WO2013009459A1

WO2013009459A1 - Urine assays, kits, and devices for typhoid fever diagnosis

Info

Publication number: WO2013009459A1
Application number: PCT/US2012/044101
Authority: WO
Inventors: Edward T. Ryan; Richelle Charles; Firdausi Qadri
Original assignee: The General Hosptial Corporation; International Centre For Diarrhoeal Disease Research, Bangladesh (Icddr,B)
Priority date: 2011-07-14
Filing date: 2012-06-26
Publication date: 2013-01-17

Abstract

Provided are methods, assays, kits, compositions and devices for detection of S. Typhi in urine samples.

Description

URINE ASSAYS, KITS, AND DEVICES FOR TYPHOID FEVER DIAGNOSIS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims benefit under 35 U.S.C. §119(e) of a U.S. provisional application No. 61/507,987, filed on July 14, 2011, the content of which are incorporated herein by reference in their entirety.

SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on June 25, 2012, is named 32587561.txt and is 64,613 bytes in size.

FIELD OF THE INVENTION

[0003] The present invention relates to immunoassays to detect presence or absence of newly identified S. Typhi antigens in a urine sample to diagnose Typhoid or enteric fever and salmonellosis in humans.

BACKGROUND OF THE INVENTION

[0004] Attempts have been made previously to create a urine diagnostic using known Salmonella proteins that have been previously purified: Vi capsular antigen, O antigen 9, and flagellar antigens (see, e.g., Barrett TJ, Snyder JD, Blake PA, Feeley JC (1982) Enzyme-linked immunosorbent assay for detection of Salmonella typhi Vi antigen in urine from typhoid patients J Clin Microbiol 15: 235-237; Chaicumpa W, Ruangkunaporn Y, Burr D, Chongsa-Nguan M, Echeverria P (1992) Diagnosis of typhoid fever by detection of Salmonella typhi antigen in urine J Clin Microbiol 30: 2513-2515; Chaicumpa W, Thin-Inta W, Khusmith S, Tapchaisri P, Echeverria P, et al (1988) Detection with monoclonal antibody of Salmonella typhi antigen 9 in specimens from patients J Clin Microbiol 26: 1824-1830; Fadeel MA, Crump JA, Mahoney FJ, Nakhla IA, Mansour AM, et al (2004) Rapid diagnosis of typhoid fever by enzyme -linked immunosorbent assay detection of Salmonella serotype Typhi antigens in urine Am J Trop Med Hyg 70: 323-328; Hatta M, Smits HL (2007) Detection of Salmonella typhi by nested polymerase chain reaction in blood, urine, and stool samples Am J Trop Med Hyg 76: 139-143; Rockhill RC, Rumans LW, Lesmana M, Dennis DT (1980) Detection of Salmonella typhi D, Vi, and d antigens, by slide coagglutination, in urine from patients with typhoid fever J Clin Microbiol 11 : 213-216; Taylor DN, Harris JR, Barrett TJ, Hargrett NT, Prentzel I, et al (1983) Detection of urinary Vi antigen as a diagnostic test for typhoid fever. J Clin Microbiol 18: 872-876; West B, Richens JE, Howard PF (1989) Evaluation in Papua New Guinea of a urine coagglutination test and a Widal slide agglutination test for rapid diagnosis of typhoid fever. Trans R Soc Trop Med Hyg 83: 715-717). However, these diagnostic approaches have not been successful.

[0005] Urine samples are much easier to collect and pose significantly less of an infection risk than collection of blood samples, particularly, in environments where typhoid fever is most prominent. Thus, identification of possible target to determine Salmonella infection and assays and kits for detecting the Salmonella and particularly Salmonella Typhi specific proteins in human urine samples would be desirable.

SUMMARY

[0006] We have developed a point-of-care urine test or assay for enteric fever also referred to a typhoid fever. The assay or test is based on identification of 14 different Salmonella Typhi proteins present in the urine of bacteremic patients which serve as antigen targets for antibodies used in a urine diagnostic test provided herein. The proteins were identified using mass spectrometry screening.

[0007] Accordingly, we provide assays, methods, kits, and devices for analysis and detection of at least one of the 14 proteins identified in urine samples of the typhoid fever patients. Presence of the protein in the urine sample indicates that the individual has typhoid fever or is infected with S. Typhi. Thus, such individuals can be subjected to appropriate care. In some aspects of all the embodiments of the invention, if at least one of the proteins, or at least two of the proteins, or at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or all 14 of the proteins is/are detected, then the subject is subjected to treatment for S. Typhi.

[0008] In one embodiment, SipC (STY3007) and PilQ (STY4545) are detected. SipC is unique to Salmonella species and is a protein important in Salmonella pathogenesis. PilQ is part of the pilus cluster within Salmonella pathogenicity island 7, an island which is rarely found in non- Typhi Salmonella species.

[0009] Accordingly, in one embodiment, the invention provides an in vitro assay or method comprising the steps of: (a) contacting in vitro a urine sample from a human patient with an isolated and purified antibody against at least one S. Typhi protein selected from the group consisting of STY3639, STY3461, STY3007, STY0777, STY2194, STY2874, STY4489, STY3710, STY0184, STY4070, STY4090, STY4545, STY3467, STY2073 and any combination and portion or fragment thereof, thereby forming an immune complex between the at least one S. Typhi protein and the antibody; and (b) detecting the bound antibodies, i.e., the immune complex, to determine whether the biological sample contains the at least one S. Typhi antigen.

[00010] In some aspects of this embodiment, the S. Typhi antigen in the in vitro assay is STY3007, STY4545 or a combination thereof.

[00011] In one embodiment, the invention provides an in vitro assay or method comprising the steps of: (a) contacting in vitro a urine sample from a human patient with an isolated and purified first antibody against STY3007 or a fragment thereof, and an isolated and purified second antibody against STY4545, or a fragment thereof, thereby forming an immune complex between STY3007 and the first antibody and STY4545 and the second antibody; and (b) detecting the bound first and second antibodies to determine whether the biological sample contains the at one of STY3007 or STY4545. [00012] In one aspect, the in vitro assays further comprise a step of diagnosis of the human patient.

[00013] Accordingly, in one aspect of the in vitro assays of the invention, the assay further comprised diagnosing salmonellosis in a patient from whom the biological sample has been obtained if STY3007 is detected in the biological sample but STY4545 is not detected.

[00014] In one aspect of the in vitro assays of the invention, the assay further comprises diagnosing typhoid fever in the patient from whom the sample has been taken if both STY3007 and STY4545 are detected.

[00015] In some aspects of the in vitro assays of the invention, the assay further comprised a step of diagnosing the human patient with typhoid fever if the at least one S. Typhi antigen is detected.

[00016] In some aspects of the assays of the invention, the diagnosing is performed using a non-human machine. The machine can include a display module to display the result of presence or absence of the analyzed proteins on a computer screen, on a print out, on an e-mail or any other type of display that can indicate the presence or absence of the proteins in the urine sample.

[00017] In some aspects of all the embodiments of the invention, the non-human machine includes a computer implemented software.

[00018] In some aspects of all the assays of the invention, the antibody is labeled.

[00019] In some aspects of all the assays of the invention the antibody is detected using a secondary antibody.

[00020] In some aspects of the assays of the invention, the antibody is bound or immobilized to a solid surface. The solid surface can be a bead, such as a magnetic, gold, or polystyrene bead or strip, or an array, such as glass, plastic or silicon chip.

[00021] In some embodiments, the invention provides a test strip comprising an antibody against at least one S. Typhi antigen selected from the group consisting of STY3639, STY3461, STY3007, STY0777, STY2194, STY2874, STY4489, STY3710, STY0184, STY4070, STY4090, STY4545, STY3467, STY2073 and any combination thereof bound to a surface.

[00022] In some aspects of this embodiment, the test strip comprises a first antibody against STY3007 and a second antibody against STY4545.

[00023] In some aspects the test strip further comprises at least one control for a negative test result and at least one control for a positive test result.

[00024] In some aspects, the test strip comprises a sold surface.

[00025] In some aspects, the solid surface is a porous surface.

[00026] In some embodiments, the invention further provides a microfluidic device comprising an antibody against at least one isolated S. Typhi antigen selected from the group consisting of STY3639, STY3461, STY3007, STY0777, STY2194, STY2874, STY4489, STY3710, STY0184, STY4070, STY4090, STY4545, STY3467, STY2073 and any combination thereof. [00027] In some aspects, the microfluidic device comprises a first antibody against STY3007 and a second antibody against STY4545.

[00028] In one embodiment, the invention provides a composition comprising at least one antibody against an isolated S. Typhi antigen selected from the group consisting of STY3639, STY3461, STY3007, STY0777, STY2194, STY2874, STY4489, STY3710, STY0184, STY4070, STY4090, STY4545, STY3467, STY2073 and any combination and fragment thereof.

[00029] In some aspects, the composition comprises a first antibody against STY3007 and a second antibody against STY4545.

[00030] In some embodiments, the invention also provides a kit comprising an antibody against an isolated S. Typhi antigen selected from the group consisting of STY3639, STY3461, STY3007, STY0777, STY2194, STY2874, STY4489, STY3710, STY0184, STY4070, STY4090, STY4545, STY3467, STY2073 and any combination and fragment thereof.

[00031] In some aspects, the kit comprises a first antibody against STY3007 and a second antibody against STY4545.

DETAILED DESCRIPTION OF THE INVENTION

[00032] The invention generally provides assays, methods, systems, devices and kits for detecting one or more of fourteen (14) S. Typhi antigens from a urine sample of a human to determine if the human has Typhoid (enteric fever) or samonellosis.

[00033] The assays and methods generally comprise the steps of contacting, in vitro, a urine sample with one or more antibodies against S. Typhi proteins selected from the group consisting of STY3639, STY3461, STY3007, STY0777, STY2194, STY2874, STY4489, STY3710, STY0184, STY4070, STY4090, STY4545, STY3467, STY2073 or fragments thereof. In some embodiments the proteins are STY3007 or STY4545 or combination or fragments thereof. The bound antibodies are detected using well known methods. If an antigen is detected in the sample, the patient can be diagnosed as having typhoid fever. The antigen can be detected using automated detection of binding such as an image reader of an ELISA assay, and if a bound antibody is detected, the data indicating that a bound antibody has been detected can be transferred, e.g., to a computer display or on a paper print out.

[00034] In some embodiments, one can assay for the difference between salmonellosis and Typhoid. This can be done, for example, using STY3007 as a marker for Salmonella because this antigen is also present in other Salmonella species, and STY4545 as a marker for S. Typhi. If the patient has salmonellosis, only STY3007 is detected. If the patient has typhoid fever, both STY3007 and STY4545 are detected.

[00035] Differential detection allows selection of treatment options for the specific disease. Thus, in one embodiment, if only STY3007 is detected and not STY4545, the patient will be given a treatment suitable for salmonellosis whereas if both STY3007 and STY4545 are detected, the patient will be given a treatment suitable for Typhoid.

[00036] The S. Typhi proteins and their fragments may be isolated and/or purified or produced synthetically or using recombinant nucleic acid technology. The purification may be partial or substantial.

[00037] As used herein, the term "comprising" means that other elements can also be present in addition to the defined elements presented. The use of "comprising" indicates inclusion rather than limitation.

[00038] Both polyclonal and monoclonal antibodies can be prepared using the entire proteins as antigens or fragments thereof.

[00039] The term "fragment" refers to any subject polypeptide having an amino acid residue sequence shorter than that of a polypeptide whose amino acid residue sequence is described herein. Typically, the antigenic fragments are at least 5 consecutive amino acids long, and can be longer. The fragments can be modified to be more antigenic using well known methods in the art.

[00040] The fragment preferably comprises at least one epitope. An "epitope" is the collective features of a molecule, such as primary, secondary and tertiary peptide structure, and charge, that together form a site recognized by an immunoglobulin, T cell receptor or HLA molecule. Alternatively, an epitope can be defined as a set of amino acid residues which is involved in recognition by a particular immunoglobulin, or in the context of T cells, those residues necessary for recognition by T cell receptor proteins and/or Major Histocompatibility Complex (MHC) receptors.

[00041] Epitopes are present in nature, and can be isolated, purified or otherwise

prepared/derived by human or non-human means. For example, epitopes can be prepared by isolating the S. Typhi peptides from a natural source, such as a bacterial culture, or they can be synthesized in accordance with standard protocols in the art.

[00042] Synthetic epitopes can comprise artificial amino acids "amino acid mimetics," such as D isomers of natural occurring L amino acids or non-natural amino acids such as cyclohexylalanine. Throughout this disclosure, the terms epitope and peptide are often used interchangeably. In some embodiments, one can use analogs of said epitopes to produce additional antibodies against the S. Typhi proteins described herein.

[00043] Protein or polypeptide molecules that comprise one or more S. Typhi peptide epitopes of the invention as well as additional amino acid(s) are still within the bounds of the invention. In certain embodiments, there is a limitation on the length of a polypeptide of the invention of, for example, not more than 120 amino acids, not more than 110 amino acids, not more than 100 amino acids, not more than 95 amino acids, not more than 90 amino acids, not more than 85 amino acids, not more than 80 amino acids, not more than 75 amino acids, not more than 70 amino acids, not more than 65 amino acids, not more than 60 amino acids, not more than 55 amino acids, not more than 50 amino acids, not more than 45 amino acids, not more than 40 amino acids, not more than 35 amino acids, not more than 30 amino acids, not more than 25 amino acids, 20 amino acids, 15 amino acids, or 14, 13, 12, 11, 10, 9 or 8 amino acids. In some instances, the embodiment that is length-limited occurs when the protein/polypeptide comprising an epitope of the invention comprises a region (i.e., a contiguous series of amino acids) having 100% identity with a native sequence.

[00044] The term "derived" when used to discuss a peptide epitope is a synonym for

"prepared." A derived epitope can be isolated from a natural source, or it can be synthesized in accordance with standard protocols in the art.

[00045] Protein or polypeptide molecules that comprise one or more S. Typhi peptide epitopes of the invention as well as additional amino acid(s) are still within the bounds of the invention.

[00046] In certain embodiments, there is a limitation on the length of a polypeptide used for producing the antibody. For example, if one uses STY3007 as a source for antigens, one can use a protein which is between about 8 and about 400 amino acids, or not more than 400 amino acids, not more than 350 amino acids, not more than 300 amino acids, not more than 250 amino acids, not more than 200 amino acids, not more than 150 amino acids, not more than 125 amino acids, not more than 110 amino acids, not more than 100 amino acids, not more than 95 amino acids, not more than 90 amino acids, not more than 85 amino acids, not more than 80 amino acids, not more than 75 amino acids, not more than 70 amino acids, not more than 65 amino acids, not more than 60 amino acids, not more than 55 amino acids, not more than 50 amino acids, not more than 45 amino acids, not more than 40 amino acids, not more than 35 amino acids, not more than 30 amino acids, not more than 25 amino acids, or a protein that is about 20-8 amino acids, such as about 20, amino acids, 15 amino acids, or 14, 13, 12, 11, 10, 9 or 8 amino acids. In some instances, the embodiment that is length- limited occurs when the protein/polypeptide comprising an epitope of the invention comprises a region (i.e., a contiguous series of amino acids) having 100% identity with a native sequence.

[00047] For a polypeptide comprising an epitope of the invention and a region with 100% identity with the native S. Typhi polypeptide sequence, the region with 100% identity to the native sequence generally has a length of: less than or equal to 120 amino acids, more often less than or equal to 100 amino acids, often less than or equal to 85 amino acids, often less than or equal to 75 amino acids, often less than or equal to 65 amino acids, and often less than or equal to 50 amino acids. In certain embodiments, the S. Typhi polypeptide comprises a peptide having a region with less than 50 amino acids that has 100% identity to a native peptide sequence, in any increment of amino acids down to 5 amino acids (i.e., 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, or 50-5 amino acids). Preferably, such S. Typhi polypeptide comprises one or more S. Typhi peptide epitopes that are capable of eliciting an immune reaction and thus production of suitable antibodies. [00048] In some embodiments embodiment, the polyclonal antibodies are generated using the whole proteins.

[00049] In some embodiments, the monoclonal antibodies are directed against the epitopes set forth in Table 1. Additional peptides can be prepared and used by routine methods well known to one skilled in the art.

[00050] An "immunogenic peptide" or "peptide epitope" is a peptide that will bind an HLA molecule and induce a cytotoxic T lymphocyte (CTL) response and/or a helper T lymphocyte (HTL) response. Thus, immunogenic peptides of the invention are capable of binding to an appropriate HLA molecule and thereafter inducing a cytotoxic T lymphocyte (CTL) response, or a helper T lymphocyte (HTL) response, to the peptide.

[00051] The term "motif" refers to a pattern of residues in an amino acid sequence of defined length, usually a peptide of from about 8 to about 13 amino acids for a class I HLA motif and from about 16 to about 25 amino acids for a class II HLA motif, which is recognized by a particular HLA molecule. Motifs are typically different for each HLA protein encoded by a given human HLA allele. These motifs often differ in their pattern of the primary and secondary anchor residues.

[00052] The term "residue" refers to an amino acid or amino acid mimetic incorporated into a peptide or protein by an amide bond or amide bond mimetic.

[00053] "Synthetic peptide" refers to a peptide that is not naturally occurring, but is man-made using such methods as chemical synthesis or recombinant DNA technology.

[00054] Antibodies, both polyclonal and monoclonal, can be produced by a skilled artisan either by them selves using well known methods or they can be manufactured by service providers who specialize making antibodies based on known protein sequences. In the present invention, the protein sequence s are known and thus production of antibodies against them is a matter of routine.

[00055] For example, production of monoclonal antibodies can be performed using the traditional hybridoma method by first immunizing mice with an isolated S. Typhi protein or fragment thereof of choice or even with a crude extract of S. Typhi and making hybridoma cell lines that each produce a specific monoclonal antibody. The antibodies secreted by the different clones are then assayed for their ability to bind to the antigen using, e.g., ELISA or Antigen Microarray Assay, or immuno-dot blot technique. To detect the antibodies that are most specific for the detection of the protein of interest can be selected using routine methods and using the antigen and other antigens as well as S. Typhi crude extract as controls. The antibody that most specifically detects the desired antigen and protein and not other antigens or proteins will be selected for the detection assays.

[00056] The best clones can then be grown indefinitely in a suitable cell culture medium. They can also be injected into mice (in the peritoneal cavity, surrounding the gut) where they produce tumors secreting an antibody-rich ascites fluid from which the antibodies can be isolated and purified.

[00057] The antibodies can be purified using techniques that are well known to one of ordinary skill in the art.

[00058] In the methods and assays of the invention, the presence of any one or any combination of the 14 5. Typhi proteins is determined using antibodies specific for the S. Typhi proteins and detecting immunospecific binding of each antibody to its respective cognate marker.

[00059] Any suitable immunoassay method may be utilized, including those which are commercially available, to determine the level of each at least one of the specific S. Typhi proteins measured according to the invention. Extensive discussion of the known immunoassay techniques is not required here since these are known to those of skill in the art. Typical suitable immunoassay techniques include sandwich enzyme-linked immunoassays (ELISA), radioimmunoassays (RIA), competitive binding assays, homogeneous assays, heterogeneous assays, etc. Various of the known immunoassay methods are reviewed, e.g., in Methods in Enzymology, 70, pp. 30-70 and 166-198 (1980).

[00060] In the assays of the invention, "sandwich-type" assay formats can be used. These typically involve mixing the test sample with detection probes conjugated with a specific binding member (e.g., antibody) for the analyte (e.g., the urine sample) to form complexes between the analyte and the conjugated probes. These complexes are then allowed to contact a receptive material (e.g., antibodies) immobilized within the detection zone. Binding occurs between the analyte/probe conjugate complexes and the immobilized receptive material, thereby localizing "sandwich" complexes that are detectable to indicate the presence of the analyte. This technique may be used to obtain quantitative or semi-quantitative results. Some examples of such sandwich-type assays are described in by U.S. Pat. No. 4,168,146 to Grubb, et al. and U.S. Pat. No. 4,366,241 to Tom, et al. An alternative technique is the "competitive -type" assay. In a competitive assay, the labeled probe is generally conjugated with a molecule that is identical to, or an analog of, the analyte. Thus, the labeled probe competes with the analyte of interest for the available receptive material. Competitive assays are typically used for detection of analytes such as haptens, each hapten being monovalent and capable of binding only one antibody molecule. Examples of competitive immunoassay devices are described in U.S. Pat. No. 4,235,601 to Deutsch, et al., U.S. Pat. No. 4,442,204 to Liotta, and U.S. Pat. No. 5,208,535 to Buechler, et al.

[00061] The antibodies can be labeled. In some embodiments, the detection antibody is labeled by covalently linking to an enzyme, label with a fluorescent compound or metal, label with a chemiluminescent compound. For example, the detection antibody can be labeled with catalase and the conversion uses a colorimetric substrate composition comprises potassium iodide, hydrogen peroxide and sodium thiosulphate; the enzyme can be alcohol dehydrogenase and the conversion uses a colorimetric substrate composition comprises an alcohol, a pH indicator and a pH buffer, wherein the pH indicator is neutral red and the pH buffer is glycine-sodium hydroxide; the enzyme can also be hypoxanthine oxidase and the conversion uses a colorimetric substrate composition comprises xanthine, a tetrazolium salt and 4,5-dihydroxy-l,3-benzene disulphonic acid. In one embodiment, the detection antibody is labeled by covalently linking to an enzyme, label with a fluorescent compound or metal, or label with a chemiluminescent compound.

[00062] Direct and indirect labels can be used in immunoassays. A direct label can be defined as an entity, which in its natural state, is visible either to the naked eye or with the aid of an optical filter and/or applied stimulation, e.g., ultraviolet light, to promote fluorescence. Examples of colored labels which can be used include metallic sol particles, gold sol particles, dye sol particles, dyed latex particles or dyes encapsulated in liposomes. Other direct labels include radionuclides and fluorescent or luminescent moieties. Indirect labels such as enzymes can also be used according to the invention. Various enzymes are known for use as labels such as, for example, alkaline phosphatase, horseradish peroxidase, lysozyme, glucose-6-phosphate dehydrogenase, lactate dehydrogenase and urease. For a detailed discussion of enzymes in immunoassays see Engvall, Enzyme Immunoassay ELISA and EMIT, Methods of Enzymology, 70, 419-439 (1980).

[00063] In some embodiments, the immunoassay method or assay comprises a double antibody technique for measuring the level of the S. Typhi proteins in the patient's body fluid, such as urine. According to this method one of the antibodies is a "capture" antibody and the other is a "detector" antibody. The capture antibody is immobilized on a solid support which may be any of various types which are known in the art such as, for example, microtiter plate wells, beads, tubes and porous materials such as nylon, glass fibers and other polymeric materials. In this method, a solid support, e.g., microtiter plate wells, coated with a capture antibody, preferably monoclonal, raised against the particular S. Typhi protein of interest, constitutes the solid phase. Patient body fluid, e.g., urine, which may be diluted or not, typically at least 1, 2, 3, 4, 5, 10, or more standards and controls are added to separate solid supports and incubated. When the S. Typhi protein is present in the body fluid it is captured by the immobilized antibody which is specific for the S. Typhi protein in question. After incubation and washing, an anti-marker protein detector antibody, e.g., a polyclonal rabbit anti- marker protein antibody, is added to the solid support. The detector antibody binds to marker protein bound to the capture antibody to form a sandwich structure. After incubation and washing an anti-IgG antibody, e.g., a polyclonal goat anti-rabbit IgG antibody, labeled with an enzyme such as horseradish peroxidase (HRP) is added to the solid support. After incubation and washing a substrate for the enzyme is added to the solid support followed by incubation and the addition of an acid solution to stop the enzymatic reaction.

[00064] The degree of enzymatic activity of immobilized enzyme is determined by measuring the optical density of the oxidized enzymatic product on the solid support at the appropriate wavelength, e.g., 450 nm for HRP. The absorbance at the wavelength is proportional to the amount of S. Typhi protein in the fluid sample. A set of marker protein standards is used to prepare a standard curve of absorbance vs. S. Typhi protein concentration. This method is useful because test results can be provided in 45 to 50 minutes and the method is both sensitive over the concentration range of interest for each S. Typhi protein and is highly specific.

[00065] The antibody can be attached to a surface. Examples of useful surfaces on which the antibody can be attached for the purposes of detecting the desired antigen include nitrocellulose, PVDF, polystyrene, and nylon. The surface or support may also be a porous support (see., e.g., U.S. Patent No. 7,939,342).

[00066] The standards may be positive samples comprising various concentrations of the at least one S. Typhi protein to be detected to ensure that the reagents and conditions work properly for each assay. The standards also typically include a negative control, e.g., for detection of

contaminants. In some aspects of the embodiments of the invention, the positive S. Typhi controls may be titrated to different concentrations, including non-detectable amounts and clearly detectable amounts, and in some aspects, also including a sample that shows a signal at the threshold level of detection in the biological sample.

[00067] The assays can be carried out in various assay device formats including those described in U.S. Pat. Nos. 4,906,439; 5,051,237 and 5,147,609 to PB Diagnostic Systems, Inc.

[00068] The diagnosis of typhoid fever can be made if the presence of any one of the S. Typhi proteins is detected in the patient's sample, such as a urine sample.

[00069] In addition to presence of the S. Typhi protein in the sample, one can also measure the quantity of the S. Typhi protein in the sample using routine methods known to one skilled in the art.

[00070] The assay devices used according to the invention can be arranged to provide a quantitative or a qualitative (present/not present) result.

[00071] The assays may be carried out in various formats including, as discussed previously, a microtiter plate or a microfluidic device format are particularly useful for carrying out the assays in a batch mode. The assays may also be carried out in automated immunoassay analyzers which are well known in the art and which can carry out assays on a number of different samples. These automated analyzers include continuous/random access types. Examples of such systems are described in U.S. Pat. Nos. 5,207,987 and 5,518,688 to PB Diagnostic Systems, Inc. Various automated analyzers that are commercially available include the OPUS® and OPUS MAGNUM® analyzers.

[00072] Another assay format which can be used according to the invention is a rapid manual test which can be administered at the point-of-care at any location. Typically, such point-of-care assay devices will provide a result which is either "positive" i.e. showing the protein is present, or

"negative" showing that the protein is absent. Typically, a control showing that the reagents worked in general is included with such point-of-care system. Point-of-care systems, assays and devices have been well described for other purposes, such as pregnancy detection (see, e.g., U.S. Patent No.

7,569,397; 7,959,875).

[00073] Accordingly, the invention also provides devices, such as point-of-care test strips and microfluidic devices to perform the in vitro assays of the present invention.

[00074] It should be recognized also that the assay devices used according to the invention can be provided to carry out one single assay for a particular marker protein or to carry out a plurality of assays, from a single volume of body fluid, for a corresponding number of different S. Typhi proteins. In some embodiments, an assay device of the latter type is one which can provide a semiquantitative result for the S. Typhi proteins measured according to the invention, i.e., STY3639, STY3461, STY3007, STY0777, STY2194, STY2874, STY4489, STY3710, STY0184, STY4070, STY4090, STY4545, STY3467, STY2073. These devices typically are adapted to provide a distinct visually detectable colored band at the location where the capture antibody for the particular marker protein is located when the concentration of the marker protein is above the threshold level. For additional detailed discussion of assay types which can be utilized according to the invention as well as various assay formats and automated analyzer apparatus see e.g., U.S. Pat. No. 5,747,274.

[00075] In other embodiments, the assays or immunoassays of the invention comprise beads coated with an antibody against an isolated S. Typhi protein or a fragment thereof, as described e.g., in Binder SR., Lupus. 2006, 15:412-21. Commonly used are polystyrene beads that can be labeled to establish a unique identity. Detection is performed by flow cytometry.

[00076] The S. Typhi antigen detection can further be performed using multiplex technologies. Other types of bead-based immunoassays are well known in the art, e.g., laser bead immunoassays and related magnetic bead assays (Fritzler, Marvin J; Fritzler, Mark L, Expert Opinion on Medical Diagnostics, 2009, pp. 3: 81-89).

[00077] In one embodiment, the invention further provides a system to facilitate the diagnosis of typhoid fever in a human subject, comprising: a determination module configured to receive and output the amount of S. Typhi antigens or proteins detected in a biological sample extracted from a human; a storage module configured to store output information from the determination module; a comparison module adapted to compare the data stored on the storage module with reference data and/or control data, and to provide a comparison content, and an output module for displaying the comparison content for the user, wherein if there is no detectable amount of S. Typhi antigens in the sample, then the subject does not have typhoid fever and if the peptide is dectected that the subject has typhoid fever.

[00078] The collected urine sample is in some embodiments undiluted. In some embodiments the sample can be diluted or concentrated depending on the detection application.

[00079] In some embodiments, one can essentially concentrate the proteins in the urine sample by using a solid surface coated with a monoclonal antibody to capture the antigen. The recovered captured antigens can then be analyzed using any suitable methods well known to one skilled in the art. The solid surface can be e.g., beads, such as magnetic beads, polystyrene beads, or gold beads, or in an array or a microarray format using a glass, a plastic or a silicon chip. Such antigen capture can be also a part of a channel in a microfluidic device.

[00080] In one embodiment, the invention provides a computer readable storage medium comprising: a storing data module containing data from a sample obtained from a subject that represents a signal level from an immunoassay for the S. Typhi antigens present in the urine sample taken from the human patient; a comparison module that compares the data stored on the storing data module with a reference data and/or control data, and to provide a comparison content, and an output module displaying the comparison content for the user, wherein the presence of a detectable amount of S. Typhi antigens relative to the reference data and/or control data indicates that the subject has typhoid fever.

[00081] Kits provided according to the present invention include kits comprising the compositions, i.e., the antibodies, and optionally comprising a device with a solid surface, such as a lateral flow assay device, to which the antibodies are bound. Kits, such as those described in U.S. Patent No. 7,932,099, comprising the component of at least one of the 14 S. Typhi specific antigens described, e.g., in Tables 2 and 3 are envisioned.

[00082] Table 2 sets fort the 14 S. Typhi proteins detected from the urine samples of humans with typhoid fever.

Table 2: 14 Salmonella Urine Proteins

Locus Protein names Gene #AA MW %Cov names (Kda)

STY3639 Thioredoxin-1 (Trx-1) trxA 109 11.8 39.45

STY3461 ATP-dependent RNA helicase (ATP- 646 72.6 4.64 dependent RNA helicase (Dead-box

protein; 'Dead' disclosed as SEQ ID

NO: 26)

STY3007 Cell invasion protein sipC sipC 409 43.1 5.87

STY0777 Succinate dehydrogenase flavoprotein sdhA 588 64.4 4.93 Table 2: 14 Salmonella Urine Proteins

Locus Protein names Gene #AA MW %Cov names (Kda)

subunit

STY2194 Cellulose synthesis regulatory protein yedQ 567 65.1 4.76

STY2874 SsrA-binding protein smpB 160 18.2 15.63

STY4489 ProP effector proQ 228 25.4 14.91

STY3710 Phosphoribosylamine— glycine ligase purD 429 45.6 10.49

STY0184 Putative uncharacterized protein ygbK ygbK 423 45.0 9.93

STY4070 3-deoxy-D-manno-octulosonic-acid kdtA 425' 47.2 6.12 transferase

STY4090 Putative uncharacterized protein 427 47.3 6.09

STY4545 Nucleotide -binding protein (PilQ) pilQ 571 62.9 5.95

STY3467 Translation initiation factor IF-2 inffl 892 97.4 3.25

STY2073 Exodeoxyribonuclease VIII recE 1042 116.7 2.4

[00083] Table 3 sets forth the sequences for the S. Typhi proteins useful according to the present invention.

Table 3

STY-Numbers Accession Numbers SEQ ID NOs

STY3639 NP_457831.1 SEQ IS NO: 1

STY3461 NP_. 457662,2 SEQ IS NO: 2

STY3007 NP_457278.1 SEQ IS NO: 3

STY0777 NP_455290.1 SEQ IS NO: 4

STY2194 NP .456547.1 SEQ IS NO: 5

STY2874 NP _.457157.1 SEQ IS NO: 6

STY4489 NP_458589.1 SEQ IS NO: 7

STY3710 NP_457899.1 SEQ IS NO: 8

STY0184 NP_...454775, 1 SEQ IS NO: 9

STY4070 NP_458202.1 SEQ IS NO: 10

STY4090 NP_458222.1 SEQ IS NO: 11

STY4545 NP_...458633.1 SEQ IS NO: 12

STY3467 NP...457668.1 SEQ IS NO: 13

STY2073 NP_„456434.1 SEQ IS NO: 14 Test Strips for Lateral Flow Assays

[00084] The invention provides simple to use point-of-care diagnostic test strips akin to pregnancy detection strips, wherein the strip comprises at least one antibody against at least one of the listed S. Typhi proteins. The test strip may comprise a positive and negative control to show the user that the reagents work properly and/or that the sample has been added to the strip properly. The strips may be provided with or without a casing and with or without additional reagents.

[00085] In its most simplest form, the strip comprises at least one antibody against at least one of the S. Typhi proteins or antigens provided herewith, wherein the at least one antibody is immobilized on the solid or porous surface of the test strip surface. "Antibody" is an

immunoglobulin, or derivative or fragment or active fragment thereof, having an area on the surface or in a cavity which specifically binds to and is thereby defined as complementary with a particular spatial and polar organization of another molecule. The antibody can be monoclonal or polyclonal and can be prepared by techniques that are well known in the art such as, for example, immunization of a host and collection of sera or hybrid cell line technology.

[00086] The test strip may include a simple indication of positive result upon detection of one or more of the proteins set forth in the specification.

[00087] Diagnostic test strips for lateral flow assays, such as the test strip assay described herein, may be constructed as described in the art, see, e.g., U.S. Patent application publication No. 20100196200; 20100129935; 20090253119; 20090111171.

[00088] A "test strip" can include one or more bibulous or non-bibulous materials. If a test strip comprises more than one material, the one or more materials are preferably in fluid

communication. One material of a test strip may be overlaid on another material of the test strip, such as for example, filter paper overlaid on nitrocellulose. Alternatively or in addition, a test strip may include a region comprising one or more materials followed by a region comprising one or more different materials. In this case, the regions are in fluid communication and may or may not partially overlap one another. Suitable materials for test strips include, but are not limited to, materials derived from cellulose, such as filter paper, chromatographic paper, nitrocellulose, and cellulose acetate, as well as materials made of glass fibers, nylon, dacron, PVC, polyacrylamide, cross-linked dextran, agarose, polyacrylate, ceramic materials, and the like. The material or materials of the test strip may optionally be treated to modify their capillary flow characteristics or the characteristics of the applied sample. For example, the sample application region of the test strip may be treated with buffers to correct the pH or specific gravity of an applied urine sample, to ensure optimal test conditions.

[00089] The material or materials can be a single structure such as a sheet cut into strips or it can be several strips or particulate material bound to a support or solid surface such as found, for example, in thin-layer chromatography and may have an absorbent pad either as an integral part or in liquid contact. The material can also be a sheet having lanes thereon, capable of spotting to induce lane formation, wherein a separate assay can be conducted in each lane. The material can have a rectangular, circular, oval, triagonal or other shape provided that there is at least one direction of traversal of a test solution by capillary migration. Other directions of traversal may occur such as in an oval or circular piece contacted in the center with the test solution. However, the main consideration is that there be at least one direction of flow to a predetermined site. In the following discussion strips will be described by way of illustration and not limitation.

[00090] The support for the test strip, where a support is desired or necessary, will normally be water insoluble, frequently non-porous and rigid but may be elastic, usually hydrophobic, and porous and usually will be of the same length and width as the strip but may be larger or smaller. The support material can be transparent, and, when a test device of the present invention is assembled, a transparent support material can be on the side of the test strip that can be viewed by the user, such that the transparent support material forms a protective layer over the test strip where it may be exposed to the external environment, such as by an aperture in the front of a test device. A wide variety of non-mobilizable and non-mobilizable materials, both natural and synthetic, and combinations thereof, may be employed provided only that the support does not interfere with the capillary action of the material or materials, or non-specifically bind assay components, or interfere with the signal producing system. Illustrative polymers include polyethylene, polypropylene, poly(4- methylbutene), polystyrene, polymethacrylate, poly(ethylene terephthalate), nylon, poly( vinyl butyrate), glass, ceramics, metals, and the like. Elastic supports may be made of polyurethane, neoprene, latex, silicone rubber and the like.

[00091] A "control zone" is a region of a test strip in which a dye as described supra can be observed to shift location, appear, change color, or optionally to disappear. Detection or observation of the control zone may be done by any convenient means, depending upon the particular choice of dye, especially, for example but not limited to, visually, fluorescently, by reflectance,

radiographically, and the like. As will be described, the dye may or may not be applied directly to the control zone, depending upon the design of the control being used.

[00092] A "label" may be any molecule bound to a specific binding member that can produce a detectable signal. In the present invention, the label may be inert and provide a signal by concentrating in the detection zone, or it may serve solely as a binding site for a member of the signal producing system, or it may spontaneously produce a detectable signal or may produce a detectable signal in conjunction with a signal producing system. The label may be isotopic or nonisotopic.

[00093] "Proximal end" of a test strip refers to the end of a test device or test strip that includes the sample application aperture of the test device and sample application zone of the test strip.

[00094] "Reagent zone" refers to a region of a test strip where reagent is provided. The reagent zone can be on a reagent pad, a separate segment of bibulous or non-bibulous material included on the test strip, or it can be a region of a bibulous or non-bibulous material of a test strip that also includes other zones, such as an analyte detection zone. The reagent zone can carry a detectable label, which may be a direct or indirect label. Preferably the reagent is provided in a form that is immobile in the dry state and mobile in the moist state. A reagent can be a specific binding member, an analyte or analyte analog, an enzyme, a substrate, indicators, components of a signal producing system, chemicals or compounds such as buffering agents, reducing agents, chelators, surfactants, etc., that contribute to the function of the test strip assay.

[00095] "Sample" is any material to be tested for the presence or amount of an analyte. The sample can be a fluid sample, preferably a liquid sample, such as a urine sample. Examples of liquid samples that may be tested using a test device of the present invention include bodily fluids including blood, serum, plasma, saliva, urine, ocular fluid, semen, and spinal fluid. Viscous liquid, semi-solid, or solid specimens may be used to create liquid solutions, eluates, suspensions, or extracts that can be samples.

[00096] The test strip may be accessed through a sample application aperture. "Sample application aperture" refers to the portion of a test device where an opening in the test device provides access to the sample application zone of the test strip. In one embodiment of the present invention, a sample application aperture is created by an open-ended channel at the proximal end of the test device. Preferably, a test strip in engaged in the open-ended channel such that sample contacted with the sample application aperture is thereby applied to the test strip. In an alternate embodiment, a sample application aperture is formed by an opening in the front of a test device, such that the sample application zone of the test strip is in fluid communication with the exterior of the test device.

[00097] "Sample application zone" is the portion of a test strip where sample can be applied. The sample application zone of a test strip of the present invention preferably occurs at the sample application aperture of a test device of the present invention, and is in fluid communication with the sample application aperture.

[00098] "Specific binding member" is one of two different molecules having an area on the surface or in a cavity which specifically binds to and is thereby defined as complementary with a particular spatial and polar organization of the other molecule. The members of the specific binding pair are referred to as ligand and receptor (antiligand). These will usually be members of an immunological pair such as antigen-antibody. In the case of binding pairs such as avidin-biotin, reagent can be labeled with one member of this pair and a detection zone can include the other member of this pair in a capture type assay. Other general types of assays using avidin-biotin pairs or binding pairs of this type are known in the art. In addition, an antigen or epitope can be labeled for use as a reagent for the detection of antibodies that bind with or specifically bind with such an antigen or epitope. In one aspect of this type of assay, an antigen or epitope can be detectably labeled and present in the reagent zone and the antigen or epitope can be present in a detection zone. This type of assay can be used, for example, to detect antibodies such as antibodies that bind with or specifically bind with an etiological agent in a sample. Other general types of assays using labeled antigen or labeled epitopes are known in the art.

[00099] The test strip may also include a test result zone. "Test results zone" is a region of a test strip that provides a detectable signal indicating the presence of the analyte. The test results zone can include an immobilized binding reagent specific for an analyte ("specific binding member"), and/or an enzyme that reacts with the analyte. A test results determination zone can include one or more analyte detection zones. Other substances that may allow or enhance detection of the analyte, such as substrates, buffers, salts, may also be provided in the test results determination zone. One or more members of a signal producing system may be bound directly or indirectly to the detection zone. A test results determination zone can optionally include one or more control zones that provide indication that the test has been performed properly.

Microfluidic devices

[000100] The invention further provides microfluidic devices for the diagnosis of typhoid fever, specifically from a urine sample. The components of the assays, namely, the antibody and the reagents needed for detection of the S. Typhi proteins in urine described herein can be used in the format of a microfluidic device. Such devices have been well described in the art, see, e.g., U.S. Patent Nos. 6,444,461 ; 6,479,299; 7,041,509, incorporated herein by reference in their entirety.

[000101] The microfluidic devices can be designed to comprise a channel or chamber that contains one or more antibodies against the S. Typhi proteins preferably immobilized on the channel surface. The device can be supplied with appropriate buffers for binding the proteins from a sample, such as a urine sample to the antibodies and detecting the bound proteins either inside the device or eluting them out and detecting them in the eluted sample.

Automation

[000102] The methods of the invention can be automated using robotics and computer directed systems. The biological sample, such as the urine sample, can be injected into a system, such as a microfluidic devise entirely run by a robotic station from sample input to output of the result.

[000103] The step of displaying the result can also be automated and connected to the same system or in a remote system. Thus, the sample analysis can be performed in one location and the comparison and the result analysis in another location, the only connection being, e.g., an internet connection in such way that the analysis result can be fed from the analysis module to the comparison module which can then either in the same location or by sending the result to a third location, which may or may not be the same location as the first location wherein the analysis was performed, to be displayed in a format suitable for either reading by a health professional or by a patient.

[000104] In one embodiment, the analysis, comparison and the result is performed in one location. Salmonellosis and typhoid fever

[000105] In some embodiments, the method further comprises selecting an individual having symptoms of fever suspected of being associated with salmonellosis or typhoid fever.

[000106] Typhoid fever is characterized by a slowly progressive fever as high as 40°C (104 °F), profuse sweating and gastroenteritis. Less commonly, a rash of flat, rose-colored spots may appear. Classically, the course of untreated typhoid fever is divided into four individual stages, each lasting approximately one week. In the first week, there is a slowly rising temperature with relative bradycardia, malaise, headache, and cough. A bloody nose (epistaxis) is seen in a quarter of cases and abdominal pain is also possible. There is leukopenia, a decrease in the number of circulating white blood cells, with eosinopenia and relative lymphocytosis, a positive reaction and blood cultures are positive for Salmonella typhi or paratyphi. The classic Widal test is negative in the first week.

[000107] In the second week of the infection, the patient lies prostrate with high fever in plateau around 40 °C (104 °F) and bradycardia (sphygmothermic dissociation), classically with a dicrotic pulse wave. Delirium is frequent, frequently calm, but sometimes agitated. This delirium gives to typhoid the nickname of "nervous fever". Rose spots appear on the lower chest and abdomen in around a third of patients. There are rhonchi in lung bases. The abdomen is distended and painful in the right lower quadrant where borborygmi can be heard. Diarrhea can occur in this stage: six to eight stools in a day, green with a characteristic smell, comparable to pea soup. However, constipation is also frequent. The spleen and liver are enlarged (hepatosplenomegaly) and tender, and there is elevation of liver transaminases. The Widal reaction is strongly positive with antiO and antiH antibodies. Blood cultures are sometimes still positive at this stage. (The major symptom of this fever is that the fever usually rises in the afternoon up to the first and second week.)

[000108] In the third week of typhoid fever, a number of complications can occur including intestinal hemorrhage due to bleeding in congested Peyer's patches (this can be very serious but is usually not fatal); intestinal perforation in the distal ileum: this is a very serious complication and is frequently fatal - it may occur without alarming symptoms until septicaemia or diffuse peritonitis sets in; encephalitis; neuropsychiatric symptoms (described as "muttering delirium" or "coma vigil"), with picking at bedclothes or imaginary objects; metastatic abscesses, cholecystitis, endocarditis and osteitis. The fever is still very high and oscillates very little over 24 hours. Dehydration ensues and the patient is delirious (typhoid state). By the end of third week the fever has started reducing this (defervescence). This carries on into the fourth and final week. A person may become an

asymptomatic carrier of typhoid fever, suffering no symptoms, but capable of infecting others.

According to the CDC approximately 5% of people who contract typhoid continue to carry the disease after they recover.

[000109] The assays of this invention can diagnose the typhoid fever both at the acute and later stages as well as in the latent stage. [000110] Salmonellosis or Salmonella enterocolitis is one of the most common types of food poisoning. It occurs when you swallow food or water that is contaminated with the salmonella bacteria. Any food can become contaminated if food preparation conditions and equipment are unsanitary.

[000111] The time between infection and symptom development is 8 - 48 hours. Symptoms include: Abdominal pain or cramping or tenderness; Chills; Diarrhea; Fever; Muscle pain; Nausea; and Vomiting. All these symptoms can also occur in some stages of typhoid fever.

[000112] Treatment of salmonellosis and typhoid fever is typically different. The goal of salmonellosis treatment is often to replace fluids and electrolytes lost by diarrhea. Antidiarrheal medications are generally not given because they may prolong the infection. Severe symptoms can be treated with antibiotics. Thus, in one embodiment, if the subject is diagnosed as having salmonellosis based on the selected presence and/or absence or proteins as described, supra, the subject is treated with fluid and electrolyte replacement and not given antidiarrheal medications.

[000113] Typhoid fever requires use of antibiotics, such as cetriaxone, chloramphenicol, or ciprofloxacin or other fluoroquinolones. In one embodiment, the invention provides a method of diagnosing a patient using the urine assays described throughout this application. In some embodiments, the method further comprises the step of administering to the patient antibiotics, such as cetriaxone, chloramphenicol, or ciprofloxacin or other fluoroquinolones if the urine sample tests positive for at least one marker selected from the group consisting of STY3639, STY3461, STY3007, STY0777, STY2194, STY2874, STY4489, STY3710, STY0184, STY4070, STY4090, STY4545, STY3467, STY2073. In one embodiment, the at least one marker is STY4545.

[000114] In one embodiment, the antibiotics are prescribed or administered if at least two of the markers are identified positively in the urine sample. In one embodiment, the two markers that are needed for a positive detection are STY3007 and STY4545.

[000115] All the references cited throughout the specification and examples are herein incorporated by reference in their entirety.

Claims

CLAIMS We claim:

1. An in vitro assay comprising the steps of:

a. contacting in vitro a urine sample from a human patient with an isolated and purified antibody against at least one S. Typhi protein selected from the group consisting of STY3639 (SEQ ID NO: 1), STY3461 (SEQ ID NO: 2), STY3007 (SEQ ID NO: 3), STY0777 (SEQ ID NO: 4), STY2194 (SEQ ID NO: 5), STY2874 (SEQ ID NO: 6), STY4489 (SEQ ID NO: 7), STY3710 (SEQ ID NO: 8), STY0184 (SEQ ID NO: 9), STY4070 (SEQ ID NO: 10), STY4090 (SEQ ID NO: 11), STY4545 (SEQ ID NO: 12), STY3467 (SEQ ID NO: 13), STY2073 (SEQ ID NO: 14) and any combination and fragment thereof, thereby forming an immune complex between the at least one S. Typhi protein and the antibody;

b. detecting the bound antibodies to determine whether the biological sample contains the at least one S. Typhi antigen.

2. The in vitro assay of claim 1, wherein the S. Typhi antigen is STY3007 (SEQ ID NO: 3), STY4545 (SEQ ID NO: 12) or a combination thereof.

3. An in vitro assay comprising the steps of:

a. contacting in vitro a biological sample from a human patient with an isolated and purified first antibody against STY3007 (SEQ ID NO: 3) or a fragment thereof, and an isolated and purified second antibody against STY4545 (SEQ ID NO: 12), or a fragment thereof, thereby forming an immune complex between STY3007 (SEQ ID NO: 3) and the first antibody and STY4545 (SEQ ID NO: 12) and the second antibody;

b. detecting the bound first and second antibodies to determine whether the biological sample contains the at one of STY3007 (SEQ ID NO: 3) or STY4545 (SEQ ID NO: 12).

4. The in vitro assay of claim 3, further comprising diagnosing salmonellosis if STY3007 (SEQ ID NO: 3) is detected and STY4545 (SEQ ID NO: 12) is not detected in the biological sample.

5. The in vitro assay of claims 3, further comprising diagnosing typhoid fever if both

STY3007 (SEQ ID NO: 3) and STY4545 (SEQ ID NO: 12) are detected in the biological sample.

6. The in vitro assay of any one of the preceding claims further comprising a step of

diagnosing the human patient with typhoid fever if the at least one S. Typhi antigen is detected.

7. The in vitro assay of any one of the claims 4-6, wherein the diagnosing is performed using a non-human machine.

8. The in vitro assay of claim 7, wherein the non-human machine is a computer

implemented software.

9. The in vitro assay of any one of the preceding claims, wherein the antibody is labeled.

10. The in vitro assay of any one of the preceding claims, wherein the antibody is detected using a secondary antibody.

11. The in vitro assay of any one of the preceding claims, wherein the antibody is bound to a solid surface.

12. The in vitro assay of any one of claims 3-11, wherein the biological sample is a urine sample.

13. A test strip comprising an antibody against at least one S. Typhi antigen selected from the group consisting of STY3639 (SEQ ID NO: 1), STY3461 (SEQ ID NO: 2), STY3007 (SEQ ID NO: 3), STY0777 (SEQ ID NO: 4), STY2194 (SEQ ID NO: 5), STY2874 (SEQ ID NO: 6), STY4489 (SEQ ID NO: 7), STY3710 (SEQ ID NO: 8), STY0184 (SEQ ID NO: 9), STY4070 (SEQ ID NO: 10), STY4090 (SEQ ID NO: 11), STY4545 (SEQ ID NO: 12), STY3467 (SEQ ID NO: 13), STY2073 (SEQ ID NO: 14) and any combination thereof bound to a surface.

14. A test strip comprising a first antibody against STY3007 (SEQ ID NO: 3) and a second antibody against STY4545 (SEQ ID NO: 12).

15. The test strip of any one of the claims 13-14 further comprising at least one control for a negative test result and at least one control for a positive test result.

16. The test strip of any one of the claim 13, wherein the surface is a sold surface.

17. The test strip of any one of the claims 13, wherein the surface is a porous surface.

18. A microfluidic device comprising an antibody against at least one isolated S. Typhi

antigen selected from the group consisting of STY3639 (SEQ ID NO: 1), STY3461 (SEQ ID NO: 2), STY3007 (SEQ ID NO: 3), STY0777 (SEQ ID NO: 4), STY2194 (SEQ ID NO: 5), STY2874 (SEQ ID NO: 6), STY4489 (SEQ ID NO: 7), STY3710 (SEQ ID NO: 8), STY0184 (SEQ ID NO: 9), STY4070 (SEQ ID NO: 10), STY4090 (SEQ ID NO: 11), STY4545 (SEQ ID NO: 12), STY3467 (SEQ ID NO: 13), STY2073 (SEQ ID NO: 14) and any combination thereof.

19. The microfluidic device of claim 18 comprising a first antibody against STY3007 (SEQ ID NO: 3) and a second antibody against STY4545 (SEQ ID NO: 12).

20. A composition comprising at least one antibody against an isolated S. Typhi antigen

selected from the group consisting of STY3639 (SEQ ID NO: 1), STY3461 (SEQ ID NO: 2), STY3007 (SEQ ID NO: 3), STY0777 (SEQ ID NO: 4), STY2194 (SEQ ID NO: 5), STY2874 (SEQ ID NO: 6), STY4489 (SEQ ID NO: 7), STY3710 (SEQ ID NO: 8), STY0184 (SEQ ID NO: 9), STY4070 (SEQ ID NO: 10), STY4090 (SEQ ID NO: 11), STY4545 (SEQ ID NO: 12), STY3467 (SEQ ID NO: 13), STY2073 (SEQ ID NO: 14) and any combination and fragment thereof.

21. The composition of claim 20 comprising a first antibody against STY3007 (SEQ ID NO:

3) and a second antibody against STY4545 (SEQ ID NO: 12).

22. A kit comprising an antibody against an isolated S. Typhi antigen selected from the group consisting of STY3639 (SEQ ID NO: 1), STY3461 (SEQ ID NO: 2), STY3007 (SEQ ID NO: 3), STY0777 (SEQ ID NO: 4), STY2194 (SEQ ID NO: 5), STY2874 (SEQ ID NO: 6), STY4489 (SEQ ID NO: 7), STY3710 (SEQ ID NO: 8), STY0184 (SEQ ID NO: 9), STY4070 (SEQ ID NO: 10), STY4090 (SEQ ID NO: 11), STY4545 (SEQ ID NO: 12), STY3467 (SEQ ID NO: 13), STY2073 (SEQ ID NO: 14) and any combination and fragment thereof.

23. The kit of claim 22 comprising a first antibody against STY3007 (SEQ ID NO: 3) and a second antibody against STY4545 (SEQ ID NO: 12).

24. A method for diagnosing typhoid fever in a human patient the method comprising the steps of (a) contacting in vitro a urine sample from the human patient with an isolated and purified antibody against at least one S. Typhi protein selected from the group consisting of STY3639 (SEQ ID NO: 1), STY3461 (SEQ ID NO: 2), STY3007 (SEQ ID NO: 3), STY0777 (SEQ ID NO: 4), STY2194 (SEQ ID NO: 5), STY2874 (SEQ ID NO: 6), STY4489 (SEQ ID NO: 7), STY3710 (SEQ ID NO: 8), STY0184 (SEQ ID NO: 9), STY4070 (SEQ ID NO: 10), STY4090 (SEQ ID NO: 11), STY4545 (SEQ ID NO: 12), STY3467 (SEQ ID NO: 13), STY2073 (SEQ ID NO: 14) and any combination and fragment thereof, thereby forming an immune complex between the at least one S. Typhi protein and the antibody; and detecting the bound antibodies to determine whether the urine sample contains the at least one S. Typhi antigen; wherein if the urine sample contains at least one S. Typhi protein, the human patient is diagnosed as having typhoid fever.

25. The method of claim 24, wherein at least two proteins are detected and the diagnosis of typhoid fever is made if the at least two proteins are detected in the urine sample.

26. The method of any one of claims 24-25, wherein if at least two markers is detected and one of them is STY3007 (SEQ ID NO: 3) protein, the patient has salmonellosis instead of typhoid fever if STY3007 (SEQ ID NO: 3) is present in the urine sample but no other S. Typhi protein is detected.