal RBS sites, TATA boxes, or Chi recombination sites, which may negatively influence expression. The DNA sequence of SEQ ID NO: 6 is optimized for expression in E. coli, with a CAI (codon adaptation index) of 0.97, which indicates that the sequence is extremely well adapted for expression in E coli. Other sequences can be used to express S100A8 in E. coli or in other organisms, including eukaryotic cells such as yeast cells or mammalian cells, as described above.

[0086] In the alternative of an immunoassay in which a platform similar to that typically employed for ELISA assays is used, the immobilized S100A9 polypeptide or the immobilized calprotectin is attached to the surface of a solid phase, such as that of a microtiter well, a magnetic particle, or a plastic bead. Typically, a microtiter well is used. Although the exact nature of the solid support is not critical to the performance of the assay, the dimensions and configuration of the solid support affect the volumes of immobilized S100A9 polypeptide or calprotectin, sample, antibody, and wash buffer used. Various formats are known in the art for such solid supports; one particularly suitable format is the use of a microwell plate with 96 wells. Additional solid supports are described, for example, in D. Wild & W. Kusnezow, "Separation Systems" in The Immunoassay Handbook (D. Wild, ed., 3^rd ed., Elsevier, Amsterdam, 2005), ch. 10, pp. 179-185, incorporated herein by this reference.

[0087] In general, this alternative of an immunoassay method according to the present invention employing the solid phase platform uses the following steps:

(1 ) providing an antigen selected from the group consisting of purified S100A9 polypeptide and purified calprotectin; (2) coating the antigen onto the solid phase;

(5) determining the quantity of labeled antibody specifically binding the antigen bound to the solid phase to determine the concentration of calprotectin in the sample;

[0088] The label of the labeled anti-S100A9 antibody or of the labeled anti- calprotectin antibody can be any conventional label as known in the art. When the assay is performed employing the solid phase platform as described above, in general, an enzyme label is used. When an enzyme label is used, the step of determining the quantity of labeled anti-S100A9 antibody or labeled anti-calprotectin antibody bound to the solid phase comprises the step of incubating the solid phase with a substrate for the enzyme of the enzyme-labeled anti-S100 antibody or anti-calprotectin antibody.

[0089] A number of suitable enzymes are known in the art for use in these assays. These enzymes include, but are not limited to, alkaline phosphatase, horseradish peroxidase, glucose 6-phosphate dehydrogenase, and β-galactosidase. Other enzyme labels are also known in the art. Such additional enzymes include, but are not necessarily limited to, acetate kinase, β-lactamase, glucose oxidase, firefly luciferase, laccase, Renilla luciferase, and xanthine oxidase. Enzyme-labeled antibodies can be prepared by covalent coupling procedures involving reagents such as, but not limited to, glutaraldehyde, N-succinimidyl 3-[pyridyl] propionate,

carbodiimides, carbonyldiimidazole, and other cross-linking reagents known in the art, such as those described in G.T. Hermanson, "Bioconjugate Techniques" (Academic Press, San Diego, 1996), pp. 630-637, incorporated herein by this reference. As another alternative, recombinant antibody conjugates can be prepared by genetic engineering techniques known in the art, the conjugates being produced by transcription and translation of gene fusions.

[0090] In many cases, the enzyme in the enzyme-labeled antibody produces a product that is detected and/or quantitated photometrically, such as by spectroscopy. However, in some alternatives, the enzyme produces a product that is monitored and/or quantitated by other means, such as detection and/or quantitation of fluorescence, bioluminescence, or chemiluminescence. For example, immunoassays that use horseradish peroxidase as the enzyme label can be assayed by the detection of chemiluminescence using a mixture of luminol, peroxide, and an enhancer such as p- iodophenol or by using an acridan derivative. Umbelliferone phosphate is a

nonfluorescent substrate that is converted to the highly fluorescent umbelliferone by the catalytic activity of the enzyme alkaline phosphatase. Another sensitive assay using an alkaline phosphatase label uses a chemiluminescent adamantyl 1 ,2-dioxetane aryl phosphate substrate, which is dephosphorylated by the catalytic activity of alkaline phosphate and decomposes with a concomitant long-lived glow of light, such that the detection limit for alkaline phosphatase using this assay can be as low as 1 zeptomole (10^"21 moles). In another alternative, an enzyme cascade is used, The advantage of the use of an enzyme cascade is that it combines the amplification properties of two enzymes— the alkaline phosphatase present in the antibody label and the second enzyme, alcohol dehydrogenase, in the assay reagent, thereby producing an extremely sensitive assay. For example, the alkaline phosphatase can dephosphorylate NADP to NAD, which then takes part in a reaction catalyzed by alcohol dehydrogenase and the enzyme diaphorase in which ethanol is oxidized to acetaldehyde and p- iodonitrotetrazolium violet is reduced to the detectable formazan by the catalytic activity of diaphorase.

[0091] Other assay techniques involving enzyme-labeled antibodies are known in the art. For example, enzyme multiplied immunoassay technique (EMIT) is a homogeneous immunoassay. In this technique, antibody against the analyte (S100A9 polypeptide or calprotectin, as described above) is added together with substrate for an enzyme label to a sample. Binding of the antibody and the antigen (S100A9

polypeptide or calprotectin) occurs. An aliquot of a conjugate of an enzyme that can catalyze a reaction involving the substrate and the antigen is added to the sample. The enzyme conjugate then binds with any excess antibody to the antigen, forming an antibody-enzyme-conjugate-antigen complex. In the absence of free S100A9 polypeptide or calprotectin in the original sample, the enzyme is inactivated, such as by the binding of the enzyme in the enzyme-conjugate to the antibody which physically blocks access of the substrate to the active site of the enzyme or by changing the conformation of the enzyme and thus altering the activity. In this assay, which is a homogeneous assay not requiring separation of bound and free label, the greater the concentration of analyte (S100A9 polypeptide or calprotectin), the greater is the quantity of enzyme activity.

[0092] Still another antibody immunoassay format known in the art is cloned enzyme donor immunoassay (CEDIA). In CEDIA, inactive fragments (the enzyme donor and acceptor) of the enzyme β-galactosidase are prepared by manipulation of the Z gene of the lac operon of the bacterium Escherichia coli. These two fragments spontaneously reassemble to form active enzyme, even if the enzyme donor is attached to an antigen, such as S100A9 polypeptide or calprotectin. However, binding of antibody to the enzyme donor inhibits reassembly, thus blocking the formation of active enzyme and preventing enzyme activity. Thus, competition between the antigen (S100A9 polypeptide) and the enzyme donor antigen conjugate for a fixed quantity of antibody in the presence of the enzyme acceptor modulates the measured enzyme activity so that high concentrations of antigen produce the least inhibition of enzyme activity and the greatest measured enzyme activity, while low concentrations of antigen produce the most inhibition of enzyme activity and the least measured enzyme activity.

[0093] Various other types of labeled antibodies can be used, such as are well known in the art. For example, immunoassays can be performed using radiolabeled antibodies, antibodies labeled with fluorescent, chemiluminescent,

electrochemiluminescent, or bioluminescent labels, antibodies labeled with colloidal metals such as gold or silver, antibodies labeled with colloidal metal oxide particles, antibodies labeled with dyes, antibodies labeled with colored particles such as antibodies labeled with colored latex particles, antibodies labeled with colored polystyrene or polypropylene particles, antibodies labeled with liposomes, as well as other labels known in the art, such as, but not limited to, Surface-Enhanced Resonant Raman Spectroscopy (SERRS) labels, or signaling aptamers. For example, for bioluminescent labels, native or recombinant apoaequorin from the bioluminescent jellyfish Aequorea can be used as the label; the label is activated by reactions with coelenterazine, and light emission at 469 nm is triggered by reaction with calcium ions, such as calcium chloride. In fluorescence excitation transfer immunoassay, a fluorophore (donor)-labeled antigen competes with an antigen in the sample for binding sites on an antibody labeled with a fluorescent dye (acceptor). The fluorescence of the donor is quenched when it is bound to the acceptor-labeled antibody. Another alternative assay format is immuno-PCR. Immuno-PCR is a heterogeneous

immunoassay in which a piece of single- or double-stranded DNA is used as a label for an antibody in a sandwich assay. Bound DNA label is amplified using PCR. The amplified DNA produced is separated by gel electrophoresis and quantitated by densitometric scanning of a gel stained with the DNA-specific stain ethidium bromide. Still another alternative assay format is luminescent oxygen channeling immunoassay (LOCI). LOCI is a homogeneous sandwich immunoassay in which an antigen (S100A9 polypeptide or calprotectin in this example) links an antibody-coated sensitizer dye- loaded particle (250-nm diameter) and an antibody-coated particle (250-nm diameter) loaded with a mixture of a precursor of a chemiluminescent compound and a

fluorophore. Irradiation produces singlet oxygen at the surface of the sensitizer dye- loaded particle. This diffuses or "channels" to the other particle held in close proximity by the immunochemical reaction between the antigen (S100A9 polypeptide or calprotectin in this example) and antibodies on the particles. The singlet oxygen reacts with the chemiluminescent compound precursor in the particle to form a

chemiluminescent dioxane compound, which then decomposes to emit light via a fluorophore-sensitized mechanism. No signal is obtained from precursor fluorophore- loaded particles that are not linked by an immunological reaction with an antigen (S100A9 polypeptide or calprotectin in this example). Yet another alternative assay format is phosphor immunoassay. Phosphor immunoassay is a heterogeneous immunoassay in which an upconverting phosphor nanoparticle is used as a label. The nanoparticle (200- to 400-nm diameter) is a crystalline lanthanide oxysulfide. It absorbs two or more photons of infrared light (980 nm) and produces light emission at a shorter wavelength (anti-Stokes shift). The phosphorescence is not influences by reaction conditions such as temperature or buffer composition and there is no upconverted signal from biological components in the sample, resulting in a low background. Still another alternative assay format is quantum dot immunoassay. Quantum dot immunoassay is a heterogeneous immunoassay in which a nanometer-sized (less than 10 nm) semiconductor quantum dot is used as a label. A quantum dot is a highly fluorescent nanocrystal composed of CdSe, ZnSe, InP or InAs or a layer of ZnS or CdS on, for example, an CdSe core. Yet another alternative assay format is solid phase, light-scattering immunoassay, in which indium spheres are coated on glass to measure an antibody binding to an antigen (S100A9 polypeptide or calprotectin in this example). Binding of antibody to antigen increases dielectric layer thickness, which produces a greater degree of scatter than in areas where only an antigen is bound; quantitation is achieved by densitometry. Still another alternative assay format is surface effect immunoassay, in which an antibody is immobilized on the surface of a waveguide (a quartz, glass, or plastic slide, or a gold- or silver-coated prism), and binding of an antigen is measured directly by total internal reflection fluorescence, surface plasmon resonance, or attenuated total reflection. Other assay formats are known in the art. [0094] Typically, the step of coating the solid support with the S100A9 polypeptide or the calprotectin is performed in Tris-buffered saline with from about 0.2 mM to 2 mM calcium present, such as calcium chloride. Preferably, the calcium concentration is from about 0.5 mM to about 1 .5 mM. More preferably, the calcium concentration is about 1 mM. Typically, the S100A9 polypeptide or the calprotectin is incubated with the solid support at a concentration of from 0.4 g/mL to about 1 .2 g/mL. Preferably, the S100A9 polypeptide or the calprotectin is incubated with the solid support at a concentration of from 0.6 g/mL to about 1 .0 g/mL. More preferably, the S100A9 polypeptide or the calprotectin is incubated with the solid support at a concentration of about 0.8 g/mL.

[0095] Preferably, for the incubation of the solid support with the S100A9 or the calprotectin polypeptide to bind the S100A9 polypeptide or the calprotectin to the solid support, the solid support is covered with vapor tight adhesive plastic and stored at a temperature from about 0° C to about 10° C, preferably about 5° C, for an incubation period of from about 6 hours to several weeks. Preferably, the incubation period is about 18 hours.

[0096] In one preferred procedure, subsequent to the step of binding the

S100A9 polypeptide or the calprotectin to the solid support, the solid support is washed with Tris-buffered saline with from about 0.2 mM to 2 mM calcium present, such as calcium chloride. Preferably, the calcium concentration is from about 0.5 mM to about 1 .5 mM. More preferably, the calcium concentration is about 1 mM.

[0097] In this procedure, subsequent to the washing step, a conditioning step is typically performed on the solid support. In the conditioning step, the solid support is incubated with a solution of sucrose and bovine serum albumin containing a phosphate buffer, referred to herein as "SBP buffer." Typically, the concentration of sucrose in the SBP buffer is from about 1 .5% to about 3.5%. Preferably, the concentration of sucrose in the SBP buffer is from about 2.0% to about 3.0%. More preferably, the concentration of sucrose in the SBP buffer is about 2.5%. Typically, the concentration of bovine serum albumin in the SBP buffer is from about 0.5% to about 1 .5%. Preferably, the concentration of bovine serum albumin in the SBP buffer is from about 0.75% to about 1 .25%. More preferably, the concentration of bovine serum albumin in the SBP buffer is about 1 .0%. Typically, the concentration of phosphate buffer in the SBP buffer is from about 5 mM to about 15 mM. Preferably, the concentration of phosphate buffer in the SBP buffer is from about 7.5 mM to about 12.5 mM. More preferably, the concentration of phosphate buffer in the SBP buffer is about 10 mM. Typically, the pH of the phosphate buffer is from about 7.5 to about 8.5. Preferably, the pH of the phosphate buffer is from about 7.8 to about 8.2. More preferably, the pH of the phosphate buffer is about 8.0. Typically, in the conditioning step, the solid support is incubated with SBP buffer at room temperature (20-25° C) for about 15 minutes to about 45 minutes, preferably about 30 minutes.

[0098] Subsequent to the conditioning step, the solid support is washed with a buffer referred to herein as "washing buffer." The washing buffer contains Tris, sodium chloride, magnesium chloride, and a compatible biocide such as Kathon®. Typically, the washing buffer contains from about 25 mM to about 75 mM of Tris. Preferably, the washing buffer contains from about 37.5 mM to about 62.5 mM of Tris. More preferably, the washing buffer contains about 50 mM of Tris. Typically, the washing buffer contains from about 100 mM to about 200 mM of sodium chloride. Preferably, the washing buffer contains from about 125 mM to about 175 mM of sodium chloride. More preferably, the washing buffer contains about 150 mM of sodium chloride. Typically, the washing buffer contains from about 0.25 mM to about 0.75 mM of magnesium chloride.

Preferably, the washing buffer contains from about 0.375 mM to about 0.625 mM of magnesium chloride. More preferably, the washing buffer contains about 0.50 mM of magnesium chloride. Typically, the washing buffer contains from about 0.5% to about 1 .5% of a compatible biocide such as Kathon®. Preferably, the washing buffer contains from about 0.75% to about 1 .25% of a compatible biocide such as Kathon®. More preferably, the washing buffer contains about 1 .0% of a compatible biocide such as Kathon®. Typically, the pH of the washing buffer is about 7.5 to about 8.5. Preferably, the pH of the washing buffer is from about 7.8 to about 8.2. More preferably, the pH of the washing buffer is about 8.0. [0099] Subsequent to the washing step, the assay is performed. In the performance of the assay, the size of the sample is typically from about 50 μΙ_ to about 150 μΙ_. The size of the sample will vary with the source of the sample and with the expected concentration of calprotectin in the sample. It is possible, and, in some cases preferable, to run the assay with multiple sample sizes, especially if there is no certain indication from the clinical condition of the patient from which was the sample was obtained about the existence of inflammatory bowel disease. As detailed below, typically, a standard curve is also constructed and the concentration of calprotectin is determined by comparison with the standard curve. In one alternative, the standard curve is constructed using a plurality of concentrations of purified calprotectin. In another alternative, as detailed further below, the standard curve is constructed using a plurality of concentrations of purified S100A9 polypeptide such as the cloned

recombinant S100A9 polypeptide described above.

[0100] After the samples and standards are added to the solid support, the labeled antibody is added to the solid support; as this is a competitive assay, the labeled antibody and the sample are present simultaneously and are in contact with the solid support to which purified S100A9 polypeptide or calprotectin has previously been bound. After the labeled antibody is added, the solid support, such as the wells of a conventional multiwall ELISA plate, is covered with a suitable covering, such as plastic sheeting or film, tape or a lid and incubated at a suitable temperature, such as room temperature (20-25° C) for about 10 minutes to about 20 minutes, preferably for about 15 minutes. Typically, the incubation is performed with horizontal shaking at about 500 rpm. Other suitable incubation conditions can be used as is known in the art.

[0101] Subsequently, the solid support is washed. Typically, the solid support is washed three times with a washing buffer such as phosphate buffered saline. If the labeled antibody is labeled with an enzyme that produces a detectable product, a substrate for the enzyme is then added; if the enzyme requires cofactors, such cofactors are also added at that time. Suitable enzymes include, but are not limited to, alkaline phosphatase, horseradish peroxidase, glucose 6-phosphate dehydrogenase, and β-galactosidase, as described above. Substrates, as well as any required cofactors, for these enzymes are well known in the art. For example, if the enzyme is alkaline phosphatase, a suitable substrate is p-nitrophenylphosphate. If the enzyme is horseradish peroxidase, a suitable substrate is tetramethylbenzidine. If the enzyme is glucose 6-phosphate dehydrogenase, a suitable substrate is glucose 6-phosphate; the cofactor NAD⁺ must also be present as a source of oxidizing power for the

dehydrogenation reaction catalyzed by the enzyme. If the enzyme is β-galactosidase, a suitable substrate is o-nitrophenyl- -D-galactoside. Other enzymes are also known in the art, such as acetate kinase, β-lactamase, glucose oxidase, firefly luciferase, laccase, Renilla luciferase, and xanthine oxidase.

[0102] The reaction of the enzyme with the substrate and any required cofactors is allowed to proceed for an period sufficient to allow the appearance of a sufficient quantity of a detectable product of the enzymatic reaction to detect or determine the quantity of calprotectin in the sample. Typically, when the enzyme is horseradish peroxidase, the reaction of the enzyme with the substrate is allowed to proceed for about 10 to about 30 minutes, preferably for about 15 to about 25 minutes, more preferably about 20 minutes. The appropriate reaction period can be determined by one of ordinary skill in the art for other substrates for horseradish peroxidase and for other combinations of enzymes and substrates, based on factors such as the

concentrations of enzyme and substrate and maximum turnover number for the enzyme.

[0103] At the end of the reaction period, the enzymatic reaction is stopped, typically by addition of an acid. When the enzyme is horseradish peroxidase, the enzymatic reaction can be stopped by addition of a stop solution. Typically, the volume of the stop solution is from about 75 μΙ_ to about 150 μΙ_; preferably, the volume of the stop solution is about 100 μΙ_. Typically, the stop solution is a dilute solution of sulfuric acid. Typically, the concentration of sulfuric acid in the stop solution is from about 0.15 M to about 0.25 M. Preferably, the concentration of sulfuric acid in the stop solution is about 0.2 M.

[0104] When the enzyme used is horseradish peroxidase, the substrate used is tetramethylbenzidine, and the stop solution includes sulfuric acid, the concentration of product is read by determining absorbance at 450 nm. Appropriate wavelengths for absorbance determinations for other substrates used with horseradish peroxidase and other combinations of enzymes and substrates are known in the art.

[0105] When the assay described above is performed in a conventional ELISA microwell plate, the absorbance can be determined using a conventional microwell ELISA reader. However, absorbance determinations can be performed by other methods known in the art and are not limited to the use of a conventional ELISA microwell reader.

[0106] The assay procedure described above is a procedure that employs an enzyme label. However, as described above, the assay procedure of the present invention is not limited to an assay employing an enzyme label. For example, a direct label such as a colloidal gold or silver label can be employed as described above. In this alternative, the quantity of the direct label bound to the solid support can be evaluated by instrumentation known in the art. Similarly, if the label is a fluorescent, chemiluminescent, or bioluminescent label, the quantity of the label bound to the solid support can be evaluated by appropriate optical instrumentation for the detection of fluorescence, chemiluminescence, or bioluminescence.

[0107] Typically, the sample is a fecal sample or a gastrointestinal (Gl) tract sample, although solid phase platform assays according to the present invention can be performed on other samples, such as whole blood, serum, plasma, urine or crevicular fluid. Testing on crevicular fluid can be used to detect or monitor the course of periodontitis (T. Nakamura et al., "The Association of Calprotectin Level in Gingival Crevicular Fluid With Gingival Index and the Activities of Collagenase and Aspartate Aminotransferase in Adult Periodontitis Patients," J. Periodontol. 71 : 361 -367 (2000). If the sample is a fecal sample or a gastrointestinal tract sample, the sample can be extracted prior to performance of the assay according to the procedure described in U.S. Patent No. 6,225,072 by Holtund et al., incorporated herein in its entirety by this reference. Briefly, this extraction procedure comprises: (1 ) mixing a small amount of sample (preferably 10 to 500 mg and more preferably 20-150 mg, optionally

preweighed) with an excess amount of aqueous extraction buffer (preferably in the region of a 50-fold excess (v/v)), comprising at least one dissociating, disaggregating, and/or chelating agent; (2) homogenizing the sample (preferably by vortexing), in a closed tube; (3) separating the solid and liquid material of the dispersion resulting from homogenization of the sample (preferably by centrifugation and additionally or optionally by filtration); and (4) recovering the substantially clear liquid extract resulting from the separation, which contains calprotectin as well as other proteins. A suitable buffer is a citrate buffer with a pH of from about pH 5 to about pH 10. The citrate buffer can be the same citrate buffer described above. In addition to or in place of citrate, other chelators could be used. The dissociating agent can be an agent such as sodium dodecyl sulfate (SDS) or urea; urea concentrations up to 1 M are particularly suitable. Additionally, the buffer can contain 0.5% to 2% of bovine serum albumin (BSA), optionally in saline.

[0108] In general, in immunoassays according to the present invention, either purified calprotectin or purified S100A9 protein (e.g., recombinant S100A9 protein) can be employed as the standard. In many cases, however, it is preferred to use purified S100A9 protein (e.g., recombinant S100A9 protein) as the standard.

[0109] Because calprotectin has proven to be an efficient marker for the detection or diagnosis of inflammatory bowel disease, another aspect of the invention is a method of detecting or diagnosing the presence of active inflammatory bowel disease using competitive immunoassays for S100A9 with the solid phase platform as described above. The suggested reference limit for distinguishing the presence of inflammatory bowel disease from its absence is 50 mg/kg, in stool samples. A value for calprotectin concentration above this limit indicates the presence of active inflammatory bowel disease, while a value for calprotectin concentration below this limit indicates the absence of active inflammatory bowel disease. Accordingly, in general, a method for diagnosing or detecting the presence of active inflammatory bowel disease comprises the steps of:

[0110] Typically, in this method, the immunoassay for calprotectin is performed as described above using the solid phase platform.

[0111] Yet another aspect of the present invention is a method for determining whether a patient with inflammatory bowel disease who has been in remission is at risk of suffering a relapse comprising the steps of:

(2) coating the antigen onto the solid phase;

(4) washing the solid phase subsequent to the reaction of any calprotectin in the sample and the labeled antibody specifically binding the antigen to remove unbound labeled antibody specifically binding the antigen from the solid phase; (5) determining the quantity of labeled antibody specifically binding the antigen bound to the solid phase to determine the concentration of calprotectin in the sample; and

[0112] Typically, in this method, the immunoassay for calprotectin is performed as described above using the solid phase platform.

[0113] Yet another aspect of the present invention is a method of performing an immunoassay for S100A9 and for a related antigen to determine whether inflammatory bowel disease exists in a subject, the method comprising the steps of:

(1 ) providing a stool sample from a subject;

(2) performing a competitive immunoassay for the antigen selected from the group consisting of calprotectin and S100A9 using: (i) an immobilized antigen selected from the group consisting of immobilized calprotectin and immobilized S100A9 polypeptide; and (ii) a labeled antibody that is anti-calprotectin antibody when the immobilized antigen is calprotectin and is labeled anti-S100A9 antibody when the immobilized antigen is S100A9 polypeptide, the competitive immunoassay employing a solid phase platform;

(3) performing an immunoassay for a related antigen;

[0114] As used, the term "related antigen" means that the antigen in question is one that is associated with the occurrence of inflammatory bowel disease and can be used to discriminate between patients who have inflammatory bowel disease and patients who do not have inflammatory bowel disease. The term "related antigen" does not necessarily imply that the antigen in questions necessarily has any defined degree of sequence identity or sequence similarity with S100A9, although a degree of sequence identity or sequence similarity may in fact exist.

[0115] In one alternative, the related protein is S100A12. A suitable cutoff value for S100A12 is 66 pg/kg (T. Kaiser et al,. "Faecal S100A12 as a Non-invasive Marker Distinguishing Inflammatory Bowel Disease from Irritable Bowel Syndrome," Gut

56:1706-13 (2007)). Typically, if the related protein is S100A12, the immunoassay is an ELISA sandwich immunoassay; the ELISA sandwich immunoassay for A12 can employ an unlabeled monoclonal anti-A12 antibody and an enzyme-labeled polyclonal anti-A12 antibody. However, other alternatives are possible for the unlabeled capture antibody and the labeled antibody in an ELISA immunoassay. Additionally, the immunoassay for the related antigen, such as S100A12, can be an immunoassay other than an ELISA immunoassay. For example, the immunoassay can be a competitive immunoassay, such as a homogeneous or heterogeneous competitive immunoassay.

[0116] In another alternative, the related antigen is lactoferrin. Immunoassays for lactoferrin are well known in the art and are described, for example, in U.S. Patent No. 7,560,240 to Boone et al, U.S. Patent No. 7,192,724 to Boone et al., U.S. Patent 6,727,073 to Moore et al., U.S. Patent No. 6,727,073 to Moore et al., U.S. Patent No. 6,174,664 to Heine, U.S. Patent No. 5,552,292 to Uchida et al., U.S. Published Patent Application No. 2009/0253155 by Boone et al., U.S. Published Patent Application No. 2009/0053736 by Mattingly et al., U.S. Published Patent Application No. 2008/0166719 by Lois, U.S. Published Patent Application No. 2008/0085524 by Lois, U.S. Published Patent Application No. 2006/0003392 by Oh et al., and U.S. Published Patent Application No. 2004/0137536 by Boone et al. Methods for assay of lactoferrin as known in the art can include sandwich immunoassays such as ELISA immunoassays and lateral flow immunoassays, as well as competitive immunoassays. A suitable cutoff value for lactoferrin is 2.2 ng/mL.

[0117] The immunoassay for the related antigen can be performed by a competitive immunoassay method such as a competitive immunoassay on a solid phase platform as described above. The immunoassay for the related antigen can also be another conventional competitive immunoassay, such as a homogeneous or heterogeneous competitive immunoassay, or a sandwich immunoassay such as a conventional ELISA or a conventional lateral flow immunoassay. Immunoassays are described in D.G. Wild, ed., "The Immunoassay Handbook" (3^rd ed., Elsevier,

Amsterdam, 2005).

[0118] As indicated above, in another alternative of an immunoassay method according to the present invention, a lateral flow platform is used.

[0119] In an immunoassay employing a lateral flow platform as described above, the assay platform is typically constructed of a solid support that provides lateral flow of a sample through the assay platform when a sample is applied to a sampling platform that is in operable contact with the assay platform. The sampling platform and the assay platform are typically constructed of a material such as nitrocellulose, glass fiber, paper, nylon, and a synthetic nanoporous polymer. Suitable materials are well known in the art and are described, for example, in U.S. Patent No. 7,256,053 to Hu, 7,214,417 to Lee et al., 7,238,538 to Freitag et al., 7,238,322 to Wang et al., 7,229,839 to Thayer et al., 7,226,793 to Jerome et al., RE39,664 to Gordon et al., 7,205,159 to Cole et al., 7,189,522 to Esfandiari, 7,186,566 to Qian, 7,166,208 to Zweig, 7,144,742 to

Boehringer et al., 7,132,078 to Rawson et al., 7,097,983 to Markovsky et al., 7,090,803 to Gould et al., 7,045,342 to Nazareth et al., 7,030,210 to Cleaver et al., 6,981 ,522 to O'Connor et al., 6,924,153 to Boehringer et al., 6,849,414 to Guan et al., 6,844,200 to Brock, 6,841 ,159 to Simonson, 6,767,714 to Nazareth et al., 6,699,722 to Bauer et al., 6,656,744 to Pronovost et al., 6,528,323 to Thayer et al., 6,297,020 to Brock, 6,140,134 to Rittenburg, 6,136,610 to Polito et al., 5,965,458 to Kouvonen et al., 5,712,170 to Kouvanen et al., 4,956,302 to Gordon et al., and 4,943,522 to Eisinger et al., all of which are incorporated herein by this reference.

[0120] Various alternatives for the construction of the lateral flow platform and device and the performance of the competitive assay for calprotectin employing either immobilized S100A9 polypeptide or immobilized calprotectin are known in the art and are described below. Common to all of these platforms, devices, and assay methods is the immobilization of S100A9 polypeptide or calprotectin on the assay platform.

Because this is a competitive assay, both the labeled anti-S100A9 antibody or labeled anti-calprotectin antibody and any calprotectin in the sample need to contact the immobilized S100A9 polypeptide or the immobilized calprotectin for the competition to occur and for the quantity of labeled anti-S100A9 antibody or labeled anti-calprotectin antibody bound to the immobilized S100A9 polypeptide or to the immobilized

calprotectin to accurately reflect the concentration of S100A9 polypeptide, and thus calprotectin, or the concentration of calprotectin directly, in the sample. Therefore, these platforms, devices, and assay methods employ a solid support that provides lateral flow. The solid support that provides lateral flow has a first end and a second end. The immobilization occurs at a defined region of the solid support that provides lateral flow, the defined region being referred to herein as the "detection zone."

Typically, the detection zone is located between the first end and the second end of the solid support that provides lateral flow, closer to the second end. As detailed below, the sample and the labeled anti-S100A9 antibody or the labeled anti-calprotectin antibody are typically applied at or near the first end of the solid support that provides lateral flow so that the sample and the labeled anti-S100A9 antibody or the labeled anti-calprotectin antibody migrate through the support to the detection zone. Various methods can be used to do this, again, as detailed below. This competitive lateral flow assay is an assay in which the quantity of label bound at the detection zone is inversely proportional to the quantity of S100A9 polypeptide, and thus calprotectin, or the quantity of calprotectin directly, in the sample. Lateral flow immunoassay devices are further described in R. Wong & H. Tse, eds., "Lateral Flow Immunoassay" (Humana Press, New York, NY, 2009). As used below, the term "labeled antibody" refers to both labeled anti-S100A9 antibody and labeled anti-calprotectin antibody unless one of these alternatives is expressly excluded.

[0121] In Alternative I for the lateral flow platform and device, the labeled antibody is located in the device before the sample is applied. The labeled antibody is dried down in the device in a position where it is contacted by sample applied to the device, such that the labeled antibody is then mobilized by the sample contacting the dried labeled antibody. The dried labeled antibody can be located, for example, in: (a) a conjugate zone located on the solid support that provides lateral flow at a position where the sample first contacts the dried labeled antibody; (b) a conjugate pad in direct or indirect operable contact with one end of the solid support that provides lateral flow such that the end is distal to the portion of the solid support that provides lateral flow in which the detection zone is located; or (c) a conjugate pad located in another position that is not initially in direct or indirect operable contact with the solid support that provides lateral flow but is moved into direct or indirect operable contact with the solid support that provides lateral flow after the commencement of the assay.

[0122] In Alternative I, the sample can be applied in a number of locations; in each case, the sample must flow in a path that results in solubilization and mobilization of the dried labeled antibody. For example, the sample can be applied to the conjugate pad, if a conjugate pad is used. This will result in solubilization and mobilization of the dried labeled antibody Alternatively, the sample can be applied to a sample pad that is either located so that it is in either direct or indirect operable contact with either the solid support that provides lateral flow (in alternatives in which the dried labeled antibody is located in a conjugate zone directly on the solid support) or the conjugate pad, or located so that it is moved into either direct or indirect operable contact with either the solid support that provides lateral flow (in alternatives in which the dried labeled antibody is located in a conjugate zone directly on the solid support) or the conjugate pad.

[0123] In Alternative II, the labeled anti-S100A9 antibody or labeled anti- calprotectin antibody is not present in the device prior to sample application. In

Alternative II, labeled anti-S100A9 antibody or labeled anti-calprotectin antibody, typically already mobile and in solution, is added to the sample just before application to the device. In Alternative II, the sample and the mobile labeled anti-S100A9 antibody or labeled anti-calprotectin antibody can be applied to: (a) a portion of the solid support that provides lateral flow that is separated from the detection zone; or (b) a

sample/conjugate pad that is in either direct or indirect operable contact with the end of the solid support that provides lateral flow separated from the detection zone when the sample and labeled anti-S100A9 antibody or labeled anti-calprotectin antibody is applied thereto, or is moved into direct or indirect operable contact with the end of the solid support that provides lateral flow separated from the detection zone after the application of the sample and the labeled S100A9 antibody or labeled anti-calprotectin antibody. In another arrangement, the device can comprise both a sample pad and a conjugate application pad and labeled antibody is applied to the conjugate application pad so that the sample migrates to the conjugate application pad, picking up antibody, and then to the detection zone.

[0124] Typically, the solid support that provides lateral flow is backed with a nonporous backing on one side, such as a plastic strip. The solid support that provides lateral flow can also be enclosed in a casing that provides openings as required for the performance of the assay. Depending upon the exact configuration of the assay device, the openings can include an opening for application of the sample and/or the labeled antibody, an opening positioned for detection at the detection zone, and an opening positioned for detection at a control zone, as described below.

[0125] The device can include a control zone to verify that the assay has been performed correctly. In one alternative, the control zone binds the labeled antibody. The control zone is typically located close to the detection zone but separated from it. Typically, the control zone includes an immobilized antibody that binds the labeled anti- antibody by binding to a portion of the antibody distinct from the paratope of the labeled antibody, such as, but not limited to, an epitope located in the constant region of the labeled antibody. The binding of the labeled antibody to the immobilized S100A9 polypeptide or calprotectin in the detection zone should not interfere with the binding of the labeled antibody to the control zone, and the binding of the labeled antibody to the control zone should not interfere with the binding of the labeled antibody to the immobilized S100A9 polypeptide or calprotectin in the detection zone. If this version of a control zone is used, additional labeled antibody should be included to account for the quantity of antibody expected to be bound to the control zone.

[0126] In another alternative, the control zone includes an unrelated antigen and the device includes a second labeled antibody that specifically binds the unrelated antigen. There should be no cross-reactivity between the labeled antibody that specifically binds S100A9 polypeptide or calprotectin and the second labeled antibody that specifically binds the unrelated antigen. The labeled antibody that specifically binds the unrelated antigen is typically present in, or added to, the device along with the the labeled antibody that specifically binds S100A9 polypeptide or calprotectin. The label of the labeled antibody that specifically binds the unrelated antigen can be the same as the label of the labeled antibody that specifically binds S100A9 polypeptide or calprotectin, or can be a different label; typically, however, for convenience in reading, the label of the labeled antibody that specifically binds the unrelated antigen is typically the same as the label of the labeled antibody that specifically binds S100A9 polypeptide or calprotectin.

[0127] A number of alternatives for an assay device that can perform such competitive immunoassays are depicted in Figures 1A, 1 B, 2A, 2B, 3A, 3B, 4, 5, 6A, 6B, 7A, 7B, and 8.

[0128] Figure 1 A depicts a top view of first alternative for an assay device that can perform a competitive immunoassay according to Alternative I as described above. In Figure 1 , the device 10 includes a solid support that provides lateral flow 12 having a first end 14 and a second end 16. The solid support that provides lateral flow 12 has a detection zone 18 and an optional control zone 20. The detection zone 18 and, if present, the optional control zone 20 are located closer to the second end 16 of the solid support that provides lateral flow 12 than to the first end 14 of the solid support that provides lateral flow 12; if present, the optional control zone 20 can be either located closer to the second end 16 of the solid support that provides lateral flow 12 than the detection zone 18, or the detection zone 18 can be located closer to the second end 16 of the solid support that provides lateral flow than the control zone 20. The solid support that provides lateral flow 12 also has a conjugate zone 22; the conjugate zone is located closer to the first end 14 of the solid support that provides lateral flow than to the second end 16. The device 10 includes a sample pad 24 for application of the sample that is in operable contact with the solid support that provides lateral flow 12 so that the sample flows from the sample pad 24 into the solid support that provides lateral flow 12 so that the labeled antibody at the conjugate zone 22 is solubilized and mobilized. As shown in Figure 1 B, the device 10 can optionally include a backing 26 for the solid support that provides lateral flow 12 and a casing 28. The casing 28 has apertures, including a first aperture 30 for application of the sample to the sample pad 20, a second aperture 32 allowing viewing and/or measurement of labeled antibody bound to the detection zone 18, and a third aperture 34 allowing viewing and/or measurement of labeled antibody or other antibody used as a control bound to the control zone 20.

[0129] Figure 2A depicts a top view of a second alternative for an assay device that can perform a competitive immunoassay according to Alternative I as described above. In Figure 2A, the device 40 includes a solid support that provides lateral flow 42 having a first end 44 and a second end 46. The solid support that provides lateral flow has a detection zone 48 and an optional control zone 50. The device 40 has a conjugate pad 52 in operable contact with the first end 44 of the solid support that provides lateral flow 42; sample is applied to the conjugate pad 52 so that the labeled antibody in the conjugate pad 52 is solubilized and mobilized. As shown in Figure 2B, the device 40 can optionally include a backing 54 for the solid support that provides lateral flow 42 and a casing 56. The casing 56 has apertures, including a first aperture 58 for application of the sample to the conjugate pad 52, a second aperture 60 allowing viewing and/or measurement of labeled antibody bound to the detection zone 48, and a third aperture 62 allowing viewing and/or measurement of labeled antibody or other antibody used as a control bound to the control zone 50.

[0130] Figure 3A depicts a top view of a third alternative for an assay device that can perform a competitive immunoassay according to Alternative I as described above. In Figure 3A, the device 100 includes a solid support that provides lateral flow 102 having a first end 104 and a second end 106. The solid support that provides lateral flow 102 has a detection zone 108 and an optional control zone 1 10. The device 100 has a conjugate pad 1 14 in operable contact with the first end 104 of the solid support that provides lateral flow 102 and a sample pad 1 16 in operable contact with the conjugate pad 1 14; sample is applied to the sample pad 1 16 and flows to the conjugate pad 1 14 so that the labeled antibody in the conjugate pad 1 14 is solubilized and mobilized. As shown in Figure 3B, the device 100 can optionally include a backing 1 18 for the solid support that provides lateral flow 102 and a casing 120. The casing 120 has apertures, including a first aperture 122 for application of the sample to the sample pad 1 16, a second aperture 124 allowing viewing and/or measurement of labeled antibody bound to the detection zone 108, and a third aperture 126 allowing viewing and/or measurement of labeled anti-S100A9 antibody or other antibody used as a control bound to the control zone 1 10.

[0131] Figure 4 depicts a fourth alternative for an assay device that can perform a competitive immunoassay according to Alternative I as described above. The device of Figure 4 is a folding device that is constructed of two substantially planar parts connected by a hinge. The device 140 includes a solid support that provides lateral flow 142. The solid support that provides lateral flow 142 has a first end 144 and a second end 146, with a detection zone 148 and, optionally, a control zone 150. The solid support that provides lateral flow 142 is attached to a first substantially planar part 152. The device 140 further includes an applicator part 154 that is a second substantially planar part; a hinge 156 flexibly connects the applicator part 154 that is a second substantially planar part and first substantially planar part 152 that includes the solid support that provides lateral flow 142. The applicator part 154 that is a second substantially planar part includes a conjugate pad 158. In use, sample is added to the conjugate pad 158 so that the labeled antibody in the conjugate pad 158 is solubilized and mobilized. The device 140 is then closed via the hinge 156 so that the conjugate pad 158 is brought into operable contact with the first end 144 of the solid support that provides lateral flow 142 so that the sample and the labeled antibody flow through the solid support that provides lateral flow 142.

[0132] Figure 5 depicts a fifth alternative for an assay device that can perform a competitive immunoassay according to Alternative I as described above. The device of Figure 5 is also a folding device that is constructed of two substantially planar parts connected by a hinge; it is generally similar to that of Figure 4, except that the applicator part includes a separate sample pad and a conjugate pad. The device 200 includes a solid support that provides lateral flow 202. The solid support that provides lateral flow 202 has a first end 204 and a second end 206, with a detection zone 208 and, optionally, a control zone 210. The solid support that provides lateral flow 202 is attached to a first substantially planar part 212. The device 200 further includes an applicator part that is a second substantially planar part; a hinge 216 flexibly connects the applicator part 214 and the first substantially planar part 212 that includes the support that provides lateral flow 202. The applicator part 214 includes a sample pad 218 and a conjugate pad 220; the sample pad 218 is in operable contact with the conjugate pad 220. In use, sample is added to the sample pad 218; the sample then flows to the conjugate pad 220 so that the labeled antibody in the conjugate pad 220 is solubilized and mobilized. The device 200 is then closed via the hinge 216 so that the conjugate pad 220 is brought into operable contact with the first end 204 of the solid support that provides lateral flow 202 so that the sample and the labeled antibody flow through the solid support that provides lateral flow 202.

[0133] Figure 6A is a top view of a first alternative for an assay device that can perform a competitive immunoassay according to Alternative II as described above. In Alternative II, the labeled anti-S100A9 antibody is not present on the device at the start of the assay, but is added to the device together with the sample at the commencement of the assay. In the device of Figure 6A, the device 300 includes a solid support that provides lateral flow 302 having a first end 304 and a second end 306. The solid support that provides lateral flow 302 has a detection zone 308 and an optional control zone 310. The device 300 includes a sample pad 312 for application of the sample and the labeled anti-S100A9 antibody that is in operable contact with the solid support that provides lateral flow 302 so that the sample and the labeled anti-S100A9 antibody flow from the sample pad 312 into the solid support that provides lateral flow 302. As shown in Figure 6B, the device 300 can optionally include a backing 314 for the solid support that provides lateral flow 302 and a casing 316. The casing 316 has apertures, including a first aperture 318 for application of the sample and the labeled antibody to the sample pad 312, a second aperture 320 allowing viewing and/or measurement of labeled anti-S100A9 antibody bound to the detection zone 308, and a third aperture 322 allowing viewing and/or measurement of labeled anti-S100A9 antibody or other antibody used as a control bound to the control zone 310.

[0134] Figure 7A is a top view of a second alternative for an assay device that can perform a competitive immunoassay according to Alternative II as described above. The device of Figure 7A is generally similar to that of Figure 6A, but the sample pad is dispensed with and the sample and the labeled antibody are applied directly to the first end of the solid support that provides lateral flow. In the device of Figure 7A, the device 340 includes a solid support that provides lateral flow 342 having a first end 344 and a second end 346. The solid support that provides lateral flow 342 has a detection zone 348 and an optional control zone 350. The device 340 is arranged so that the sample and the labeled anti-S100A9 antibody are applied directly to the first end 344 of the solid support that provides lateral flow 342. As shown in Figure 7B, the device 340 can optionally include a backing 352 for the solid support that provides lateral flow 342 and a casing 354. The casing 354 has apertures, including a first aperture 356 for application of the sample and the labeled antibody to the first end 344 of the solid support that provides lateral flow 342, a second aperture 358 allowing viewing and/or measurement of labeled antibody bound to the detection zone 348, and a third aperture 360 allowing viewing and/or measurement of labeled anti-S100A9 antibody or other antibody used as a control bound to the control zone 350.

[0135] Figure 8 is a third alternative for an assay device that can perform a competitive immunoassay according to Alternative II as described above. The device of Figure 8 is a folding device that is constructed of two substantially planar parts connected by a hinge. The device 400 includes a solid support that provides lateral flow 402. The solid support that provides lateral flow 402 has a first end 404 and a second end 406, with a detection zone 408 and, optionally, a control zone 410. The solid support that provides lateral flow 402 is attached to a first substantially planar part 412. The device 400 further includes a applicator part 414 that is a second substantially planar part; a hinge 416 flexibly connects the applicator part 414 that is a second substantially planar part and the first substantially planar part 412 that includes the solid support that provides lateral flow 402. The applicator part 414 includes a sample pad 418. In use, sample and labeled anti-S100A9 antibody are added to the sample pad 418. The device 400 is then closed via the hinge 416 so that the sample pad 418 is brought into operable contact with the first end 404 of the solid support that provides lateral flow 402 so that the sample and the labeled antibody flow through the solid support that provides lateral flow 404.

[0136] Yet another alternative for an assay device that can perform a

competitive immunoassay according to Alternative II as described above is constructed according to Figure 5 above, under Alternative I, except that the conjugate pad 220 of Figure 5 is replaced by a conjugate application pad to which labeled antibody is applied at the commencement of the assay. The assay then proceeds in such a way that the sample migrates to the conjugate application pad, picking up antibody, and then to the detection zone.

[0137] Other assay devices can be constructed on similar principles for performing competitive immunoassays for S100A9 polypeptide or calprotectin. These devices can, for example, include additional elements for controlling flow between components. It is also possible to construct multiplex devices operating on similar principles for assay of multiple samples and/or standards in a single assay.

[0138] When an immunoassay is performed on a device for performing a lateral flow competitive immunoassay according to the present invention, reading the result can be done either by eye, for a qualitative estimate of the concentration of the analyte being assayed, such as S100A9 polypeptide or calprotectin, or by a reader for a lateral flow immunoassay device. Readers for lateral flow immunoassay devices are known in the art and are described, for example, in B. O'Farrell, "Evolution in Lateral Flow-Based Immunoassay Systems" in Lateral Flow Immunoassay (R. Wong & H. Tse, eds., Humana Press, New York, NY, 2009), ch.1 , pp. 23-28, incorporated herein by this reference. The choice of a reader will depend on the label of the labeled antibody and the signal produced by that label; however, readers include charge coupled device (CCD) cameras, confocal or other optical sensors for detection of fluorescent particles subsequent to LED excitation, and detection of paramagnetic monodisperse latexes using magnetic assay reader technology. Other reader systems are known in the art.

[0139] In general, therefore, another aspect of the invention is a device for performing a lateral flow competitive immunoassay for S100A9 polypeptide or calprotectin comprising a solid support for lateral flow, a labeled anti-S100A9 antibody or anti-calprotectin antibody that is either present on the device before the

commencement of the immunoassay or is added to the device at the commencement of the immunoassay, and a detection zone on the solid support comprising immobilized S100A9 polypeptide or immobilized calprotectin such that any S100A9 polypeptide or calprotectin in the sample competes with the immobilized S100A9 polypeptide or with the immobilized calprotectin in the detection zone.

[0140] These aspects are described further below in terms of devices including the significant elements for performing such a lateral flow competitive immunoassay.

[0141] Accordingly, another aspect of the invention is a device for performing a lateral flow competitive immunoassay for S100A9 or for calprotectin comprising:

(4) a sample pad for application of a sample in operable contact with the first end of the solid support; wherein a sample applied to the sample pad migrates to the conjugate zone to solubilize and mobilize the mobilizable labeled antibody and the sample and the mobilized labeled antibody migrate to the detection zone, such that any calprotectin or S100A9 polypeptide in the sample competes for binding of the labeled antibody with the immobilized calprotectin or immobilized S100A9 polypeptide bound at the detection zone to perform a competitive immunoassay.

[0142] The device can further include an optional control zone and a casing with apertures as described above. If the control zone is used, and the control zone includes an unrelated antigen, the device includes a labeled antibody specifically binding the unrelated antigen as described above.

[0143] Yet another aspect of the invention is a device for performing a lateral flow competitive immunoassay for S100A9 or for calprotectin comprising:

[0144] The device can further include an optional control zone and a casing with apertures as described above.

[0145] Still another aspect of the invention is a device for performing a lateral flow competitive immunoassay for S100A9 or for calprotectin comprising: (1 ) a solid support providing lateral flow having a first end and a second end;

[0146] Yet another aspect of the invention is a device for performing a lateral flow competitive immunoassay for S100A9 or for calprotectin comprising:

(2) a second substantially planar part, the second substantially planar part comprising a conjugate pad having a mobilizable labeled antibody selected from the group consisting of anti-calprotectin antibody and anti-S100A9 antibody; and

[0147] Yet another aspect of the invention is a device for performing a lateral flow competitive immunoassay for S100A9 or for calprotectin comprising:

[0148] Still other alternatives employ a mobile labeled anti-S100A9 antibody or a mobile labeled anti-calprotectin antibody, typically in solution as described above, that is added to the device with the sample and is not originally present in the device. [0149] Accordingly, yet another aspect of the invention is a device for performing a lateral flow competitive immunoassay for S100A9 polypeptide or calprotectin comprising:

(3) a sample pad in operable contact with the first end of the solid support;

wherein, after a sample and a mobile labeled antibody selected from the group consisting of anti-calprotectin antibody and anti-S100A9 antibody is applied to the sample pad, the sample and the mobile labeled antibody migrate to the detection zone, such that any calprotectin or S100A9 polypeptide in the sample competes for binding of the labeled antibody with the immobilized calprotectin or immobilized S100A9

polypeptide bound at the detection zone to perform a competitive immunoassay.

[0150] Accordingly, yet another aspect of the invention is a device for performing a lateral flow competitive immunoassay for S100A9 polypeptide or calprotectin comprising:

polypeptide bound at the detection zone to perform a competitive immunoassay. [0151] Yet another aspect of the invention is a device for performing a lateral flow competitive immunoassay for S100A9 polypeptide or calprotectin comprising:

wherein, after a sample is applied to the sample pad and a mobile labeled antibody is applied to the conjugate application pad, the hinge connecting the first substantially planar part with the second substantially planar part is closed so that the sample pad and the conjugate application pad are placed into operable contact with the solid support and the sample and the mobile labeled antibody migrate to the detection zone, such that any calprotectin or S100A9 polypeptide in the sample competes for binding of the labeled antibody with the immobilized calprotectin or immobilized S100A9 polypeptide bound at the detection zone to perform a competitive immunoassay.

[0152] Yet another aspect of the invention is a device for performing a lateral flow competitive immunoassay for S100A9 polypeptide or calprotectin comprising:

(1 ) a first substantially planar part, the first substantially planar part comprising a solid support that provides lateral flow, the solid support that provides lateral flow comprising a detection zone having an immobilized protein or polypeptide selected from the group consisting of immobilized calprotectin and immobilized S100A9 polypeptide; (2) a second substantially planar part, the second substantially planar part comprising a sample pad for application of a sample and a mobile labeled antibody selected from the group consisting of anti-calprotectin antibody and anti-S100A9 antibody to the sample pad;

[0153] Still other devices can be constructed according to these general principles; modifications of these devices according to general principles known in the art. Such devices can include multiplex devices capable of assaying a plurality of samples and/or standards in a single device. In general, such multiplex devices are constructed so that the multiple solid supports that provide lateral flow, one for each sample, are positioned in parallel so that the detection zones, and, if present, control zones, are at the same position relative to the end of the device for each sample and/or standard to be assayed.

[0154] Typically, the label of the mobilizable or mobile labeled anti-S100A9 antibody or anti-calprotectin antibody is a directly detectable label, such as a radioactive label, a fluorescent label, a chemiluminescent label, an electrochemiluminescent label, a bioluminescent label, a colloidal metal label such as a colloidal gold or colloidal silver label, a colloidal metal oxide label, a dye label, a colored latex particle label, a colored polystyrene or polypropylene particle label, a liposome label, a Surface-Enhanced Resonant Raman Spectroscopy (SERRS) label, or a signaling aptamer label. A preferred label is a colloidal metal label. A particularly preferred label is a colloidal gold or colloidal silver label. A more particularly preferred label is a colloidal gold label.

Additional preferred labels are dye labels or colored latex particle labels.

[0155] As another alternative, an enzyme label such as those described above can be used. However, because the use of an enzyme label requires an additional incubation step, it is generally preferred to use a direct label such as those described above, especially a colloidal gold or colloidal silver label, a dye label, or a colored latex particle label.

[0156] Methods for immobilizing the S100A9 polypeptide or the calprotectin at the detection zone of the solid support that provides lateral flow are well known in the art. Immobilization can be either by covalent coupling to the solid support or non- covalent immobilization on the solid support. Suitable immobilization techniques are described, for example, in P. Tijssen, "Practice and Theory of Enzyme Immunoassays" (Elsevier, Amsterdam, 1985), ch. 13, pp. 297-328, incorporated herein by this reference.

[0157] Devices for reading direct labels such as colloidal gold or colloidal silver labels are known in the art. Such devices are described, for example, in R.C. Wong & H. Tse, eds, "Lateral Flow Immunoassay" (Humana Press, New York, NY, 2009).

[0158] Accordingly, yet another aspect of the present invention is a method for determining the concentration of calprotectin in a sample comprising the steps of:

(1 ) applying the sample to a device for performing a lateral flow competitive immunoassay for S100A9 polypeptide or for calprotectin comprising a solid support for lateral flow, a labeled anti-S100A9 antibody or anti-calprotectin antibody, and a detection zone on the solid support comprising immobilized S100A9 polypeptide or calprotectin such that any S100A9 polypeptide or calprotectin in the sample competes with the immobilized S100A9 polypeptide or calprotectin in the detection zone;

(2) allowing the sample and the labeled antibody to migrate through the solid support for lateral flow and to the detection zone at which any calprotectin or S100A9 polypeptide in the sample competes with the immobilized calprotectin or S100A9 polypeptide in the detection zone; and (3) determining the quantity of labeled antibody bound to the detection zone to determine the concentration of calprotectin in the sample, wherein the quantity of calprotectin or S100A9 polypeptide immobilized at the detection zone and the quantity of labeled calprotectin antibody or labeled anti-S100A9 antibody are chosen such that the calprotectin or S100A9 polypeptide immobilized at the detection zone and any calprotectin in the sample compete for the labeled anti-calprotectin antibody or labeled anti-S100A9 antibody such that the quantity of labeled anti-calprotectin antibody or labeled anti-S100A9 antibody immobilized to the detection zone is related inversely to the concentration of calprotectin in the sample.

[0159] In this assay, any of the alternatives for the lateral flow platform as described above can be used. In this assay, as described above, the label of the labeled anti-S100A9 antibody or labeled anti-calprotectin antibody can be any of the labels described above; however, typically, the label is a direct label, and, preferably, the label is a colloidal gold or silver label. Methods for determining the quantity of the labeled anti-S100A9 antibody or anti-calprotectin antibody bound to the detection zone are described above and are well known in the art.

[0160] The sample volume applied to the device is typically from about 10 μί to about 150 μΙ_; preferably, the sample volume is from about 20 μΙ_ to about 50 μΙ_.

[0161] Suitable immobilized S100A9 polypeptides are those described above with respect to the solid phase platform. Typically, the immobilized S100A9 polypeptide is a recombinant S100A9 polypeptide as described above. Alternatively, the

immobilized S100A9 polypeptide is a purified and isolated naturally occurring S100A9 polypeptide. Suitable immobilized calprotectin molecules are also those described above.

[0162] Suitable anti-S100A9 antibodies are those described above with respect to the solid phase platform. A particularly suitable anti-S100A9 antibody is an anti- Si 00A9 monoclonal antibody such as the monoclonal antibody designated as Calpro Mab CAL1 -4H1/2/2. Alternatively, the anti-S100A9 antibody can be a polyclonal anti- Si 00A9 antibody. Similarly, suitable anti-calprotectin antibodies are those described above. [0163] In performance of the assay, sample, and, if necessary, labeled antibody, are added to the appropriate portion of the device (i.e., a sample pad if present or one end of the solid support that provides lateral flow), the sample and the labeled antibody (either in liquid form as added to the device or solubilized by the sample) migrate through the device to the detection zone, and the quantity of labeled antibody bound at the detection zone is determined. Typically, the assay takes from about 5 minutes to about 15 minutes to perform. Typically, the assay is performed at room temperature, but can be performed at a lower or higher temperature without interference with the performance of the assay.

[0164] Typically, a standard curve is prepared with multiple concentrations of S100A9 polypeptide standards or purified calprotectin standards as described above. The results from the standard curve are then used to determine the calprotectin concentration.

[0165] Similarly, yet another aspect of the present invention is a method of detecting or diagnosing the presence of active inflammatory bowel disease using competitive immunoassays for S100A9 or for calprotectin with the lateral flow platform described above. In general, this method comprises the steps of:

(2) allowing the sample and the labeled antibody to migrate through the solid support for lateral flow and to the detection zone at which any calprotectin or S100A9 polypeptide in the sample competes with the immobilized calprotectin or S100A9 polypeptide in the detection zone;

(3) determining the quantity of labeled antibody bound to the detection zone to determine the concentration of calprotectin in the sample, wherein the quantity of calprotectin or S100A9 polypeptide immobilized at the detection zone and the quantity of labeled calprotectin antibody or labeled anti-S100A9 antibody are chosen such that the calprotectin or S100A9 polypeptide immobilized at the detection zone and any calprotectin in the sample compete for the labeled anti-calprotectin antibody or labeled anti-S100A9 antibody such that the quantity of labeled anti-calprotectin antibody or labeled anti-S100A9 antibody immobilized to the detection zone is related inversely to the concentration of calprotectin in the sample; and

(4) determining the presence or absence of active inflammatory bowel disease in the patient according to whether the calprotectin concentration in the sample is at least 50 mg/kg, in which case active inflammatory bowel disease is present in the patient, or is less than 50 mg/kg, in which case active inflammatory bowel disease is absent in the patient.

[0166] Suitable devices, samples, antibodies, and methods for performing the assay are as described above.

[0167] Similarly, yet another aspect of the present invention is a method for determining whether a patient with inflammatory bowel disease who has been in remission is at risk of suffering a relapse, again using competitive immunoassays for S100A9 polypeptide or calprotectin with the lateral flow platform described above, the method comprising the steps of:

(2) allowing the sample and the labeled antibody to migrate through the solid support for lateral flow and to the detection zone at which any calprotectin or S100A9 polypeptide in the sample competes with the immobilized calprotectin or S100A9 polypeptide in the detection zone; (3) determining the quantity of labeled antibody bound to the detection zone to determine the concentration of calprotectin in the sample, wherein the quantity of calprotectin or S100A9 polypeptide immobilized at the detection zone and the quantity of labeled calprotectin antibody or labeled anti-S100A9 antibody are chosen such that the calprotectin or S100A9 polypeptide immobilized at the detection zone and any calprotectin in the sample compete for the labeled anti-calprotectin antibody or labeled anti-S100A9 antibody such that the quantity of labeled anti-calprotectin antibody or labeled anti-S100A9 antibody immobilized to the detection zone is related inversely to the concentration of calprotectin in the sample; and

(4) determining the risk of relapse of inflammatory bowel disease in the patient according to whether the calprotectin concentration in the sample is at least 50 mg/kg, in which case a risk of relapse of inflammatory bowel disease exists, or is less than 50 mg/kg, in which case a risk of relapse of inflammatory bowel disease does not exist.

[0168] Suitable devices, samples, antibodies, and methods for performing the assay are as described above.

[0169] Similarly, yet another aspect of the present invention is a method of performing an immunoassay for S100A9 polypeptide or for calprotectin and for a related antigen to determine whether inflammatory bowel disease exists in a subject, the method comprising the steps of:

(1 ) providing a stool sample from a subject;

(2) performing a competitive immunoassay for S100A9 polypeptide or calprotectin as described above using immobilized S100A9 polypeptide and labeled anti-S100A9 antibody, or immobilized calprotectin and labeled anti-calprotectin antibody, the competitive immunoassay employing a lateral flow platform, wherein the performance of the competitive immunoassay occurs as described above;

(3) performing an immunoassay for a related antigen;

(4) determining the concentration of calprotectin in the stool sample from the results of the competitive immunoassay for S100A9 polypeptide or for calprotectin employing the lateral flow platform as described above; (5) determining the concentration of the related antigen in the stool sample from the results of the immunoassay for the related antigen; and

(6) comparing the concentration of calprotectin and the concentration of the related antigen in the stool sample with the cutoff values for S100A9 polypeptide and the related antigen to determine whether or not inflammatory bowel disease exists in the subject.

[0170] Suitable devices, samples, antibodies, and methods for performing the assay for S100A9 polypeptide or for calprotectin on the lateral flow platform are as described above.

[0171] The term "related antigen" is defined above with respect to the analogous assay method employing a solid phase platform described above; the same related antigens that are used in that assay method are used in this alternative.

[0172] In this alternative, suitable related antigens include lactoferrin and

S100A12, as described above.

[0173] Yet another aspect of the present invention is a kit for immunoassay of S100A9 polypeptide and diagnosis, screening, or monitoring of inflammatory bowel disease comprising, separately packaged:

(2) a labeled anti-S100A9 antibody to be applied to the solid support.

[0174] The quantity of the labeled anti-S100A9 antibody can be such that the quantity is intended to be used in a single assay, in which case the kit includes a single solid support. Alternatively, the quantity can be such that it is intended to be used in a plurality of individual assays, such as 2, 3, 5, 10, 15, 20, 25, 30, 40, 50, or 100 individual assays, and the appropriate quantity for a single assay measured out by the individual performing the assay, in which case the kit includes a plurality of solid supports, and the quantity of labeled anti-S100A9 antibody is sufficient for a plurality of assays such that the quantity of labeled anti-S100A9 antibody is sufficient for use with each of the solid supports included in the kit. [0175] In addition, a kit according to the present invention can further include one or more of the following items, all separately packaged:

(1 ) in the event that the label of the labeled anti-S100A9 antibody is an enzyme label, a substrate for the enzyme label; and

(2) a set of S100A9 polypeptide standards of differing concentrations so that a standard curve for S100A9 polypeptide can be established.

[0176] Similarly, a kit for immunoassay of calprotectin and diagnosis, screening, or monitoring of inflammatory bowel disease comprises, separately packaged:

(b) a labeled anti-calprotectin antibody to be applied to the solid support.

[0177] This kit can also contain the other items described above with respect to the kit for immunoassay of S100A9 polypeptide, and can contain appropriate quantities of the labeled antibody as described above with respect to the kit for immunoassay of S100A9 polypeptide.

[0178] Although the use of the solid phase platform and the lateral flow platform for performing competitive immunoassays have been described above with respect to immunoassays for S100A9 polypeptide and calprotectin, these immunoassay formats can also be used to perform competitive immunoassays for other antigens, including proteins, glycoproteins, lipoproteins, carbohydrates, or small molecules such as steroids, alkaloids, terpenes, or pharmacologically active compounds, using appropriate monoclonal or polyclonal antibodies, labels, and controls. Virtually any antigen to which antibodies of sufficient specificity can be prepared can be assayed by solid phase or lateral flow competitive immunoassays analogous to those described above for S100A9 polypeptide.

[0179] Additionally, although these assays have been described with particular attention to the use of calprotectin as a marker for inflammatory bowel disease, calprotectin can be used as a marker for the existence of inflammation in general.

Specifically, calprotectin can be used as a marker for many inflammatory diseases and conditions, including, but not limited to, allograft rejection, coronary heart disease, atheromatosis, sepsis, preeclampsia, cirrhosis, periodontitis, obesity, type 2 diabetes, acne vulgaris, asthma, autoimmune disorders, chronic prostatitis, glomerulonephritis, pelvic inflammatory disease, sarcoidosis, and vasculitis. Because inflammatory processes associated with calprotectin contribute to the etiology of cancer, including pancreatic cancer, ovarian cancer, and uterine cancer, and are especially prominent in inflammatory breast cancer, assay of calprotectin concentration can also be used in the diagnosis and monitoring of cancer.

[0180] The invention is illustrated by the following Examples. These Examples are included for illustrative purposes only, and are not intended to limit the invention.

EXAMPLES

Example 1

Competitive ELISA Immunoassay Employing Solid Support Platform

[0181] Microwells of standard 96 well microplate format from Costar, USA were used. To the microwells were added 150 μΙ of a solution of recombinant S100A9, 0.8 g/ml in Tris-buffered saline with 1 mM calcium chloride (TBS-Ca). The wells were covered by plastic tape and left at 5° C for at least 18 hours. Before use, the wells were washed once, 250 μΙ per well, with TBS-Ca; to each well was then added 250 μΙ of a solution containing 1 % BSA, 2.5 % sucrose in 10 mM potassium phosphate buffer pH 8; the wells were then left at room temperature (22 C) for 30 minutes. The wells were then washed three times with a buffer containing 50 mM Tris, 150 mM NaCI, 0.5 mM magnesium chloride, 1 % Kathon® and 0.5 mL/L Tween 20, pH 8.0.

[0182] In different wells were 50 μΙ of calibrators and samples followed by 50 μΙ of a HRP-conjugated monoclonal anti-S100A9 antibody. Three different dilutions of the HRP-conjugated monoclonal anti-S100A9 antibody were used, 1 :60,000, 1 :120,000, and 1 :240,000, prepared by serial dilutions. The wells were covered by plastic film and incubated with shaking at 500 rpm, for 15 minutes. Subsequently, all wells were washed three times; 100 μΙ HRP tetramethylbenzidine (TMB) substrate was added to each well. After a predetermined time interval, 100 μΙ stop solution, 0.2 M sulfuric acid, was added to each well and the color intensity read at 450 nm in a microwell ELISA reader. The predetermined time interval was 10 minutes for the 1 :60,000 dilution, 20 minutes for the 1 :120,000 dilution, and 40 minutes for the 1 :240,000 dilution.

[0183] In Figure 9 is shown the standard curve obtained by this procedure.

Example 2

Correlation Between Results Obtained by Assay of Example 1 and Original Sandwich

ELISA Assay for Extracts of Stool Samples

[0184] A prerequisite for alternative calprotectin assays is that the results obtained correspond to those from the original ELISA immunoassay for calprotectin when stool extracts are tested.

[0185] The results of this comparison testing are shown in Table 1 .

TABLE 1

[0186] In Table 1 , the leftmost column (Standard ELISA) refers to an ELISA assay performed as previously. The middle column (NG ELISA) refers to a variation of an ELISA assay in which the antibody immobilized on the solid support is a monoclonal anti-S100A9 antibody, and the labeled antibody is a polyclonal anti-S100A9 antibody. The rightmost column (Competitive A9 ELISA) refers to the competitive ELISA immunoassay of the present invention.

[0187] In Figure 10 it is shown that a satisfactory correlation between the two was found, validating the accuracy of the competitive immunoassay for S100A9 performed on a solid phase platform as performed in Example 1 .

Example 3

Lateral Flow Competitive Immunoassay for S100A9 Polypeptide

[0188] Nitrocellulose papers were striped with recombinant S100A9 polypeptide in concentration from 0.4 to 1 .6 mg/ml; an animal protein was used for the control line. Colloidal gold particles were conjugated onto the anti-S100A9 monoclonal antibody (the same as used in the ELISA in Example 1 ), and tried in dilutions between 1 :10 and 1 :80 in a Lumiquick Inc. proprietary buffer. The conjugate was mixed with a suitable gold- labeled antibody against the animal protein in the control line. Recombinant calprotectin standards were prepared by dilution in Tris-buffered saline with 10 g/L bovine serum albumin and 0.05 % Tween-40, pH 7.5. In separate microplate wells were added 50 μΙ of each of the S100A9 polypeptide standards, with concentrations between 16 and 1024 ng/ml, followed by 50 μί of gold conjugate. Without any delay (or incubation) strips were put in each well and the diffusion allowed to proceed for 10 minutes. Excess reagents (standard/conjugate mixture) sticking to the ends of the strips were removed by pressing against absorbent paper. The strips were subsequently put on a document scanner connected to a computer and the staining intensities of the test lines were read using the apparatus shown in Figure 1 1 . The standards were tested as singlets. [0189] The results are shown in Figures 12-13. Figure 12 shows the stained results with samples at 16 ng/mL, 64 ng/mL, 256 ng/mL, and 1024 ng/mL. Figure 13 shows a standard curve based on these results. As shown in Figure 1 1 , decreasing T line staining intensities were found with increasing concentration of A9. In the same manner as the competitive ELISA (i.e., the assay performed above with the solid phase platform), the staining intensity of the test line approaches zero with increasing concentration of A9 so that a hook effect will not appear. The staining intensities were recorded from the scanner in arbitrary OD units. Optimal S100A9 coating was at 1 .6 mg/mL; optimal conjugate dilution was 1 :80. The sensitivity of this rapid test is comparable to that of the ELISA (i.e., the solid phase platform assay of Example 1 ). Reliable readings of test lines can be obtained down to about 8 ng/ml corresponding to an OD value of about 80.

ADVANTAGES OF THE INVENTION

[0190] The present invention provides an efficient, accurate, and reproducible immunoassay for calprotectin, and thus an improved bioassay for a marker that is useful for the detection, diagnosis, or monitoring of inflammatory bowel disease. The immunoassay is particularly useful for assay of calprotectin in stool samples and is reproducible and less subject to interference from components of stool samples than existing assays. The immunoassay also avoids falsely low values when stool samples with high concentrations of calprotectin are tested, which is a problem that frequently occurs with existing assays.

[0191] Methods and kits according to the present invention possess industrial applicability for the in vitro assay of calprotectin.

[0192] The inventions illustratively described herein can suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms "comprising," "including," "containing," etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the future shown and described or any portion thereof, and it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the inventions herein disclosed can be resorted by those skilled in the art, and that such modifications and variations are considered to be within the scope of the inventions disclosed herein. The inventions have been described broadly and

generically herein. Each of the narrower species and subgeneric groupings falling within the scope of the generic disclosure also form part of these inventions. This includes the generic description of each invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised materials specifically resided therein.

[0193] In addition, where features or aspects of an invention are described in terms of the Markush group, those schooled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group. It is also to be understood that the above description is intended to be illustrative and not restrictive. Many embodiments will be apparent to those of in the art upon reviewing the above description. The scope of the invention should therefore, be determined not with reference to the above description, but should instead be determined with reference to the appended claims, along with the full scope of equivalents to which such claims are entitled. The disclosures of all articles and references, including patent publications, are incorporated herein by reference.

Claims

What is claimed is:

1 . A method for determining the concentration of calprotectin in a sample comprising the steps of:

(a) providing an antigen selected from the group consisting of purified S100A9 polypeptide and purified calprotectin;

(b) coating the antigen onto the solid phase;

(c) reacting the solid phase onto which the antigen has been coated with: (i) a sample that may contain calprotectin; and (ii) a labeled antibody specifically binding the antigen;

(d) washing the solid phase subsequent to the reaction of any calprotectin in the sample and the labeled antibody specifically binding the antigen to remove unbound labeled antibody specifically binding the antigen from the solid phase; and

(e) determining the quantity of labeled antibody specifically binding the antigen bound to the solid phase to determine the concentration of calprotectin in the sample;

2. The method of claim 1 wherein the antigen is S100A9 polypeptide and the labeled antibody specifically binding the antigen is a labeled anti-S100A9 antibody.

3. The method of claim 2 wherein the anti-S100A9 antibody is a monoclonal antibody.

4. The method of claim 2 wherein the anti-S100A9 antibody is a polyclonal antibody.

5. The method of claim 2 wherein the antibody to S100A9 polypeptide is produced using a recombinant S100A9 polypeptide as immunogen.

6. The method of claim 5 wherein the recombinant S100A9

polypeptide has the sequence

(SEQ ID NO: 1 ).

7. The method of claim 6 wherein the recombinant A9 polypeptide of SEQ ID NO: 1 is produced by expression of a DNA molecule with the sequence

GGTACCATATGACCTGCAAAATGAGCCAGCTGGAACGTAACATTGAAACCATCATC AACACCTTTCATCAGTATAGCGTGAAACTGGGCCATCCGGATACCCTGAACCAGG GCGAATTTATGATCGAACACATCATGGAAGATCTGGATACCAACGCGGATAAACAG CTGTCTTTCGAAGAAGAACTGGTGCGTAAAGATCTGCAGAACTTCCTGAAAAAAGA AAACAAAAACGAAAAAGAATTTATTATGCTGATGGCGCGTCTGACCTGGGCGAGCC ATGAAAAAATGCATGAAGGCGATGAAGGCCCGGGTCATCATCATAAACCGGGCCT GGGCGAAGGCACCCCGTGATAACTCG (SEQ ID NO: 3).

8. The method of claim 2 wherein the S100A9 polypeptide coated onto the solid phase is a purified and isolated naturally occurring S100A9 polypeptide.

9. The method of claim 2 wherein the S100A9 polypeptide coated onto the solid phase is a recombinant S100A9 polypeptide.

10. The method of claim 9 wherein the recombinant A9 protein has the sequence

(SEQ ID NO: 1 ).

1 1 . The method of claim 10 wherein the recombinant A9 protein of SEQ ID NO: 1 is produced by expression of a DNA molecule with the sequence

12. The method of claim 1 wherein the antigen is calprotectin and the labeled antibody specifically binding the antigen is a labeled anti-calprotectin antibody.

13. The method of claim 12 wherein the calprotectin is recombinantly produced calprotectin.

14. The method of claim 12 wherein the calprotectin is purified naturally occurring calprotectin.

15. The method of claim 1 wherein the label of the labeled antibody is an enzyme label.

16. The method of claim 1 wherein the enzyme label is selected from the group consisting of alkaline phosphatase, horseradish peroxidase, glucose 6- phosphate dehydrogenase, and β-galactosidase.

17. The method of claim 1 wherein the label of the labeled antibody is a label selected from the group consisting of a radioactive label, a fluorescent label, a chemiluminescent label, an electrochemiluminescent label, a bioluminescent label, a colloidal metal label, a colloidal metal oxide label, a dye label, a colored latex particle label, a colored polystyrene or polypropylene label, and a liposome label.

18. The method of claim 1 wherein the step of coating the solid support with the antigen is performed in Tris-buffered saline with from about 0.2 mM to 2 mM calcium present.

19. The method of claim 18 wherein the calcium is calcium chloride.

20. The method of claim 1 wherein the antigen is incubated with the solid support at a concentration of from 0.4 g/mL to about 1 .2 g/mL.

21 . The method of claim 1 wherein the incubation of the antigen with the solid support occurs at a temperature of from about 0° C to about 10° C.

22. The method of claim 1 wherein the incubation of the antigen with the solid support occurs for an incubation period of from about 6 hours to several weeks.

23. The method of claim 1 wherein subsequent to the step of binding the antigen to the solid support, the solid support is washed with Tris-buffered saline with from about 0.2 mM to 2 mM calcium present.

24. The method of claim 23 wherein subsequent to the washing step, a conditioning step is performed on the solid support, wherein the conditioning step comprises incubation of the solid support with a solution of sucrose and bovine serum albumin containing a phosphate buffer.

25. The method of claim 24 wherein the concentration of sucrose in the solution of sucrose and bovine serum albumin containing a phosphate buffer is from about 1 .5% to about 3.5%.

26. The method of claim 24 wherein the concentration of bovine serum albumin in the solution of sucrose and bovine serum albumin containing a phosphate buffer is from about 0.75% to about 1 .25%.

27. The method of claim 24 wherein the concentration of phosphate in the solution of sucrose and bovine serum albumin containing a phosphate buffer is from about 5 mM to about 15 mM.

28. The method of claim 24 wherein the pH of the phosphate buffer in the solution of sucrose and bovine serum albumin containing a phosphate buffer is from about 7.5 to about 8.5.

29. The method of claim 24 wherein the conditioning step is performed at a temperature from about 20° C to about 25° C for a period from about 15 minutes to about 45 minutes.

30. The method of claim 24 wherein, subsequent to the conditioning step, the solid support is washed with a washing buffer comprising Tris, sodium chloride, magnesium chloride, and a compatible biocide.

31 . The method of claim 30 wherein the concentration of Tris in the washing buffer is from about 25 mM to about 75 mM.

32. The method of claim 30 wherein the concentration of sodium chloride in the washing buffer is from about 100 mM to about 200 mM.

33. The method of claim 30 wherein the concentration of magnesium chloride in the washing buffer is from about 0.25 mM to about 0.75 mM.

34. The method of claim 30 wherein the washing buffer contains from about 0.5% to about 1 .5% of a compatible biocide.

35. The method of claim 30 wherein the pH of the washing buffer is from about 7.5 to about 8.5.

36. The method of claim 1 wherein the sample is a sample selected from the group consisting of a stool sample, a gastrointestinal tract sample, a whole blood sample, a plasma sample, a serum sample, a urine sample, and a crevicular fluid sample.

37. The method of claim 36 wherein the sample is a stool sample.

38. The method of claim 1 wherein the size of the sample is from about 50 μΙ_ to about 150 μΙ_.

39. The method of claim 1 wherein, subsequent to the addition of the sample and the labeled antibody to the solid support, the solid support is incubated at a temperature of from about 20-25° C for from about 10 minutes to about 20 minutes.

40. The method of claim 1 wherein the method further comprises the steps of constructing a standard curve and determining the concentration of calprotectin by comparison with the standard curve.

41 . The method of claim 40 wherein the standard curve is constructed using a plurality of concentrations of purified calprotectin.

42. The method of claim 40 wherein the standard curve is constructed using a plurality of concentrations of purified S100A9 polypeptide.

43. The method of claim 42 wherein the purified S100A9 polypeptide is a cloned recombinant S100A9 polypeptide.

44. The method of claim 15 wherein the enzyme produces a product that is detected and/or quantitated photometrically.

45. The method of claim 15 wherein the enzyme produces a product that is detected and/or quantitated by a technique selected from the group consisting of detection and/or quantitation of fluorescence, detection and/or quantitation of chemiluminescence, and detection and/or quantitation of bioluminescence.

46. A method for diagnosing or detecting the presence of active inflammatory bowel disease comprising the steps of:

(b) coating the antigen onto the solid phase;

(d) washing the solid phase subsequent to the reaction of any calprotectin in the sample and the labeled antibody specifically binding the antigen to remove unbound labeled antibody specifically binding the antigen from the solid phase;

(e) determining the quantity of labeled antibody specifically binding the antigen bound to the solid phase to determine the concentration of calprotectin in the sample; and

(f) determining the presence or absence of active inflammatory bowel disease in the patient according to whether the calprotectin concentration in the sample is at least 50 mg/kg, in which case active inflammatory bowel disease is present in the patient, or is less than 50 mg/kg, in which case active inflammatory bowel disease is absent in the patient.

47. A method for determining whether a patient with inflammatory bowel disease who has been in remission is at risk of suffering a relapse comprising the steps of:

(b) coating the antigen onto the solid phase; (c) reacting the solid phase onto which the antigen has been coated with: (i) a sample that may contain calprotectin; and (ii) a labeled antibody specifically binding the antigen;

(f) determining the risk of relapse of inflammatory bowel disease in the patient according to whether the calprotectin concentration in the sample is at least 50 mg/kg, in which case a risk of relapse of inflammatory bowel disease exists, or is less than 50 mg/kg, in which case a risk of relapse of inflammatory bowel disease does not exist.

48. A method of performing an immunoassay for an antigen selected from the group consisting of calprotectin and S100A9 and for a related antigen to determine whether inflammatory bowel disease exists in a subject, the method comprising the steps of:

(a) providing a stool sample from a subject;

(b) performing a competitive immunoassay for the antigen selected from the group consisting of calprotectin and S100A9 using: (i) an immobilized antigen selected from the group consisting of immobilized calprotectin and immobilized S100A9 polypeptide; and (ii) a labeled antibody that is anti-calprotectin antibody when the immobilized antigen is calprotectin and is labeled anti-S100A9 antibody when the immobilized antigen is S100A9 polypeptide, the competitive immunoassay employing a solid phase platform;

(c) performing an immunoassay for a related antigen;

(d) determining the concentration of the antigen selected from the group consisting of calprotectin and S100A9 in the stool sample from the results of the competitive immunoassay for the antigen selected from the group consisting of calprotectin and S100A9 employing the solid phase platform;

(e) determining the concentration of the related antigen in the stool sample from the results of the immunoassay for the related antigen; and

(f) comparing the concentration of the antigen selected from the group consisting of calprotectin and S100A9 and the concentration of the related antigen in the stool sample with the cutoff values for the antigen selected from the group consisting of calprotectin and S100A9 and for the related antigen to determine whether or not inflammatory bowel disease exists in the subject.

49. The method of claim 48 wherein the related antigen is S100A12.

50. The method of claim 48 wherein the related antigen is lactoferrin.

51 . The method of claim 48 wherein the immunoassay for the related antigen is performed in a competitive immunoassay.

52. The method of claim 48 wherein the immunoassay for the related antigen is performed in a sandwich immunoassay.

53. A device for performing a lateral flow competitive immunoassay for an antigen selected from the group consisting of calprotectin and S100A9 comprising:

(a) a solid support for lateral flow;

(b) a labeled antibody specifically binding the antigen selected from the group consisting of calprotectin and S100A9 comprising that is either present on the device before the commencement of the immunoassay or is added to the device at the commencement of the immunoassay; and

(c) a detection zone on the solid support comprising either: (i) immobilized calprotectin when the labeled antibody is an antibody specifically binding calprotectin or (ii) immobilized S100A9 polypeptide when the labeled antibody is an antibody specifically binding S100A9 polypeptide such that any calprotectin or S100A9 polypeptide in the sample competes with the immobilized calprotectin or the

immobilized S100A9 polypeptide in the detection zone.

54. The device of claim 53 wherein the antigen is calprotectin and the labeled antibody is an antibody specifically binding calprotectin.

55. The device of claim 53 wherein the antigen is S100A9 polypeptide and the labeled antibody is an antibody specifically binding S100A9 polypeptide.

56. The device of claim 53 wherein the solid support is constructed of a material selected from the group consisting of nitrocellulose, glass fiber, paper, nylon, and a synthetic nanoporous polymer.

57. The device of claim 56 wherein the solid support is nitrocellulose.

58. The device of claim 53 wherein the labeled antibody is present on the device before the commencement of the immunoassay.

59. The device of claim 53 wherein the labeled antibody is added to the device at the commencement of the immunoassay.

60. The device of claim 53 further comprising a control zone.

61 . The device of claim 60 wherein the control zone comprises an immobilized antibody capable of specifically binding the labeled antibody by binding to a portion of the antibody distinct from the paratope of the labeled antibody.

62. The device of claim 60 wherein the control zone comprises an immobilized unrelated antigen and the device comprises a labeled antibody specifically binding the immobilized unrelated antigen.

63. The device of claim 53 further comprising a casing having at least one aperture therein.

64. The device of claim 53 wherein the device comprises:

(a) a solid support providing lateral flow having a first end and a second end;

(b) a detection zone having an immobilized protein or polypeptide selected from the group consisting of immobilized calprotectin and immobilized S100A9 polypeptide, the detection zone located on the solid support;

(c) a conjugate zone having a mobilizable labeled antibody selected from the group consisting of anti-calprotectin antibody and anti-S100A9 antibody located on the solid support; and

(d) a sample pad for application of a sample in operable contact with the first end of the solid support; wherein a sample applied to the sample pad migrates to the conjugate zone to solubilize and mobilize the mobilizable labeled antibody and the sample and the mobilized labeled antibody migrate to the detection zone, such that any calprotectin or S100A9 polypeptide in the sample competes for binding of the labeled antibody with the immobilized calprotectin or immobilized S100A9 polypeptide bound at the detection zone to perform a competitive immunoassay.

65. The device of claim 53 wherein the device comprises:

(a) a solid support providing lateral flow having a first end and a second end;

(b) a detection zone having an immobilized protein or polypeptide selected from the group consisting of immobilized calprotectin and immobilized S100A9 polypeptide, the detection zone located on the solid support; and

(c) a conjugate pad having a mobilizable labeled antibody selected from the group consisting of anti-calprotectin antibody and anti-S100A9 antibody in operable contact with the first end of the solid support;

66. The device of claim 53 wherein the device comprises:

(a) a solid support providing lateral flow having a first end and a second end;

(c) a conjugate pad having a mobilizable labeled antibody selected from the group consisting of anti-calprotectin antibody and anti-S100A9 antibody in operable contact with the first end of the solid support; and (d) a sample pad for application of the sample in operable contact with the conjugate pad;

67. The device of claim 53 wherein the device comprises:

(a) a first substantially planar part, the first substantially planar part comprising a solid support that provides lateral flow, the solid support that provides lateral flow comprising a detection zone having an immobilized protein or polypeptide selected from the group consisting of immobilized calprotectin and immobilized S100A9 polypeptide;

(b) a second substantially planar part, the second substantially planar part comprising a conjugate pad having a mobilizable labeled antibody selected from the group consisting of anti-calprotectin antibody and anti-S100A9 antibody; and

(c) a hinge connecting the first substantially planar part with the second substantially planar part;

68. The device of claim 53 wherein the device comprises:

(b) a second substantially planar part, the second substantially planar part comprising a sample pad for application of a sample and a conjugate pad having a mobilizable labeled antibody selected from the group consisting of anti-calprotectin antibody and anti-S100A9 antibody in operable contact with the sample pad; and

69. The device of claim 53 wherein the device comprises:

(a) a solid support providing lateral flow having a first end and a second end;

(c) a sample pad in operable contact with the first end of the solid support;

wherein, after a sample and a mobile labeled antibody selected from the group consisting of anti-calprotectin antibody and anti-S100A9 antibody are applied to the sample pad, the sample and the mobile labeled antibody migrate to the detection zone, such that any calprotectin or S100A9 polypeptide in the sample competes for binding of the labeled antibody with the immobilized calprotectin or immobilized S100A9 polypeptide bound at the detection zone to perform a competitive immunoassay.

70. The device of claim 53 wherein the device comprises: (a) a solid support providing lateral flow having a first end and a second end; and

polypeptide bound at the detection zone to perform a competitive immunoassay.

71 . The device of claim 53 wherein the device comprises:

(b) a second substantially planar part, the second substantially planar part comprising a sample pad for application of a sample and a conjugate application pad for application of a mobile labeled antibody selected from the group consisting of anti-calprotectin antibody and anti-S100A9 antibody in operable contact with the sample pad; and

72. The device of claim 53 wherein the device comprises:

(b) a second substantially planar part, the second substantially planar part comprising a sample pad for application of a sample and a mobile labeled antibody selected from the group consisting of anti-calprotectin antibody and anti-S100A9 antibody to the sample pad;

73. The device of claim 53 wherein the label of the labeled anti-S100A9 antibody is a directly detectable label.

74. The device of claim 53 wherein the directly detectable label is selected from the group consisting of a radioactive label, a fluorescent label, a chemiluminescent label, an electrochemiluminescent label, a bioluminescent label, a colloidal metal label, a colloidal metal oxide label, a dye label, a colored latex particle label, a colored polystyrene or polypropylene particle label, a liposome label, a Surface- Enhanced Resonant Raman Spectroscopy (SERRS) label, or a signaling aptamer label.

75. The device of claim 74 wherein the directly detectable label is a colloidal metal label.

76. The device of claim 75 wherein the colloidal metal label is selected from the group consisting of a colloidal gold label and a colloidal silver label.

77. The device of claim 53 wherein the immobilized protein or polypeptide selected from the group consisting of immobilized calprotectin and immobilized S100A9 polypeptide is covalently immobilized at the detection zone.

78. The device of claim 53 wherein the immobilized protein or polypeptide selected from the group consisting of immobilized calprotectin and immobilized S100A9 polypeptide is non-covalently immobilized at the detection zone.

79. The device of claim 55 wherein the S100A9 polypeptide immobilized at the detection zone is a recombinant S100A9 polypeptide.

80. The device of claim 79 wherein the immobilized S100A9

polypeptide has the sequence

(SEQ ID NO: 1 ).

81 . The device of claim 80 wherein the recombinant A9 protein of SEQ ID NO: 1 is produced by expression of a DNA molecule with the sequence

82. The device of claim 55 wherein the S100A9 polypeptide immobilized at the detection zone is a purified and naturally occurring S100A9 polypeptide.

83. The device of claim 54 wherein the labeled anti-calprotectin antibody is an anti-calprotectin monoclonal antibody.

84. The device of claim 54 wherein the labeled anti-calprotectin antibody is an anti-calprotectin polyclonal antibody.

85. The device of claim 55 wherein the labeled anti-S100A9 antibody is an anti-S100A9 monoclonal antibody.

86. The device of claim 55 wherein the labeled anti-S100A9 antibody is an anti-S100A9 polyclonal antibody.

87. A method for determining the concentration of calprotectin in a sample comprising the steps of:

(a) applying the sample to the device of claim 53;

(b) allowing the sample and the labeled antibody to migrate through the solid support for lateral flow and to the detection zone at which any calprotectin or S100A9 polypeptide in the sample competes with the immobilized calprotectin or S100A9 polypeptide in the detection zone; and

(c) determining the quantity of labeled antibody bound to the detection zone to determine the concentration of calprotectin in the sample, wherein the quantity of calprotectin or S100A9 polypeptide immobilized at the detection zone and the quantity of labeled calprotectin antibody or labeled anti-S100A9 antibody are chosen such that the calprotectin or S100A9 polypeptide immobilized at the detection zone and any calprotectin in the sample compete for the labeled anti-calprotectin antibody or labeled anti-S100A9 antibody such that the quantity of labeled anti-calprotectin antibody or labeled anti-S100A9 antibody immobilized to the detection zone is related inversely to the concentration of calprotectin in the sample.

88. The method of claim 87 wherein the method further comprises the steps of constructing a standard curve and determining the concentration of calprotectin by comparison with the standard curve.

89. The method of claim 88 wherein the standard curve is constructed using a plurality of concentrations of purified calprotectin.

90. The method of claim 88 wherein the standard curve is constructed using a plurality of concentrations of purified S100A9 polypeptide.

91 . The method of claim 90 wherein the purified S100A9 polypeptide is a cloned recombinant S100A9 polypeptide.

92. A method of detecting or diagnosing the presence of active inflammatory bowel disease comprising the steps of:

(a) applying a sample that may contain calprotectin to the device of claim 53;

(b) allowing the sample and the labeled antibody to migrate through the solid support for lateral flow and to the detection zone at which any calprotectin or S100A9 polypeptide in the sample competes with the immobilized calprotectin or S100A9 polypeptide in the detection zone;

(c) determining the quantity of labeled antibody bound to the detection zone to determine the concentration of calprotectin in the sample, wherein the quantity of calprotectin or S100A9 polypeptide immobilized at the detection zone and the quantity of labeled calprotectin antibody or labeled anti-S100A9 antibody are chosen such that the calprotectin or S100A9 polypeptide immobilized at the detection zone and any calprotectin in the sample compete for the labeled anti-calprotectin antibody or labeled anti-S100A9 antibody such that the quantity of labeled anti-calprotectin antibody or labeled anti-S100A9 antibody immobilized to the detection zone is related inversely to the concentration of calprotectin in the sample; and

(d) determining the presence or absence of active inflammatory bowel disease in the patient according to whether the calprotectin concentration in the sample is at least 50 mg/kg, in which case active inflammatory bowel disease is present in the patient, or is less than 50 mg/kg, in which case active inflammatory bowel disease is absent in the patient.

93. The method of claim 92 wherein the method further comprises the steps of constructing a standard curve and determining the concentration of calprotectin in the sample by comparison with the standard curve.

94. The method of claim 93 wherein the standard curve is constructed using a plurality of concentrations of purified calprotectin.

95. The method of claim 93 wherein the standard curve is constructed using a plurality of concentrations of purified S100A9 polypeptide.

96. The method of claim 95 wherein the purified S100A9 polypeptide is a cloned recombinant S100A9 polypeptide.

97. A method of determining whether a patient with inflammatory bowel disease who has been in remission is at risk of suffering a relapse comprising the steps of:

(a) applying a sample that may contain calprotectin to the device of claim 53;

(d) determining the risk of relapse of inflammatory bowel disease in the patient according to whether the calprotectin concentration in the sample is at least 50 mg/kg, in which case a risk of relapse of inflammatory bowel disease exists, or is less than 50 mg/kg, in which case a risk of relapse of inflammatory bowel disease does not exist.

98. The method of claim 97 wherein the method further comprises the steps of constructing a standard curve and determining the concentration of calprotectin in the sample by comparison with the standard curve.

99. The method of claim 98 wherein the standard curve is constructed using a plurality of concentrations of purified calprotectin.

100. The method of claim 98 wherein the standard curve is constructed using a plurality of concentrations of purified S100A9 polypeptide.

101 . The method of claim 100 wherein the purified S100A9 polypeptide is a cloned recombinant S100A9 polypeptide.

102. A method of performing an immunoassay for: (i) calprotectin or S100A9 polypeptide and (ii) a related antigen to determine whether inflammatory bowel disease exists in a subject, the method comprising the steps of:

(a) providing a stool sample from a subject;

(b) applying the sample to the device of claim 53;

(c) allowing the sample and the labeled antibody to migrate through the solid support for lateral flow and to the detection zone at which any calprotectin or S100A9 polypeptide in the sample competes with the immobilized calprotectin or S100A9 polypeptide in the detection zone;

(d) performing an immunoassay for a related antigen;

(e) determining the quantity of labeled antibody bound to the detection zone to determine the concentration of calprotectin in the sample, wherein the quantity of S100A9 polypeptide or calprotectin immobilized at the detection zone and the quantity of labeled anti-S100A9 antibody or labeled anti-calprotectin antibody are chosen such that the S100A9 polypeptide or calprotectin immobilized at the detection zone and any calprotectin in the sample compete for the labeled antibody such that the quantity of labeled antibody immobilized to the detection zone is related inversely to the concentration of calprotectin in the sample;

(f) determining the concentration of the related antigen in the stool sample from the results of the immunoassay for the related antigen; and (g) comparing the concentration of calprotectin and the concentration of the related antigen in the stool sample with the cutoff values for calprotectin and the related antigen to determine whether or not inflammatory bowel disease exists in the subject.

103. The method of claim 102 wherein the method further comprises the steps of constructing a standard curve and determining the concentration of calprotectin in the sample by comparison with the standard curve.

104. The method of claim 103 wherein the standard curve is constructed using a plurality of concentrations of purified calprotectin.

105. The method of claim 103 wherein the standard curve is constructed using a plurality of concentrations of purified S100A9 polypeptide.

106. The method of claim 105 wherein the purified S100A9 polypeptide is a cloned recombinant S100A9 polypeptide.

107. The method of claim 102 wherein the related antigen is S100A12.

108. The method of claim 102 wherein the related antigen is lactoferrin.

109. The method of claim 102 wherein the immunoassay for the related antigen is performed in a competitive immunoassay.

1 10. The method of claim 102 wherein the immunoassay for the related antigen is performed in a sandwich immunoassay.

1 1 1 . A kit for immunoassay of S100A9 polypeptide and diagnosis, screening, or monitoring of inflammatory bowel disease comprising, separately packaged:

(a) a solid support for performing a lateral flow competitive immunoassay for detection of S100A9 polypeptide; and

(b) a labeled anti-S100A9 antibody to be applied to the solid support.

1 12. The kit of claim 1 1 1 wherein the kit includes a single solid support and a quantity of labeled anti-S100A9 antibody sufficient for a single assay.

1 13. The kit of claim 1 1 1 wherein the kit includes a plurality of solid supports and a quantity of labeled anti-S100A9 antibody sufficient for a plurality of assays such that the quantity of labeled anti-S100A9 antibody is sufficient for use with each of the solid supports included in the kit.

1 14. The kit of claim 1 1 1 wherein the label of the labeled anti-S100A9 antibody is an enzyme label and wherein the kit further comprises, separately

packaged, a substrate for the enzyme label.

1 15. The kit of claim 1 1 1 further comprising, separately packaged, a set of S100A9 polypeptide standards of differing concentrations so that a standard curve for S100A9 polypeptide can be established.

1 16. A kit for immunoassay of calprotectin and diagnosis, screening, or monitoring of inflammatory bowel disease comprising, separately packaged:

(b) a labeled anti-calprotectin antibody to be applied to the solid support.

1 17. The kit of claim 1 16 wherein the kit includes a single solid support and a quantity of labeled anti-calprotectin antibody sufficient for a single assay.

1 18. The kit of claim 1 16 wherein the kit includes a plurality of solid supports and a quantity of labeled anti-calprotectin antibody sufficient for a plurality of assays such that the quantity of labeled anti-calprotectin antibody is sufficient for use with each of the solid supports included in the kit.

1 19. The kit of claim 1 16 wherein the label of the labeled anti- calprotectin antibody is an enzyme label and wherein the kit further comprises, separately packaged, a substrate for the enzyme label.

120. The kit of claim 1 16 further comprising, separately packaged, a set of calprotectin standards of differing concentrations so that a standard curve for calprotectin can be established.