WO2014071455A1 - Diagnostic, prognostic, therapeutic and screening protocols with respect to infectious mycobacterium - Google Patents

Abstract

A diagnostic process for detecting the TB status of a test subject, the process comprising contacting a biological sample comprising antibodies from the subject with a dIgA binding agent, or two or three specific antibody binding agents selected from IgA, IgG and dIgA-binding agents, to allow formation of test complexes between TB antigen specific antibody and antibody binding agent, and measuring the level of each test complex and comparing the level of the test complexes formed to corresponding levels formed in control samples, wherein the levels of at least two test complexes are substantially independent and contribute to enhancing the diagnostic power of the process.

Description

DIAGNOSTIC, PROGNOSTIC, THERAPEUTIC AND SCREENING

PROTOCOLS WITH RESPECT TO INFECTIOUS MYCOBACTERIUM

PRIORITY CLAIM

[0001] The present application claims priority from Australia Provisional Patent Application No. 2012904886 filed on 8 November 2012, the disclosure of which is included herein by reference.

FIELD

[0002] The present specification relates generally to diagnostic, prognostic, screening and therapeutic protocols with respect to pathogenic Mycobacterium species such as Mycobacterium tuberculosis or tuberculosis (TB).

BACKGROUND

[0003J Bibliographic details references referred to in this specification are listed at the end of the specification.

[0004] The reference to any prior art is not and should not be taken as an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in any country.

[0005] Tuberculosis (TB) is a major health problem and is one of the most devastating diseases in terms of mortality and morbidity. This potentially lethal and highly infectious disease is caused by infection with bacteria of the genus Mycobacterium, primarily M. tuberculosis but also: M. bovis, M. africanum, M. canetti and M.microti. It is estimated that approximately one third of the world's population is latently affected with the TB bacterium, a significant proportion of whom may develop active and infectious disease. The active disease generally affects the lungs (pulmonary TB) or other organs (extrapulmonary) of a subject and active infection may be lethal if not treated early enough or in the face of drug resistant strains of the bacteria. TB and diagnosis of TB is a problem particularly in developing countries where TB is endemic. One diagnostic test involves examining sputum samples for acid-fast bacilli by microscopy. Multiple specimens and visits of the patient are required and this significantly increases the drop-out rate of subjects who might be infected and thus leading to untreated TB. More recently PCR based or serologic tests have been developed. Serologic tests for infection are preferred because of their potentially straightforward translation into both laboratory-based and point of care diagnosis. However, the available serologic tests for TB are associated with mixed results and generally low sensitivities. Illustrative serologic tests screen for one or more of IgA (which is predominantly monomeric IgA, the predominant IgA structural form in blood), IgG and IgM (Ghadiri et. al. Iranian Journal of Clinical Infectious Disease 3(4): 205-208, 2008). Heterogeneous antibody responses provide a further problem, as described by Lyashchenko et. al. Infection and Immunity 66(8): 3936, 1998. Drug treatment regimes may be complex which also leads to treatment failure due to non-compliance and facilitates the emergence of single drug-resistant, multidrug resistant and extensively drug- resistant strains of M. tuberculosis. However if diagnosed early, TB can be treated, leading to fewer deaths and reduced onward transmission to new subjects.

(0006] The detection of specific antibody (immunoglobulin (Ig)) classes is recognized as an important step in diagnostic and research methods for human and animal diseases. For example, detection of antigen-specific IgM-class antibodies is widely used as a diagnostic test for infection with viruses such as hepatitis A virus, hepatitis E virus, West Nile virus, dengue viruses, measles virus, rubella virus; and for infection with bacteria such as syphilis (Treponema pallidum) because IgM class antibodies are typically made in the body of an infected host during the acute phase of infection and are detectable for only a few months.

[0007] Conversely, IgG-class antibodies commonly persist for life and may indicate either current or past infection with a specific agent. For chronic infections such as the human immunodeficiency virus (HIV) where patients do not spontaneously clear the virus, detection of IgG-class antibodies is diagnostic for infection, whereas for others such as hepatitis C virus (HCV) where a proportion of patients do clear the virus either spontaneously or following treatment, the detection of antigen-specific IgG is not diagnostic of current or ongoing infection. IgG-class antibodies are primarily responsible for antibody-mediated immunity within the plasma compartment of the body. [0008] IgA-class antibodies have also been used to aid diagnosis of infections including hepatitis E virus, hepatitis A virus, and dengue viruses, as well as in the study of vaccines and immunity to infections. IgA is attractive for diagnostic purposes, because it is predominantly made during the acute phase of infection, and high levels of antigen- specific IgA can provide a marker of current infection, with or without the concurrent detection of IgM. In addition, because IgA is the predominant antibody class that is secreted at mucosal epithelial surfaces, its presence there is considered as a marker of mucosal immunity.

[0009] The role of different IgA structural forms as biomarkers for TB infection, such as specifically dlgA, is not understood. The role of SIgA in TB infection and TB antigens that engender SIgA responses have not been explored nor have diagnostic and prognostic protocols been developed that are designed to rapidly and conveniently assess these responses in patient sera.

[0010] In most animals, IgA is synthesized almost exclusively as dimeric or higher polymeric forms, herein described collectively as dlgA, which are able to interact with the polymeric Ig receptor (plg ). This interaction in vivo results in secretion of large amounts of secretory IgA (SIgA) into the lumen of epithelial tissues (see Figures 1 and 2). In contrast, in humans and higher primates the dlgA is only a minor fraction of the total IgA, with monomeric IgA (mlgA) representing around 90% of the total IgA, and dimeric or higher polymeric forms of IgA representing around 10% of the total IgA.

[0011] Detection of IgA, IgM, IgG and other antibody classes or isotypes is usually performed using antibody reagents prepared in another species, for example rabbit antibodies specific for human IgM, or mouse monoclonal antibodies specific for human IgA, or monoclonal antibodies specific for individual antibody subclasses such as IgA l , IgA2 or IgGl, IgG2a, IgG2b, IgG3, IgG4. Antibody based capture assay are associated with levels of non-specific binding which are generally minimised through optimisation protocols.

[0012] There is a need for improved protocols for rapid and convenient diagnosis or monitoring of TB infections in a subject, and for agents that can be used in diagnostic laboratory or point of care assays or kits to assess antigen-specific dlgA antibody production in subjects who may have active or latent TB, or who have had some exposure to TB infection or who may have had some exposure to TB infection. Such protocols will facilitate early or appropriate treatment of subjects during or following infection or exposure to TB.

SUMMARY OF CERTAIN EMBODIMENTS

[0013] The specification broadly enables processes and kits, for diagnosing TB in a subject, which assess the level of individual antibody isotypes, including dimeric/polymeric IgA (dlgA) or multiple antibody isotypes/subclasses (selected from IgA, IgG and dlgA), against one or more TB antigen preparations. The subject process may be used, inter alia, in a personalized or a population medicine approach and in the management of pathology platforms.

[0014] In one embodiment, a diagnostic process for detecting the TB status of a test subject, the process comprising obtaining a biological sample comprising antibodies from the subject and contacting same with a dlgA binding agent, to allow formation of a test complex between TB antigen specific antibody and antibody binding agent, and measuring the level of the test complex and comparing the level of the test complex formed to a corresponding level formed in a control sample.

[0015] In one embodiment, a diagnostic process for detecting the TB status of a test subject, the process comprising contacting a biological sample comprising antibodies from the subject with two or three specific antibody binding agents selected from IgA, IgG and dlgA-binding agents, to allow formation of test complexes between TB antigen specific antibody and antibody binding agent, and measuring the level of each test complex and comparing the level of the test complexes formed to corresponding levels formed in control samples, wherein the levels of at least two test complexes are substantially independent and contribute to enhancing the diagnostic power of the process.

[0016] In one embodiment, the at least two antibody-type binding agents are IgA and IgG-binding agents, or IgA and dlgA binding agents, or IgG and dlgA binding agents. [0017] In another embodiment, the at least two antibody-type binding agents are IgG and dlgA binding agents.

[0018] In one embodiment, the antibody-type binding agents are IgA, IgG and dlgA- binding agents.

[0019] ' As illustrated herein, in some embodiments, the TB status is active TB or non-TB.

[0020] Furthermore, in one embodiment, sensitivity of the diagnostic process is at least 70%, or at least 80%, or at least 88% sensitivity.

[0021] The biological sample may be any biological sample comprising antibodies. However, in one embodiment the biological sample is whole blood or a blood product such as heparinised (or other anticoagulant) blood or a serum sample.

[0022] In some embodiments, one or more TB antigens recognised by dlgA or two or three of IgA, IgG and dlgA are directly or indirectly bound to a solid surface and captures TB antigen specific antibody from the biological sample applied to the surface to form immobilised TB antigen-TB antigen specific antibody complexes. An illustrative assay is represented diagrammatically in Figure 9.

[0023] In other embodiment, the specific dlgA-binding agent is directly or indirectly bound to a solid surface, and the TB antigen specific dlgA-dlgA binding agent complex is probed with TB antigen. Many different antibody formats are well known to those of skill in the art. TB antigen may be labelled for detection using art recognised methods, including but not limited to direct labeling with a reagent such as biotin, or indirect labeling with monoclonal or polyclonal antibodies and subsequent detection of these antibodies. In this embodiment, the antibody binding agent is a TB antigen.

[0024] In some embodiments, the IgA and/or IgG-binding agent is directly or indirectly bound to a solid surface, and the TB antigen specific antibody-and IgA and/or IgG binding agent complex is probed with TB antigen. TB antigen may be labelled for detection using art recognised methods. In this embodiment, the antibody binding agent is a TB antigen.

[0025] Any TB antigen known in the art or determined using the present disclosure may be developed and employed in the subject process. In some illustrative embodiments, the TB antigen is selected from the group comprising or consisting of SCWP, LAM, CWF, CFP, CYT, PstSl, Anda A60, Omega, TDM, mAGP, PIM6, LM, 38KD-LPS and SL1.

[0026] In one, non-limiting example, the TB antigen is Anda A60, SCWP and LPS.

[0027] In one important embodiment, the dlgA-binding agent is plgR or a dlgA- binding variant thereof. Antibodies or other dlgA-binding agents (e.g., anti-J chain antibodies) may also be employed.

[0028] In one embodiment, the following antigen-isotype combinations are included in the process or kit to maximise diagnostic power (sensitivity) Anda A60-sIgA, Anda A60-IgA, SCWP-IgA, 38 D-LPS-IgG and SCWP-IgG.

[0029] Rule out markers can be useful for improving the specificity of diagnostic marker assays. In one embodiment, the present process uses an anti-CRP binding agent to assess the level of C-Reactive protein in the biological sample.

[0030] In one embodiment, the process includes the use of an anti-procalcitonin binding agent to assess the level of a procalcitonin in order to detect or rule out pneumonia. Procalcitonon levels are elevated in community acquired pneumonia and less elevated in active TB.

[0031] In one embodiment, active TB is diagnosed where IgA or IgG are positive at 2 standard deviations cut off or two out of three from IgA or IgG at 1 standard deviation cut off and CRP is more than 39 μg/ml, and not CRP less than 6 μg ml.

[0032] Binding agents are conveniently antibodies or antigen-binding derivatives as known in the art. Antibody binding agents include receptors and antigens.

[0033] Immunoassays, detection methods and tags/labels are all routine in the art. [0034] Any suitable assay format may be employed. Illustrative immunoassay formats include ELISA, or chromatographic process or equivalent formats.

[0035] In one embodiment, the process comprises diagnosis and treatment, wherein the treatment comprises administering treatment to the subject if they are diagnosed with active TB.

[0036] Also provided is a method of treating active TB in a subject, the method comprising requesting a test for diagnosis of active TB of any one of claims 1 to 16 and administering treatment to the diagnosed subject if the test is positive for active TB. TB treatment agents are known in the art.

[0037] In one embodiment, the specification provides a diagnostic process for determining TB status of a test subject, the process comprising contacting a biological sample comprising antibodies from the subject with a specific dlgA binding agent to allow formation of a TB antigen specific dlgA-dlgA-binding agent complex, measuring the level of the dlgA-dlgA-binding agent test complex formed and comparing the level of the dlgA- dlgA-binding agent text complex formed to a corresponding level formed in a control sample.

[0038] In one embodiment, the specification provides a process wherein one or more TB antigens recognised by dlgA is directly or indirectly bound to a solid surface and captures TB antigen specific dlgA from the biological sample applied to the surface to form immobilised TB antigen-dlgA complexes.

[0039] In one embodiment, the specification provides a process wherein the specific dlgA-binding agent is directly or indirectly bound to a solid surface, and the TB antigen specific dlgA-dlgA binding agent complex is probed with TB antigen.

[0040] In one embodiment, the specification provides a process wherein the TB antigen as an antigen binding agent is selected from one, two or three of the group comprising SCWP, LAM, CWF, CFP, CYT, PstSl, Anda A60, Omega, TDM, mAGP, and PIM6, LM, and SLl. [0041] In one embodiment, the TB antigen is Anda A60, SCWP and LPS. [0042] In one embodiment the TB antigen is Anda A60.

[0043] In one embodiment, the following antigen-isotype combinations are included: two, or three or four or five of Anda A60-sIgA, Anda A60-IgA, Anda A60-IgA, SCWP- IgA, 38KD-LPS-IgG and SCWP-IgG.

[0044] In one embodiment, the dlgA-specific binding agent is a recombinant plgR or a dlgA-binding part or variant thereof.

[0045] In one embodiment, the recombinant plgk comprises a domain from rabbit, mouse or rat homologs and substantially fails to bind IgM or wherein IgM antibodies are removed from the sample prior to contacting plgR.

[0046] In one embodiment, the specification provides a process wherein the subject may be diagnosed with active TB if an elevated level of TB antigen-specific dlgA is detected in the sample relative to the level in a non-TB control sample and wherein the subject may be diagnosed as not having active TB if the level is not elevated relative to a non-TB control.

[0047] In one embodiment, the specification provides a process which includes determining the level of CRP or procalcitonin in the biological sample.

[0048] In one embodiment, the specification provides a process of determining TB status of a subject, the process comprising contacting a biological sample comprising antibodies from a subject being tested for TB status with a recombinant plgR or plgA- binding variant thereof to allow formation of a TB antigen specific dlgA-pIgR complex, measuring the level of the dlgA-pIgR complex formed and comparing the level of the dlgA-pIgR complex formed to a corresponding level formed in a control sample.

[0049] In one embodiment, the specification provides a process wherein the level of TB specific IgA and/or IgG is also measured. [0050] In one embodiment, the specification provides a method of identifying dIgA binding TB antigens, or characterisation of TB antigens that engender dIgA and secretory IgA (SIgA) responses at mucosal surfaces including pulmonary or extrapulmonary mucosal surfaces, the method comprising screening TB antigens or fragments thereof for their ability to bind to dIgA from TB subjects or engender dIgA responses in a suitable subject.

(0051] In one embodiment, kits or immunochromatographic devices comprise, for example, reverse-flow or lateral-flow formats.

[0052] Kits for diagnosing TB status are contemplated comprising reagents for conducting the processes as described and defined herein.

[0053] In one embodiment, the kit comprises a dIgA binding agent, or two or three specific antibody binding agents selected from IgA, IgG and dlgA-binding agents, to allow formation of test complexes between TB antigen specific antibody and antibody binding agent.

[0054] In one embodiment, the at least two antibody binding agents are IgA and IgG- binding agents, or IgA and dIgA binding agents, or IgG and dIgA binding agents.

[0055] In another embodiment, the at least two antibody binding agents are IgG and dIgA binding agents.

[0056] In one embodiment, the binding agents are IgA, IgG and dlgA-binding agents.

[0057] As illustrated herein, in some embodiments, the TB status is active TB or non-TB.

[0058] Furthermore, in one, embodiment, sensitivity of the diagnostic process conducted by the kit is at least 70%, or at least 80% or at least 88%.

[0059] The biological sample may be any biological sample comprising antibodies. However, in one embodiment the biological sample is whole blood or a blood product such as heparinised (or other anticoagulant) blood or a serum sample. [0060] In some embodiments, one or more TB antigens recognised by dlgA or two or three of IgA, IgG and dlgA is directly or indirectly bound to a solid surface and captures TB antigen specific antibody from the biological sample applied to the surface to form immobilised TB antigen-TB antigen specific antibody complexes. An illustrative assay is represented diagrammatically in Figure 9.

[0061] In other embodiment, the specific dlgA-binding agent is directly or indirectly bound to a solid surface, and the TB antigen specific dlgA-dlgA binding agent complex is probed with TB antigen. Many different antibody formats are well known to those of skill in the art.

[0062] In some embodiments, the IgA and/or IgG-binding agent is directly or indirectly bound to a solid surface, and the TB antigen specific antibody and IgA and/or IgG binding agent complex is probed with TB antigen.

[0063] Any TB antigen known in the art or determined using the present disclosure may be developed and employed as an antibody binding agent in the subject process. In one embodiment, the TB antigen is selected from one, two or three of the group comprising or consisting of SCWP, LAM, CWF, CFP, CYT, PstSl, Anda A60, Omega, TDM, mAGP, PIM6, LM, 38 D-LPS and SL1.

[0064] In one embodiment, the TB antigen is one, or two or three of Anda A60, SCWP and LPS.

[0065] In one embodiment, the TB antigen is Anda A60.

[0066] In one important embodiment, the dlgA-binding agent is plgR or a dlgA- binding variant thereof. Antibodies or other dlgA-binding agents may also be employed.

[0067] In one embodiment, the following antigen-isotype combinations are included in the process or kit to maximise diagnostic power (sensitivity) two or three or four or five of Anda A60-dIgA, Anda A60-IgA, Anda A 60 IgG, SCWP-IgA, 38KD-LPS-IgG and SCWP-IgG. [0068] Rule out markers can be useful for improving the specificity of diagnostic kits. In one embodiment, the kit comprises an anti-CRP binding agent to assess the level of C- Reactive protein in the biological sample.

[0069] In one embodiment, the kit comprises an anti-procalcitonin binding agent to assess the level of a procalcitonin in order to detect or rule out pneumonia. Procalcitonin levels are elevated in community acquired pneumonia and less elevated in active TB.

[0070] In some embodiments, active TB is diagnosed where IgA or IgG are positive at 2 standard deviations cut off or two out of three from IgA or IgG at 1 standard deviation cut off and CRP is more than 39 g/ml and not CRP less than 6 μg/ml. Different exact CRP values may be obtained using different sample sets or kits. In other embodiments, the rule-out CRP values is less than the minimum observed in a representative control population of active TB patients, and indicative of active TB when it is more than a level of experimentally determined alongside IgA, IgG and/or dig A levels for this purpose. A "control" may be a sample taken from the subject at an earlier time point. Controls are known in the art. The cut off values (positive or negative) may be determined by statistical techniques involving one or more of ±250, ROC curves, and discriminant score methods.

[0071] In one embodiment, the specification provides a kit for assessing TB status in a biological sample from a subject which employs one or more TB antigens recognised by dlgA from subjects with active TB and employs a dlgA-binding reagent.

[0072] In one embodiment, the specification provides a kit for diagnosing active TB in a subject, comprising: i) an immunographic device comprising a porous membrane operably connected to a sample portion, a test portion, and optionally a control portion; and further comprising a sucker portion, portion comprising a dlgA-binding agent, a portion comprising a TB antigen and optionally a conjugate portion; and (ii) instructions for using the immunographic device to detect the presence of antigen specific dlgA antibody in the sample.

[0073] In one embodiment, the dlgA binding agent is recombinant plgR or a dlgA- binding variant thereof as described herein. [0074] In one embodiment, the dlgA binding agent is an antibody or antigen binding fragment thereof or anti-J chain.

[0075] In one embodiment, two or three or four or five of the Anda A60-dIgA, Anda A60-IgA, Anda A60 IgG, SCWP-IgA, 38kD-LPS-IgG, SCWP-IgG are assessed.

[0076] In one embodiment, the specification provides a use of a dlgA-binding agent in a screening method to identify dlgA-reactive Tb antigens.

[0077] In one embodiment, the specification provides a dlgA-binding agent, plgR or a dlgA-binding variant for use in diagnosing active TB or non-TB in a subject.

[0078] In one embodiment, the specification provides a dlgA-binding agent, plgR or a dlgA-binding variant, when used or for use in assessing dlgA responses in a subject with TB.

[0079] In another aspect, recombinant plgR is provided which is suitable for use in capturing or detecting dlgA and/or IgM. Illustrative recombinant plgRs include R/HpIgR or HpIgR or a dlgA and/or IgM binding variant of R/HpIgR or HpIgR. Illustrative amino acid and nucleotide sequences are set out in SEQ ID NO:l to 20, bearing in mind that some of these sequence encode or provide a CD4 cytoplasmic domain which is entirely optional and may be deleted, modified, supplemented or replaced with other binding or detection molecules known in the art. Once the subject invention is contemplated, useful variants of recombinant plgR will be apparent to . the skilled person and readily made and tested.

[0080] In some embodiments, the process further comprises: (a) generating data using a process as described herein; (b) transforming the data into computer-readable form; and (c) operating a computer to execute an algorithm, wherein the algorithm determines closeness-of-fit between the computer-readable data and data indicating a diagnosis of a kidney disease or a negative diagnosis. In some embodiments, the algorithm comprises an artificial intelligence program, such as a fuzzy logic, cluster analysis or neural network. The subject methods may also be used in a personalized or a population medicine approach in the management of pathology platforms. DETAILED DESCRIPTION OF THE FIGURES SUPPORTING AND DESCRIBING THE PRESENT INVENTION

[0081] If figures contain colour representations or entities, coloured versions of the figures are available from the Patentee upon request or from an appropriate Patent Office. A fee may be imposed if obtained from a Patent Office.

[0082] Figure 1 provides a schematic representation of the production of dlgA and its secretion at mucosal surfaces as SIgA. Most dlgA produced in the submucosal tissues is subsequently bound to plgR and transcytosed to the mucosal surface, where plgR is cleaved to produce SIgA (or free SC), with SIgA constituting a first layer of defense against pathogens. plgR binding is dependent on the presence of J-chain in polymeric Ig, and binding occurs to both IgM and dlgA in humans.

[0083] Figure 2 provides a representation of the structure of dlgAl and its interaction with plgR which is then cleaved to give SIgAl . The associated Secretory component portion of plgR interacts with both J-chain and the Fc domains of both individual IgA molecules.

[0084] Figure 3 provides a representation of the structure of chimeric R/HpIgR relative to human (HpIgR), and the R/HpIgR that is fused to the cytoplasmic domain of human CD4 at its C-terminus. The R/HpIgR and other forms are expressed and secreted at high levels in 293T cells; shown by the detection of R/HpIgR using coomassie brilliant blue staining of SDS-PAGE gel of the crude supernatant (SN) from transiently transfected cells. R HpIgR-cyto is readily purified to homogeneity and high concentration by affinity chromatography with an immobilised matrix of monoclonal antibody 4B4 directed against the cytoplasmic domain of CD4 (Pure). Either pure plgR or crude SN can be used in detection or binding of dlgA as preferred.

[0085] Figure 4 provides a schematic illustration of the structure of full-length (native) plgR (TOP), relative to the recombinant form HpIgR-cyto (bottom), in which the transmembrane domain (TM) and cytoplasmic domain (cyto) of plgR have been replaced with the cytoplasmic domain of human CD4. Because of the deletion of the TM domain, the product is secreted from cells rather than being retained at the cell surface.

[0086] Figure 5 provides a schematic of the structure of full-length (native) rabbit pIgR (TOP), relative to the recombinant form RpIgR-cyto (bottom), in which the transmembrane domain (TM) and cytoplasmic domain (cyto) of rabbit pIgR have been replaced with the cytoplasmic domain of human CD4. Because of the deletion of the TM domain, the product is secreted from cells rather than being retained at the cell surface.

[0087] Figure 6 provides a schematic of the structure HpIgR cyto, RpIgR-cyto, and chimeric R/HpIgR-cyto. These forms of pIgR demonstrate highly efficient binding to solid surfaces such as polystyrene ELISA plates (Nunc Immulon or similar) through interaction of the CD4 cyto domain with the plastic or other solid surfaces.

[0088] Figure 7 provides a schematic of the structure RpIgR and chimeric R/HpIgR. In the absence of the CD4 cyto domain, the RpIgR can be detected by reactivity with antibodies against the rabbit pIgR, and the chimera R/HpIgR can be detected by reactivity with antibodies against the rabbit (domain 1) and/or human (domain 2-5) pIgR. The preferred antibodies must be able to interact with pIgR when it is bound to dlgA, not only to free pIgR.

[0089] Figure 8 illustrates the results of ELISA comparing the binding of HpIgR and R/HpIgR to human IgM and dlgA. HpIgR or R/HpIgR were immobilised on 96-well Nunc Immulon plates overnight at 4°C. Dilutions of purified human IgM or dlgA in PBS were bound to the immobilised pIgR forms overnight. After washing, the captured IgM or dlgA were detected using anti-IgM or anti-IgA conjugated to horseradish peroxidase (HRP) and colorimetric substrate TMB. The results demonstrate that HpIgR shows preferential binding to IgM (magenta) as well as binding to dlgA (green), whereas R/HpIgR shows greatly reduced binding to IgM (yellow) but retains strong binding to dlgA (blue).

[0090] Figure 9 provides a schematic of one preferred method for detection of antigen-specific dlgA (or IgA, IgG or IgM), in which M. tuberculosis antigen is coated directly onto the ELISA plate. Serum samples are applied to the plate and antigen-specific antibodies, including IgA, IgM, IgG and dlgA, bind to the antigens and are then detected with either anti-isotype HRP, or R/HpIgR and anti-human SC HRP. After final washing, signal is generated with TMB substrate. Note that IgM is not considered valuable in TB diagnosis and has not been examined here.

[0091] Figure 10 illustrates the results of ELISA comparing dlgA, IgA and IgG reactivities against MTb antigen, SDS-soluble cell wall protein (SCWP), BEI Resources, NIH. (A) Antigen-specific dlgA is readily detected in 5/60 patients with confirmed pulmonary TB and 0/50 matched controls, using serum samples from Vietnam (Foundation for Innovative New Diagnostics, FIND). (B). IgA is also readily detected in patients, but is present in a significant number of controls. (C) IgG is readily detected but is also present in a large proportion of the controls, with a large degree of overlap between the two patient populations. Although the IgA and IgG responses show clear statistical differences between the TB and non-TB patient populations (Mann- Whitney test), the high degree of overlap between the populations and the high standard deviation among the non-TB population mean that only the most highly reactive TB patients can be classified on the basis of their IgA or IgG reactivity alone. In contrast a smaller number of TB patients show any dlgA reactivity against this antigen, but the extremely low background reactivity among control non-TB patients means that the assay cutoff (mean plus 2 standard deviations) is a low OD, and the reactive samples are generally well separated from the assay cutoff.

[0092] Figure 11 illustrates the results of ELISA comparing dlgA and IgG reactivities against MTb antigen, lipoarabinomannan (LAM), BEI Resources, NIH. (A) Antigen- specific dlgA is readily detected in 1 1/60 patients with confirmed pulmonary TB but also in 2/50 matched controls, using serum samples from Vietnam (FIND). (B). IgG is readily detected but is also present in a large proportion of the controls, with a large degree of overlap between the two patient populations. Although the IgG responses show clear statistical differences between the TB and non-TB patient populations (Mann- Whitney test), the high degree of overlap between the populations and the high standard deviation among the non-TB population mean that only the most highly reactive TB patients can be classified on the basis of their IgG reactivity. In contrast a smaller number of TB patients show any dlgA reactivity against this antigen, but the low background reactivity and standard deviation among control non-TB patients means that the assay cutoff (mean plus 2 standard deviations) is a low OD, and the reactive samples are generally well separated from the assay cutoff.

[0093] Figure 12 illustrates the results of ELISA comparing dlgA and IgA reactivities against MTb antigen, culture filtrate protein (CFP), BEI Resources, NIH. (A) Antigen- specific dlgA is readily detected in 12/60 patients with confirmed pulmonary TB but also in 3/50 matched controls, using serum samples from Vietnam (FIND). (B). IgA is readily detected but is also present in a large proportion of the controls, with a large degree of overlap between the two patient populations. Although the IgA responses show clear statistical differences between the TB and non-TB patient populations (Mann- Whitney test), the high degree of overlap between the populations and the high standard deviation among the non-TB population mean that only the most highly reactive TB patients can be classified on the basis of their IgA reactivity. In contrast a smaller number of TB patients show any dlgA reactivity against this antigen, but the low background reactivity and standard deviation among control non-TB patients means that the assay cutoff (mean plus 2 standard deviations) is a low OD, and the reactive samples are generally well separated from the assay cutoff.

[0094] Figure 13 illustrates the results of ELISA comparing dlgA and IgA reactivities against MTb antigen, Anda A60 (commercial IgA ELISA kit plate), Anda Biologicals, France. (A) Antigen-specific dlgA is readily detected in 14/60 patients with confirmed pulmonary TB but also in 1/50 matched controls, using serum samples from Vietnam (FIND). (B). IgA is readily detected but is also present in a large proportion of the controls, with a significant degree of overlap between the two patient populations. Although the IgA responses show clear statistical differences between the TB and non-TB patient populations (Mann- Whitney test), the high degree of overlap between the populations and the high standard deviation among the non-TB population mean that only the most highly reactive TB patients can be classified on the basis of their IgA reactivity. In contrast a smaller number of TB patients show any dlgA reactivity against this antigen, but the low background reactivity and standard deviation among control non-TB patients means that the assay cutoff (mean plus 2 standard deviations) is a low OD, and the reactive samples are generally well separated from the assay cutoff.

[0095] Figure 14 illustrates the correlation between IgA and dlgA reactivity using commercial Anda A60 antigen-coated plates. A proportion of the IgA reactive and dlgA reactive samples (with cutoff set at mean plus 2 standard deviations of control non-TB patients) show some correlation, and the 9 samples that are reactive for both IgA and dlgA demonstrate that the dlgA and IgA reactivity, combined can provide confirmation of TB infection in this population. However a number of IgA reactive samples show no dlgA reactivity, and a number of dlgA reactive samples show no IgA reactivity, demonstrating that the dlgA and IgA responses are at least partly independent, and highlighting the additional value of using dlgA rather than only IgA for TB diagnosis.

[0096] Figure 15 illustrates the antibody response to MTb antigens is heterogeneous among both TB patients and control non-TB patients. Each colour column shows different antigen-isotypes: yellow dlgA, green IgA, and blue IgG. The shading of cells represents the strength of reactivity, as shown on the left, being greater than the mean plus either 2, 3 or 6 SD of the non-TB patient population. The rows represent individual patient samples. Some patients are very reactive to many antigens across multiple antibody types, while others show unique reactivity with only one or two antigens/isotypes. Strikingly, it can be seen that most of the dlgA reactive samples among TB patients are positive at the highest cutoff (mean + 6 SD), with a smaller proportion of IgA reactive samples positive at this cutoff, and the lowest proportion of IgG reactive samples positive at this cutoff.

[0097] Figure 16 illustrates the very high reactivity of dlgA reactive samples allows the use of the very high cutoff of mean plus 6 SD of control non-TB patients. Across the limited number of antigens tested in the experiments shown in Figure 15, this provides a sensitivity of 27% and specificity of 98% for dlgA detection, but only 8% sensitivity for IgG detection and 15% sensitivity for IgA detection, with 98% and 100% specificity respectively. This demonstrates that dlgA is a useful component of serological testing strategies for diagnosis of active TB. [0098] Figure 17 illustrates the heterogeneity of the antibody response to multiple MTb antigens has been observed previously in studies focused on the IgG response to multiple TB protein antigens. The heat map reproduced from Kunnath-Velayudhan et al, PNAS 107: 14703-14708, 2010 shows that when using the 13 antigens that were found to be most highly reactive out of the entire MTb proteome (rows), IgG responses between patients (columns) were very heterogeneous and it is necessary to combine a large number of antigens to achieve acceptable sensitivity using IgG antibody responses. The relative reactivity of antigens with dlgA or IgA rather than IgG has not been evaluated.

[0099J Figure 18 illustrates the correlation between IgA, dlgA and IgG reactivity on a subset of MTb antigens (pure antigens LAM, PstS l , LM, TDM, mAGP, SL1 , PIM6 and crude antigens or mixtures, CWF, CYT, CFP, AndaA60 and Omega commercial kits). dlgA reactivity is shown to have some correlation with IgA reactivity, and IgA to have some correlation with IgG reactivity, but dlgA shows no significant correlation with IgG reactivity. Most attempts at screening different MTb antigens for their potential use as diagnostic reagents has focused on IgG or to a lesser extent IgA. The independence of dlgA reactivity versus IgG (and to a lesser extent, IgA) highlights the utility of dlgA as an independent diagnostic marker for MTb infection.

[0100] Figure 19 provides the results of screening 5 selected antigens for reactivity of dlgA, IgA or IgG among FIND patient samples, using a cutoff that gives 100% specificity among FIND non-TB control samples. Only the TB patient reactivities are shown because of this selected cutoff rendering all non-TB patients non-reactive. Using the antigens LAM, CWF, CFP, CYT, PstS l (all BEI Resources), IgA and IgG reactivity combined are able to detect 24/60 samples (40%) with 100% specificity. The inclusion of dlgA detects a further 7 samples that are uniquely reactive for dlgA (marked with red highlights; 3 of the samples reactive for dlgA against more than one antigen and 1 sample reactive against all five antigens), for a total of 31/60 (51.7%) sensitivity or a 29% increase over the 40% sensitivity seen with IgA and IgG alone. These results demonstrate that inclusion of dlgA detection using the R HpIgR reagent improves the sensitivity of antibody-based tests for active TB infection. [0101) Figure 20 illustrates the combination screening results using an expanded panel of MTb antigens for dlgA, IgA and IgG identifies unique reactivities, based on a cutoff of the mean plus 2 standard deviations of the non-TB sample population. Using 1 1 different antigens, 55/60 of the patients demonstrate reactivity for one or more of dlgA, IgA or IgG, with similar numbers being uniquely reactive only for dlgA (8), IgA (6) or IgG (7). Consistent with the results shown in Figure 15, this suggests that dlgA reactivity contributes as much as IgG or IgA to the sensitivity of improved serological tests for MTb- specific antigens in diagnosis of active TB. An optimal combination of assays from this set of 11 antigens would use dlgA with Anda A60, IgA with Anda A60, IgA with SCWP, IgG with 38kd-LPS (Omega) and IgG with SCWP, with a total 50/60 TB patients detected (82% sensitivity) and 6/50 non-TB patients (88% specificity). Notably, it is anticipated that the screening of additional MTb antigens will increase the sensitivity of a serological diagnostic algorithm when using dlgA alone or in combination with IgA and/or IgG, while the specificity of the assays may be improved by inclusion of a "rule-out" test in the diagnostic algorithm, such as C-reactive protein (CRP). CRP is always elevated in patients with active TB, and the absence of elevated CRP can be used to rule out active TB in at least that proportion of non-TB patients who do not have elevated CRP for other medical reasons.

[0102] Figure 21 provides the nucleotide sequence and amino acid sequence of CHIMERA-CD4 cyto (R HpIgR-cyto) showing rabbit sequences underlined and human sequences in black, and CD4 cyto sequences in red.

[0103] Figure 22 provides the nucleotide sequence and amino acid sequence of CHIMERA (R/HpIgR) showing rabbit sequence underlined and human sequence in black.

[0104] Figure 23 provides graphical illustrations of data showing the independence of IgA, IgG and dlgA reactivities can also be demonstrated with the Anda A60 antigen, where samples uniquely positive for IgA versus IgG, dlgA vs IgG, and dlgA vs IgA can be seen in pairwise comparisons. The cutoff for the respective assays (mean plus 2SD of non- TB patients, labelled "FIND-samples") is shown by the dotted line parallel to that axis, with samples having values greater than cutoff for one analyte (such as IgA) but less than cutoff for the other analyte (such as IgG) being readily apparent. Combining the different antibody types therefore increases assay sensitivity, even using a single antigen (Anda A60) instead of the full range of 1 1 antigens described previously. The sensitivity and specificity of these assay combinations for Anda A60 are described in the following graphs.

[0105] Figure 24 provides a graphical illustration of data showing independence of antibody responses to a single antigen type such as A60 can also be demonstrated at the level of antibody subtypes. In this example, IgM, IgG and IgA were measured as well as IgG subtypes IgG3, IgG2 and IgGl, and IgA subtypes IgAl and IgA2, as well as dlgA. The Heat map of A60-specific antibody responses across active TB (n=60) and non TB controls (n=50) is shown. Each column represents a serum sample and each row represents an antibody isotype/subclass. Level of reactivity illustrated by color spectrum, determined as signal to cutoffs at 1SD, 2SD, 3SD and 6SD from mean of non TB controls. This demonstrates that there is additional diversity in the antibody repertoire against TB which may prove useful for further improving assay sensitivity or specificity using methods substantially similar to those described herein for the total antibody classes of IgA, IgG and dlgA.

[0106] Figure 25 provides a graphical illustration of data illustrating C-Reactive Protein concentration in active TB and non TB controls. The utility of C-reactive protein (CRP) for rule-out of active TB is well known in the art, taking advantage of the fact that almost all patients with active TB have elevated levels of CRP, although many patients with other inflammatory conditions that may present as suspected TB can also have elevated CRP and so it is not as useful as a positive marker of TB when used alone. In this example using the FIND active TB and non-TB patients, the active TB patients were found to have no lower than 6 g/ml of CRP, whereas 30% of the non-TB patients were found to have less than this amount, and could therefore be excluded from a TB diagnosis even if their antibody profile suggested that they were infected. This can potentially reduce the false-positive rate by 30%, even among symptomatic patients who are initially suspected of having TB. Other host-specific markers such as procalcitonin may also prove useful for this purpose, since procalcitonin is less elevated in active TB, and more elevated in community-acquired pneumonia.

[0107] Figure 26 illustrates an algorithm: Active TB = IgA or IgG positive at 2 SD cutoff OR 2 out of 3 from (IgA or IgG at 1 SD cutoff and CRP >39 g/ml) AND NOT CRP <6 μg ml (rule-out). Although CRP is not useful by itself for diagnosis of active TB, it can be used in combination with specific antibody responses to improve the sensitivity of an algorithm for specific diagnosis. In this example, samples are identified as being active TB on the basis of their IgA, IgG and CRP reactivity as follows. Samples positive for IgG or IgA at a cutoff 2 SD above the mean of non-TB controls are defined as active TB (purple boxes). Samples having 2 out of 3 of the following: being positive for IgG or IgA at a cutoff 1 SD above the mean of non-TB controls, or having CRP >39 g/ml, are defined as active TB (orange boxes). This takes advantage of the partial independence of IgG and IgA reactivity, such that most "false positive" samples for IgG or IgA will not be false-positive in both assays, but instead it is more likely that true positive (active TB) patients will have both these antibodies, or one of these antibodies together with CRP, whereas CRP alone is not diagnostics of active TB. Assay specificity is improved by "ruling out" any samples with CRP less than 6 g ml (blue box).

[0108J Figure 27 provides graphical illustrations of data showing an algorithm combining IgA, IgG and dlgA reactivity improves sensitivity of TB ELISA (A60 antigen, Anda Biologicals) without compromising specificity. In this example, the assay cutoff for individual Ig types were based on reactivity of 50 suspected TB subjects proven not to have active TB (FIND). For IgA and IgG, the cutoff was set at the mean plus 1 standard deviation, for dlgA it was set at the mean plus 3 standard deviations. The result for combined Ig types was calculated by the sum of individual antibody positive or negative results. The sensitivity is the percentage of active TB patients (FIND, n=60) who were reactive in each Ig or combined Ig assay algorithm. While IgA and IgG had poor sensitivity by themselves (60 and 63% respectively), combining IgA and IgG increased sensitivity to 78%, while combining A, IgG and dlgA increased sensitivity significantly to 82%. Specificity was not significantly affected by combining Ig types. All samples were tested for C-reactive protein (CRP) and samples with less than 6 μί*/ι 1 CRP were "ruled out" as being active TB.

[0109] Figure 28 illustrates an algorithm combining IgA, IgG and dlgA reactivity improves sensitivity of TB ELISA (A60 antigen, Anda Biologicals) without compromising specificity. In this example, the assay cutoff for individual Ig types were based on reactivity of 50 suspected TB subjects proven not to have active TB (FIND). For IgA and IgG, the cutoff was set at the mean plus 1 standard deviation, for dlgA it was set at the mean plus 3 standard deviations. The assay cutoff for combined Ig types was based on the sum of individual antibody sample to cutoff ratios, which were then added together and a new cutoff determined based on the calculated value of the 50 non-TB samples (FIND). The sensitivity is the percentage of active TB patients (FIND, n=60) who were reactive in each Ig or combined Ig assay algorithm. While IgA and IgG had poor sensitivity by themselves (60 and 63% respectively), combining IgA and IgG increased sensitivity to 80% (approaching significance), while combining A, IgG and dlgA increased sensitivity significantly to 85%. Specificity was not significantly affected by combining Ig types. All samples were tested for C-reactive protein (CRP) and samples with less than 6 μg/ml CRP were "ruled out" as being active TB.

DESCRIPTION OF PARTICULAR EMBODIMENTS

[0110] Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or element or method step or group of integers or elements or method steps but not the exclusion of any other integer or element or method step or group of integers or elements or method steps.

[0111] By "consisting of is meant including, and limited to, whatever follows the phrase "consisting of. Thus, the phrase "consisting of indicates that the listed elements are required or mandatory, and that no other elements may be present. By "consisting essentially of is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase "consisting essentially of indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending upon whether or not they affect the activity or action of the listed elements.

[0112] As used herein the singular forms "a", "an" and "the" include plural aspects unless the context clearly dictates otherwise.

[0113] Nucleotide and amino acid sequences are referred to by a sequence identifier number (SEQ ID NO:). The SEQ ID NOs: correspond numerically to the sequence identifiers <400>1 (SEQ ID NO: l), <400>2 (SEQ ID NO:2), etc. A summary of sequence identifiers is provided in Table 1. A sequence listing is provided after the claims.

[0114] In the work leading up to present disclosure, the inventor/s considered that dlgA is predominantly synthesized by B-cells in the sub-mucosal tissues, where a large proportion of the antibody is secreted as SIgA following its interaction with plgR, but a proportion of dlgA enters the general circulation. As proposed herein, dlgA responses and levels are likel to be strongest/highest during infections of mucosal surfaces and closely associated tissues including the upper and lower respiratory tracts, the gut and the gut- associated lymphoid tissue (GALT), the genital tract and the liver. These dlgA responses are the precursor to SIgA, and as proposed herein may be most important for immunity to TB infectious agents at the mucosal surface. Conversely, monovalent IgA, mlgA is synthesized by B-cells in the circulation and in the bone marrow, and might not as accurately reflect exposure to TB antigens or infectious agents at the mucosal surface and associated tissues such as GALT, or immunity thereto.

[01 IS] As discussed below, the present disclosure provides a process for assessing the TB infection status of a subject including determining mucosal immune responses to TB comprising determining the level of antigen-specific dimeric or polymeric IgA (dlgA) in a biological sample from the subject.

[0116] Furthermore, the invention is predicated upon technical results indicting that antibody responses and TB antigens for IgA, IgG and dlgA are substantially independent and can therefore be used in an algorithm to improve assay performance and in the design of pathology platforms and diagnostic kits for TB.

[0117] dlgA is the precursor for SIgA, and as described herein, assaying the level of antigen-specific dlgA in a biological sample from a subject is indicative of the TB status of a subject independent of other antibody isotypes or forms such as IgG, or total IgA which is predominantly monomeric IgA (weak correlation only may be present). In some embodiments, the subject may have active or latent TB, or the subject has had some exposure to TB infection or may have had some exposure to TB infection. The process enables the development inter alia of improved diagnostic, prognostic and therapeutic protocols. The process allows a user to determine the TB infection status of a subject via a communications network. In some embodiments, the process facilitates the identification or characterisation of TB antigens that engender dlgA and secretory IgA (SIgA) responses at mucosal surfaces including pulmonary or extrapulmonary mucosal surfaces. Further, such one or more antigens may be selected for use in diagnostic or prognostic assays or for use in prophylactic or therapeutic vaccines.

[0118] The present process is predicated, in part, upon the ability to detect dlgA with high sensitivity and specificity in binding assays using a recombinant polymeric Ig receptor (plgR). In some embodiments, the present process employs recombinant plgR or recombinant variants of plgR that bind dlgA and IgM, as well as recombinant plgR or variants of plgR that preferentially bind dlgA and substantially fail to bind IgM. In some embodiments, , the recombinant plgR is selected to bind dlgA and substantially not bind IgM as disclosed herein. Although the invention is exemplified using recombinant plgR other equivalent agents or functional comparable agents may be employed. Furthermore, as described herein, the present process is predicated in part upon the finding and use of dlgA as an independent diagnostic marker for TB infection. Specifically the ability to detect dlgA responses to one or more TB antigens in infected subjects that do not correlate with IgG responses to the same antigens. Furthermore, dlgA responses correlate only very weakly with total IgA which is predominantly monomeric. As shown in the Examples, although the IgA and IgG responses with various antigens show clear statistical differences between the TB and non-TB patient populations, the high degree of overlap between the populations and the high standard deviation among the non-TB population mean that only the most highly reactive TB patients can be classified on the basis of their IgA or IgG reactivity alone. In contrast a smaller number of TB patients show any dlgA reactivity against this antigen, but the extremely low background reactivity among control non-TB patients means that the assay cutoff (mean plus 2 standard deviations) is a low value and the reactive samples are generally well separated from the assay cutoff point.

[0119] Reference to "TB" or "TB antigen" includes any species of Mycobacterium which causes tuberculosis in a human subject, primarily M. tuberculosis and M. tuberculosis antigens but also homologs derived from other TB causative strains such as, without limitation, those belonging to the one or more of the following species: M. bovis, M. africanum, M. canetti and M. microti. Reference to "TB status" or "TB infection status" includes active TB, pulmonary TB, infection with TB, non-TB infection status. Various TB antigens known to engender antibody responses generally are employed in the Examples and are encompassed herein. Illustrative antigens known in the art includes without limitation SCWP, LAM, CWF, CFP, CYT, PstSl , Omega, TDM, mAGP, PIM6, and Anda A60 (see also Example 2). Although Anda A60 is in fact prepared from M. bovis (BCG) it contains antigens that are also present in M. tuberculosis and has been used in serological diagnostic assays for TB for more than 20 years. The present process further facilitates the characterisation of TB antigens in terms of their ability to engender enhanced dlgA production which antigen are then suitable for use in the diagnostic and prognostic protocols and kits of the invention, employing dlgA detection.

[0120] Accordingly, the present specification provides a process for determining the TB status of a subject, the process comprising assessing the level of TB antigen-specific dlgA.

[0121] In another embodiment, the specification enables screening TB antigens for their ability to engender dlgA responses. Such antigens are useful in diagnostic, prognostic and therapeutic assays and kits. [0122] In another embodiment, the specification enables kits or substrates comprising TB antigens selected using the protocols described herein for their ability to engender dIgA, for use in assessing the TB status of a subject.

[0123] In an illustrative embodiment, the TB antigens include Anda-60, which is shown herein to distinguish between TB and non TB subjects, either alone or in combination with other TB antigens, in the present assays employing at least a determination of dIgA levels independent of monomeric IgA levels.

[0124] The Examples and Figures provided herein demonstrate that the inclusion of dIgA detection improves the sensitivity of antibody based tests for active TB infection status.

[0125] The use of recombinant pIgR is also highly advantageous not least because the reagent displays low background (at least 50% less background compared to antibody based reagents) in binding assays, unlike most antibody based binding agents. In addition, recombinant pIgR displays high thermal stability. For example, lyophilised recombinant pIgR retained 50% activity at 60°C and 100% activity at 45°C after three weeks prior to reconstitution, which compares favourably to the rapid loss of activity for dried anti-IgM antibody under the same conditions.

[0126] The present invention provides recombinant polymeric immunoglobulin receptor (pIgR) for use in detecting dIgA in subject samples and therefore for providing and for use in improved protocol for determining TB status in a subject. Alternative dlgA- binding agents include antibodies to the J-chain of dIgA. Although this reagent does not distinguish between dIgA and IgM, the latter may be depleted from samples prior to assessment, as illustrated herein. Different isotypes can readily be depleted using antibodies or other binding agents including R HpIgR.

[0127] Determining the presence or level of dIgA or pIgR or a complex between dIgA and recombihant pIgR or a complex between recombinant dIgA and an antigen may be by any convenient protocol. [0128] A diverse range of assays are used in research, analysis, development and clinically to detect analytes of interest. Immunoassays are a particularly useful form of assay that exploits the specificity, strength and diversity of antibody-antigen type or protein-protein reactions to analyse samples and detect specific components therein. A wide range of immunoassay techniques are available, such as those described in Wild D. " The Immunoassay Handbook" Nature Publishing Group, 2001.

[0129] In a preferred embodiment, exemplified herein, the process comprises the step of (i) detecting or capturing antigen specific dlgA with a recombinant polymeric immunoglobulin (Ig) receptor (plgR) or an dlgA-binding variant thereof wherein the recombinant plgR substantially does not bind monomeric IgA. In some embodiments, the plgR or dlgA-binding variant binds dlgA and Ig . In preferred embodiments, the recombinant plgR or dlgA-binding variant binds dlgA and substantially fails to bind IgM. In preferred embodiments, the process comprises (ii) determining the level or presence of dlgA that has bound to recombinant plgR. In some embodiments, the step (ii) optionally includes detecting a complex between dlgA and plgR or a complex between bound dlgA and an antigen.

[0130] In some embodiments, recombinant plgR binds weakly to SIgA and in accordingly, if required, the process comprises a washing step to abolish SlgA binding such as washing with 3.5M urea or an equivalent agent which does not substantially affect plgR binding to dlgA. plgR does not bind to free J-chain but the presence of J-chain which is common to dlgA and pentameric IgM is essential for binding thereto.

[0131] Methods of detecting antibody complexes, antigens or antibody-ligand complexes are well known in the art. For example, the enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA) are routinely used in laboratories. These methods generally require some level of skill in laboratory techniques. A variety of methods have also been developed which require little skill and are rapid to perform, and which are therefore suitable for the detection of antibody to specific antigens at the point of care or analysis. In particular, immunochromatographic or dipstick enzyme-linked immunosorbent kits have been developed to assay for a number of infections agents. [0132] Immunochromatographic devices are expressly contemplated comprising dlgA-binding reagents such as recombinant plgR and further comprising TB antigens identified as described herein as binding dlgA from TB infected subjects.

[0133] Kits or immunochromatographic devices comprise, for example, reverse-flow or lateral-flow formats.

[0134] In an illustrative embodiment, a kit for assessing TB status in a biological sample from a subject is provided which employs one or more TB antigens recognised by dlgA from subjects with active TB and employs a dlgA-binding reagent. dlgA-binding agents substantially fail to bind monomeric IgA.

[0135] In some embodiments, the kit comprises:

[0136] a) an immunochromatographic device comprising a porous membrane operably connected to a sample portion, a test portion, and optionally a control portion; and further comprising a sucker portion, portion comprising a dlgA-binding agent, a portion comprising a TB antigen as described herein and optionally a conjugate portion; and

[0137] b) instructions for using the immunochromatographic device to detect the presence of antigen specific dlgA antibody in the sample.

[0138] In some embodiments, the dlgA binding agent is recombinant plgR or a dlgA- binding variant thereof as described herein.

[0139] The subject assays may employ a wide range of suitable labels, tags, detection markers and detection reagents known in the art. In some embodiments, the detection marker may be detected using detectable characteristics of the detection marker and a wide range of detection protocols using detectable markers are well known to those of ordinary skill in the art. In some embodiments, the detection marker is directly or indirectly bound or otherwise associated with an TB antigen. In other embodiments, the dlgA binding agent, such as plgA comprises or is designed to interact with a detection marker. In some embodiments, the detection marker is connected the antigen or dlgA binding agent using binding partners known in the art such as without limitation biotin:avidin or anti-biotin antibody:biotin.

(0140] In one embodiment, and as illustrated in Figure 9, an ELISA format may be employed for the detection of antigen-specific dlgA (or in combination with IgA, IgG or IgM), in which M. tuberculosis antigen is coated directly onto an ELISA plate. Serum samples are applied to the plate and antigen-specific antibodies, including IgA, IgM, IgG and dlgA, bind to the antigens and are then detected with either anti-isotype HRP, or R/HpIgR and anti-human SC HRP. After final washing, signal is generated with TMB substrate. Note that IgM is not considered valuable in TB diagnosis. In some embodiments, samples may be depleted of IgM. These interactions may be reproduced in a range of different laboratory or point of care based (rapid) protocols.

[0141] In some embodiments, recombinant plgR comprises a CD4 cytoplasmic tail which may be used for efficient binding to solid surfaces such as polystyrene plates. In some embodiments, the CD4 tail may be used as an epitope tag.

{0142] The specification describes illustrative TB antigens and provides a convenient process for identifying further dlgA reactive TB antigens. It is proposed to use combinations of TB antigens as any one antigen tends to pick up only 10% or so of samples (but with very high signal/cutoff). In preferred examples therefore dlgA is used in combination with IgA and/or IgG - IgA and IgG to provide enhanced sensitivity and specificity. An algorithm based on different antigen/antibody combinations is proposed to be useful. In one example (see Example 2 and Figure 20) in order to assess TB status the following antigen-antibody combination is proposed: Anda A60-dIgA + Anda A60-IgA + SCWP-IgA + 38kD-LPS-IgG + SCWP-IgG.

[0143] As shown in Example 1, this combination showed 50/60 true positives = 82% sensitivity, and 6/50 false positives = 88% specificity.

[0144] Further combinations will be readily apparent to the skilled addressee based upon the present disclosure. In some embodiments, the TB antigen is not AndaA60. [0145] Accordingly, in one embodiment, the present invention provides a process for determining TB status in a subject, the process comprising the step of (i) contacting a biological sample from the subject with recombinant plgR or a dlgA-binding part or variant thereof as described herein wherein the recombinant plgR binds dIgA in the sample and substantially does not bind monomeric IgA and (i) determining the level of dIgA that has bound to plgR.

[0146] In some embodiments, TB antigen is directly or indirectly bound to a solid surface and captures dIgA in a sample applied to the surface, which is then detected using recombinant plgR or a dlgA-binding part or variant thereof.

[0147] In another embodiment, plgR or a dlgA-binding part or variant is directly or indirectly bound to a solid surface, dIgA from a subject sample is contacted therewith and a complex between plgR and dIgA is probed with one or more selected TB antigens of interest.

[0148] Reference herein to a "sample" means a biological sample comprising antibody, directly or indirectly derived from a subject. In some embodiments, the sample is whole blood or serum or plasma. In other embodiments, the sample is urine, stool, saliva, tears and milk etc. Blood may, in some embodiments, be maintained in the presence of an anticoagulant such as heparin, sodium citrate or ethylene diamine tetra acetic acid (EDTA).

[0149] Reference to "subject" includes humans and a wide range of mammalian or other animals including wild and domesticated animals, pets, pests and potential vehicles for emerging infectious diseases. In relation to subjects, these may have an infection, they may have had exposure to infection or they have had exposure to an infectious agent.

[0150] Polymeric immunoglobulin receptor (plgR) is encoded by the P1GR gene and is expressed in mucoal epithelial cells where it facilitates uptake of dIgA and secretion of SIgA. plgR has five immunoglobulin-like domains which bind to dIgA including to the J- chain thereof. plgR also binds to pentameric IgM.

[0151] As determined herein it is possible to detect both dIgA and IgM with high sensitivity and specificity using recombinant human polymeric Ig receptor and parts and variants thereof (see Figure 3). In one particular non-limiting embodiment it is shown herein that dlgA can be selectively detected using a recombinant form of the polymeric Ig receptor having at least domain 1 derived from the rabbit pIgR, for example, a chimera of rabbit (domain 1) and human (domain 2-5) plgRs, or with all domains from rabbit pIgR. In some embodiments, the recombinant pIgR described herein are designed to bind preferentially to dlgA, and can be used either to capture dlgA specifically to a solid phase for reaction with an antigen of interest, in which case the pIgR does not need to have an associated detection reagent, or alternatively to detect the presence of dlgA bound to an antigen of interest immobilized on a solid phase, in which case the pIgR may be conveniently detected using antibodies or other reagents directed against the pIgR itself, or against epitope tags or other sites introduced into the recombinant pIgR using methods well known in the art. A further advantage of pIgR is that it shows very low background reactivity in assays, unlike typical antibody-based detection reagents.

[0152J Roe et al, J Immunol 162: 6046-52, 1999 describe a chimeric pIgR comprising immunoglobulin-like domain 1 (Dl) derived from rabbit, and D2-5 derived from human pIgR which has preferential binding to dlgA over IgM. However, they do not disclose or suggest the use of this form or any other pIgR variant for detection or binding only of dlgA for diagnostic purposes or the advantages of the recombinant plgA or dlgA binding variants disclosed herein. The substitution of human for rabbit (or mouse or rat) Dl provides preferential binding of dlgA, but it would be expected that substitution of any one or more of D2-D5 may also be substituted with the rabbit sequence to give a molecule that preferentially binds to dlgA and these variants are also encompassed. Accordingly, in some embodiments, any one or more of Dl , D2, D3, D4 or D5 is substituted with rabbit, mouse or rat homologs.

[0153] In some embodiments, the recombinant pIgR lacks a transmembrane domain (ΔΤΜ). In other embodiments, the recombinant pIgR lacks a cytoplasmic domain. In some embodiments, the recombinant pIgR lacks a TM domain and a cytoplasmic domain (ACYT). In some embodiments, recombinant pIgR comprises a substitution in the cytoplasmic domain and provides a CD4 cytoplasmic domain. Various forms of recombinant pIgR are contemplated and illustrative examples are illustrated in Figures 4 to 7, further described in the figure legends. The ability to design and test recombinant plgR having a desired level of specific dIgA is illustrated in Figure 8 and described in the legend to Figure 8.

[0154] Phillips-Quagliata et al, J Immunol 165: 2544-2555, 2000 indicate that both rat and mouse bind predominantly dIgA, but that for mouse there is a form expressed on B- cells that has only a single amino acid change but binds both IgM and dIgA - to quote from page 2552:

[0155] "Although human plgR and the T560 mouse plgR bind both plgA and IgM, rabbit (44) and rat (47) hepatocyte plgR bind only plgA well and do not translocate IgM into bile. Because mouse liver similarly translocates plgA but not IgM into bile (48, 49), it is generally assumed that mouse hepatocyte plgR resembles rat and rabbit plgR and binds IgM poorly or not at all. If this is true, then the difference between the mouse hepatocyte and the T560 plgR that makes the latter behave more like human plgR must be explained. Given that the amino acid sequences of the mouse hepatocyte and T560 B cell plgRs are the same except for the Val to Ala change in domain 2, the difference most likely reflects differential folding or glycosylation of the plgR, probably the latter. It is easy to imagine that a bulky carbohydrate on hepatocyte-derived plgR could interfere with IgM but not with IgA binding. Furthermore, it has already been shown that deglycosylation of human SC allows it to inhibit binding of biotinylated native SC to plgA with 10 times greater efficiency than native SC itself (50), suggesting that some of the carbohydrate moieties on human plgR may actually impede binding even of plgA."

[0156J Accordingly, in some embodiments, a deglycosylated variant of the recombinant plgR including R HpIgR is used to improve binding affinity to dIgA. In some embodiments, this may be achieved by expressing the plgR in a glycan-deficient cell line known in the art such as, for example, a glycan deficient CHO cell line.

[0157] In plural embodiments, recombinant plgR comprises a deletion in the transmembrane domain (ΔΤΜ) to allow for convenient secretion of the recombinant protein and ease of use as a diagnostic/prognostic/screening agent. [0158] In some embodiments, the recombinant plgR comprises a heterologous detection domain.

[0159] Multiple detection domains are known in the art and are encompassed.

[0160] In some embodiments, the recombinant plgR comprises a heterologous binding domain.

[0161] Multiple binding domains are known in the art and are encompassed.

[0162] In other embodiments, the recombinant plgR is bound to a solid support. Solid supports include plates, wells, beads, agarose particles, nitrocellulose strips, etc.

[0163] In some embodiments, recombinant plgR is produced in glycan deficient cells such as glycan deficient CHO cells to enhance preferential binding to dlgA over IgM.

[0164] In some embodiments, the recombinant plgR is derived from a primate such as human plgR and comprises at least one immunoglobulin-like domain derived from a non- primate such as rabbit, mouse, rat.

[0165] In some embodiments, the recombinant plgR comprises an amino acid sequence set out in SEQ ID NO:2, or SEQ ID NO: 4, or SEQ ID NO: 6, or SEQ ID NO: 12, or SEQ ID NO: 14; or SEQ ID NO: 16, or an dlgA-binding part thereof or and a dlgA binding variant thereof. Illustrative variants comprise at least 70% amino acid sequence identity to one of SEQ ID NO: 2, 4, 6,12, 14 or 16 or deletion variants thereof lacking a cytoplasmic domain.

[0166] Variants include deletion, substitution and insertional variants. Illustrated herein are human derived plgR varied by one or more immunoglobulin domains (D). Variants include "parts" which includes fragments comprising from about 50%, 60%, 70%, 80%, 85%, 90%, 95% of the reference sequence. Substitution for an equivalent domain from a lower mammal such as a rat, mouse or rabbit domain.

[0167] "Variants" of the recited amino acid sequences are also contemplated. Variant molecules are designed to retain the dlgA binding functional activity of the pre-modified recombinant plgR or to exhibit enhanced activity. Polypeptide variants according to the invention can be identified either rationally, or via established methods of mutagenesis (see, for example, Watson, J. D. et al., "Molecular Biology of the Gene", Fourth Edition, Benjamin/Cummings, Menlo Park, California, 1987). Random mutagenesis approaches require no a priori information about the sequence that is to be mutated. This approach has the advantage that it assesses the desirability of a particular mutant based on its function, and thus does not require an understanding of how or why the resultant mutant protein has adopted a particular conformation. Indeed, the random mutation of target gene sequences has been one approach used to obtain mutant proteins having desired characteristics (Leatherbarrow R., J. Prot. Eng., 7:7-16, 1986; nowles J. R., Science, 256:1252-1258, 1987; Shaw W. V., Biochem. J., 246Λ-Π, 1987; Gerit J. A., Chem. Rev., 57:1079-1 105, 1987). Alternatively, where a particular sequence alteration is desired, methods of site-directed mutagenesis can be employed. Thus, such methods may be used to selectively alter only those amino acids of the protein that are believed to be important (Craik C. S., Science, 228:291 -297, 1985; Cronin et al., Biochem., 27: 4572-4579, 1988; Wilks et al, Science, 242:1541-1544, 1988). Illustrative amino acids affect glycosylation of the recombinant plgR. Polypeptides, resulting from rational or established methods of mutagenesis or from combinatorial chemistries, may comprise conservative amino acid substitutions. It is well understood in the art that some amino acids may be changed to others with broadly similar properties without changing the nature of the activity of the polypeptide (conservative substitutions, see Table 3).

[0168] Variant plgR polypeptides comprises at least 50% sequence identity to herein amino acid sequence at least over the immunoglobulin-like domain region.

[0169] The terms and "sequence identity" as used herein refer to the extent that sequences are identical or functionally or structurally similar on an amino acid-by-amino acid basis over a window of comparison. Thus, a "percentage of sequence identity", for example, is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical amino acid residue (e.g. Ala, Pro, Ser, Thr, Gly, Val, Leu, He, Phe, Tyr, Tip, Lys, Arg, His, Asp, Glu, Asn, Gin, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. For the purposes of the present invention, "sequence identity" will be understood to mean the "match percentage" calculated by the DNASIS computer program (Version 2.5 for windows; available from Hitachi Software engineering Co., Ltd., South San Francisco, California, USA) using standard defaults as used in the reference manual accompanying the software. Similar comments apply in relation to sequence similarity which counts as identical, substitutions involving conservative substitutions.

[0170] Preferably, the percentage similarity between a particular sequence and a reference sequence (nucleotide or amino acid) is at least about 60% or at least about 70% or at least about 80% or at least about 90% or at least about 95% or above such as at least about 96%, 97%, 98%, 99% or greater. Percentage similarities or identities between 60% and 100% are also contemplated such as 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%.

[0171] In another embodiment there is provided recombinant plgR encoded by the sequence of nucleotides set out in SEQTD NO:l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO: l l, SEQ ID NO: 13 or SEQ ID NO:15, or a dlgA-binding and optionally IgM-non binding variant thereof having at least 60% nucleotide sequence identity thereto or at least 60% nucleotide sequence identity to deletion variants thereof lacking a cytoplasmic domain.

[0172] In some convenient embodiments, the recombinant plgR is a human recombinant plgR variant comprising at least one immunoglobulin-like domain derived from a rabbit.

[0173] In examining patients with MTb infection, it is shown herein that the dlgA response to individual antigens does not correlate with the IgG response to the same antigens. As such, the dlgA response to MTb antigens is independent of the IgG response, and examination of the MTb proteome and non-proteinaceous antigens for preferentially dlgA-reactive antigens is likely to reveal antigens that are useful for serodiagnosis of MTb, and/or antigens that are useful for development of vaccines that give strong mucosal r

immune responses to MTb.

[0174] In some embodiments, the process further comprises: (a) generating data using a process as described herein; (b) transforming the data into computer-readable form; and (c) operating a computer to execute an algorithm, wherein the algorithm determines closeness-of-fit between the computer-readable data and data indicating a diagnosis of a disease or condition. In some embodiments, the algorithm comprises an artificial intelligence program, such as a fuzzy logic, cluster analysis or neural network. The subject methods may also be used in a personalized or a population medicine approach in the management of pathology platforms.

[0175] The present disclosure provides a computer program and hardware for diagnosis in a subject once off, over time or in response to treatment or other affectors. Values are assigned to complex levels which are stored in a machine readable storage medium. A computer program product is one able to convert such values to code and store the code in a computer readable medium and optionally capable of assessing the relationship between the stored data and incoming data and optionally a knowledge database to assess a potential TB status and/or pneumonia.

[0176] The present specification therefore provides a web-based system where data on levels of complex are provided by a client server to a central processor which analyses and compares to a control and optionally considers other information such as patient age, sex, weight and other medical conditions and then provides a diagnostic report.

[0177] The assay may, therefore, be in the form of a kit or computer-based system which comprises the reagents necessary to form and detect the herein described antibody complexes and the computer hardware and/or software including an algorithm to facilitate determination and transmission of reports to a clinician.

[0178] The present invention contemplates a method of allowing a user to determine the status of a subject with respect to TB, the method including: [0179] (a) receiving data from the conduct of the process as herein described from the user via a communications network;

[0180] (b) processing the subject data via multivariate analysis to provide a diagnostic index value;

[0181] (c) determining the status of the subject in accordance with the index value in comparison with predetermined values; and

[0182] (d) transferring an indication of the status of the subject to the user via the communications network.

[0183] Conveniently, the method generally further includes:

[0184] (a) having the user determine the data using a remote end station; and

[0185] (b) transferring the data from the end station to the base station via the communications network.

[0186] As used herein, the term "binds specifically," and the like when referring to an antigen-binding molecule refers to a binding reaction which is determinative of the presence of an antigen in the presence of a heterogeneous population of proteins and other biologies. Thus, under designated immunoassay conditions, the specified antigen-binding molecules bind to a particular antigen and do not bind in a significant amount to other proteins or antigens present in the sample. Specific binding to an antigen under such conditions may require an antigen-binding molecule that is selected for its specificity for a particular antigen. For example, antigen-binding molecules can be raised to a selected protein antigen, which bind to that antigen but not to other proteins present in a sample. A variety of immunoassay formats may be used to select antigen-binding molecules specifically immuno-interactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies specifically immuno- interactive with a protein. See Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York, for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity. [0187] For example, specific recognition is provided by a primary antibody (polyclonal or monoclonal) and a secondary detection system is used to detect presence (or binding) of the primary antibody. Detectable labels can be conjugated to the secondary antibody, such as a fluorescent label, a radiolabel, or an enzyme (e.g., alkaline phosphatase, horseradish peroxidase) which produces a quantifiable, e.g., colored, product. In another suitable method, the primary antibody itself can be detectably labeled. For example, a protein-specific monoclonal antibody, can be used both as an immunoadsorbent and as an enzyme-labeled probe to detect and quantify complexes formed in the present process or kit.

[0188] The amount of such protein present in a sample can be calculated by reference to the amount present in a standard or reference preparation using a linear regression computer algorithm (see Lacobilli et ai, (1988) Breast Cancer Research and Treatment 11 :19-30). In other embodiments, two different monoclonal antibodies to the protein of interest can be employed, one as the immunoadsorbent and the other as an enzyme-labeled probe.

[0189] Assays illustrated in the Examples are done in ELISA format with a single antigen per well, per single antibody form or class or isotype. However there are well known methods where they could be combined into a single assay for example using Luminex beads or similar where multiple individual antigens are coated on beads having different intensity of fluorescent label that can be discriminated in an instrument, and the amount of antibody binding to antigen on each bead can be separately measured from the single sample. Similarly the Luminex beads can be coated with antibody or other reagents to capture the individual antibody forms or isotypes from a sample, and then labelled antigen (or antigens) is added and the different isotype reactivities are assessed. The same can be done in micro-arrays or other arrays. Having established useful parameters in ELISA, it is then routine to transfer these findings to multiplex formats. In lateral flow and other point of care devices, where the sample flows across a membrane, it is easy to have the separate antigens present on the membrane as separate stripes or spots, and then detect the antibodies of one or more isotypes together; or else have different capture antibodies for the antibody isotopes, and then detect the (labelled) antigen binding to each of the immobilized antibody stripes or spots. The latter method (isotype capture, detection of labelled antigen bound by the immobilised patient antibody) is a most preferred approach.

[0190] Additionally, recent developments in the field of protein capture arrays permit the simultaneous detection and/or quantification of a large number of proteins. For example, low-density protein arrays on filter membranes, such as the universal protein array system (Ge (2000) Nucleic Acids Res. 28(2):e3) allow imaging of arrayed antigens using standard ELISA techniques and a scanning charge-coupled device (CCD) detector. Immuno-sensor arrays have also been developed that enable the simultaneous detection of clinical analytes. It is now possible using protein arrays, to profile protein expression in bodily fluids, such as in sera of healthy or diseased subjects, as well as in subjects pre- and post-drug treatment.

(01911 Protein capture arrays typically comprise a plurality of protein-capture agents each of which defines a spatially distinct feature of the array. The protein-capture agent can be any molecule or complex of molecules which has the ability to bind a protein and immobilize it to the site of the protein-capture agent on the array. The protein-capture agent may be a protein whose natural function in a cell is to specifically bind another protein, such as an antibody or a receptor. Alternatively, the protein-capture agent may instead be a partially or wholly synthetic or recombinant protein which specifically binds a protein. Alternatively, the protein-capture agent may be a protein which has been selected in vitro from a mutagenized, randomized, or completely random and synthetic library by its binding affinity to a specific protein or peptide target. The selection method used may optionally have been a display method such as ribosome display or phage display, as known in the art. Alternatively, the protein-capture agent obtained via in vitro selection may be a DNA or RNA aptamer which specifically binds a protein target (see, e.g. , Potyrailo et al., (1998) Anal. Chem. 70:3419-3425; Cohen et al. (1998) Proc. Natl. Acad. Sci. USA 95:14272-14277; Fukuda, et al. (1997) Nucleic Acids Symp. Ser. 37:237-238; available from SomaLogic). For example, aptamers are selected from libraries of oligonucleotides by the Selex™ process and their interaction with protein can be enhanced by covalent attachment, through incorporation of brominated deoxyuridine and UV- activated crosslinking (photoaptamers). Aptamers have the advantages of ease of production by automated oligonucleotide synthesis and the stability and robustness of DNA; universal fluorescent protein stains can be used to detect binding. Alternatively, the in vitro selected protein-capture agent may be a polypeptide (e.g., an antigen) (see, e.g., Roberts and Szostak (1997) Proc. Natl. Acad. Sci. USA 94:12297-12302).

[0192] An alternative to an array of capture molecules is one made through 'molecular imprinting' technology, in which peptides (e.g., from the C-terminal regions of proteins) are used as templates to generate structurally complementary, sequence-specific cavities in a polymerisable matrix; the cavities can then specifically capture (denatured) proteins which have the appropriate primary amino acid sequence (e.g., available from ProteinPrint™ and Aspira Biosy stems).

[0193] Exemplary protein capture arrays include arrays comprising spatially addressed TB antigens or antibody binding agents, which can facilitate extensive parallel analysis of numerous antigens and antibodies. Such arrays have been shown to have the required properties of specificity and acceptable background, and some are available commercially (e.g., BD Biosciences, Clontech, BioRad and Sigma). Various methods for the preparation of arrays have been reported (see, e.g., Lopez et al. (2003) J. Chromatogr. B 787:19-27; Cahill (2000) Trends in Biotechnology 7:47-51 ; U.S. Pat. App. Pub. 2002/0055186; U.S. Pat. App. Pub. 2003/0003599; PCT publication WO 03/062444; PCT publication WO 03/077851 ; PCT publication WO 02/59601 ; PCT publication WO 02/39120; PCT publication WO 01/79849; PCT publication WO 99/39210).

[0194] Immunoglobulin antigen-binding molecules are made either by conventional immunization (e.g., polyclonal sera and hybridomas), or as recombinant fragments, usually expressed in E. coli, after selection from phage display or ribosome display libraries (e.g., available from Cambridge Antibody Technology, Biolnvent, Affitech and Biosite). Alternatively, 'combibodies' comprising non-covalent associations of VH and VL domains, can be produced in a matrix format created from combinations of diabody- producing bacterial clones (e.g., available from Domantis). Exemplary antigen-binding molecules for use as protein-capture agents include monoclonal antibodies, polyclonal antibodies, Fv, Fab, Fab' and F(ab')₂ immunoglobulin fragments, synthetic stabilized Fv fragments, e.g., single chain Fv fragments (scFv), disulfide stabilized Fv fragments (dsFv), single variable region domains (dAbs) minibodies, combibodies and multivalent antibodies such as diabodies and multi-scFv, single domains from camelids or engineered human equivalents.

{0195] Individual spatially distinct protein-capture agents are typically attached to a support surface, which is generally planar or contoured. Common physical supports include glass slides, silicon, microwells, nitrocellulose or PVDF membranes, and magnetic and other microbeads.

[0196] While microdrops of protein delivered onto planar surfaces are widely used, related alternative architectures include CD centrifugation devices based on developments in microfluidics (e.g., available from Gyros) and specialized chip designs, such as engineered microchannels in a plate (e.g. , The Living Chip™, available from Biotrove) and tiny 3D posts on a silicon surface (e.g. , available from Zyomyx).

[0197] Particles in suspension can also be used as the basis of arrays, providing they are coded for identification; systems include color coding for microbeads (e.g., available from Luminex, Bio-Rad and Nanomics Biosystems) and semiconductor nanocrystals (e.g. , QDots™, available from Quantum Dots), and barcoding for beads (UltraPlex™, available from Smartbeads) and multimetal microrods (Nanobarcodes™ particles, available from Surromed). Beads can also be assembled into planar arrays on semiconductor chips (e.g., available from LEAPS technology and BioArray Solutions). Where particles are used, individual protein-capture agents are typically attached to an individual particle to provide the spatial definition or separation of the array. The particles may then be assayed separately, but in parallel, in a compartmentalized way, for example in the wells of a microtiter plate or in separate test tubes.

[0198] In operation, a protein sample (see, e.g., U.S. Pat. App. Pub. 2002/0055186), is delivered to a protein-capture array under conditions suitable for protein or peptide binding, and the array is washed to remove unbound or non-specifically bound components of the sample from the array. Next, the presence or amount of protein or peptide bound to each feature of the array is detected using a suitable detection system. The amount of protein bound to a feature of the array may be determined relative to the amount of a second protein bound to a second feature of the array. In certain embodiments, the amount of the second or subsequent protein in the sample is already known or known to be invariant.

[0199] In an illustrative example, fluorescence labeling can be used for detecting protein bound to the array. The same instrumentation as used for reading DNA microarrays is applicable to protein-capture arrays. For differential display, capture arrays (e.g. antibody arrays) can be probed with fluorescently labeled proteins from or are labeled with different fluorophores (e.g., Cy-3 and Cy-5) and mixed, such that the color acts as a readout for changes in target abundance. Fluorescent readout sensitivity can be amplified 10-100 fold by tyramide signal amplification (TSA) (e.g., available from PerkinElmer Lifesciences). Planar waveguide technology (e.g., available from Zeptosens) enables ultrasensitive fluorescence detection, with the additional advantage of no washing procedures. High sensitivity can also be achieved with suspension beads and particles, using phycoerythrin as label (e.g., available from Luminex) or the properties of semiconductor nanocrystals (e.g., available from Quantum Dot). Fluorescence resonance energy transfer has been adapted to detect binding of unlabelled ligands, which may be useful on arrays (e.g., available from Affibody). Several alternative readouts have been developed, including adaptations of surface plasmon resonance (e.g., available from HTS Biosystems and Intrinsic Bioprobes), rolling circle DNA amplification (e.g., available from Molecular Staging), mass spectrometry (e.g., available from Sense Proteomic, Ciphergen, Intrinsic and Bioprobes), resonance light scattering (e.g., available from Genicon Sciences) and atomic force microscopy (e.g., available from BioForce Laboratories). A microfluidics system for automated sample incubation with arrays on glass slides and washing has been co-developed by NextGen and Perkin Elmer Life Sciences.

[0200] In certain embodiments, the techniques used for detection of dlgA or other preselected products will include internal or external standards to permit quantitative or semiquantitative determination of those products, to thereby enable a valid comparison of the level or functional activity of these expression products in a biological sample with the corresponding expression products in a reference sample or samples. Such standards can be determined by the skilled practitioner using standard protocols. In specific examples, absolute values for the level or functional activity of individual expression products are determined. Controls may include - individual and population control and samples from diagnostic tests - an earlier time point.

[0201] In specific embodiments, the diagnostic method is implemented using a system as disclosed, for example, in International Publication No. WO 02/090579 and in copending PCT Application No. PCT/AU03/01517 filed November 14, 2003, comprising at least one end station coupled to a base station. The base station is typically coupled to one or more databases comprising predetermined data from a number of individuals representing the level TB antigen specific antibodies and their isotype structure (dimeric/polymeric) or subclass, when the predetermined data was collected. In operation, the base station is adapted to receive from the end station, typically via a communications network, subject data representing a measured or normalized level of at least one antibody type in a biological sample obtained from a test subject and to compare the subject data to the predetermined data stored in the database(s). Comparing the subject and predetermined data allows the base station to determine the status of the subject in accordance with the results of the comparison. Thus, the base station attempts to identify individuals having similar parameter values to the test subject and once the status has been determined on the basis of that identification, the base station provides an indication of the diagnosis to the end station. In an embodiment, recombinant plgR is sub-licensed for use in TB antigen screening or TB serological diagnosis.

[0202] Each embodiment in this specification is to be applied mutatis mutandis to every other embodiment unless expressly stated otherwise.

[0203] Functionally equivalent methods and kits employing such methods are clearly within the scope of the invention as described herein.

[0204] The present invention is further described by the following non-limiting Examples. EXAMPLE 1

(0205] The invention is illustrated using ELISA assays to test antibody-capture capabilities of various reagents and antigens.

[0206] In one embodiment, an ELISA is performed comparing dlgA, IgA and IgG reactivities against MTb antigen, SDS-soluble cell wall protein (SCWP), BEI Resources, NIH. As shown in Figure 10, (A) Antigen-specific dlgA is readily detected in 5/60 patients with confirmed pulmonary TB and 0/50 matched controls, using serum samples from Vietnam (Foundation for Innovative New Diagnostics, FIND). (B). IgA is also readily detected in patients, but is present in a significant number of controls. (C) IgG is readily detected but is also present in a large proportion of the controls, with a large degree of overlap between the two patient populations. Although the IgA and IgG responses show clear statistical differences between the TB and non-TB patient populations (Mann- Whitney test), the high degree of overlap between the populations and the high standard deviation among the non-TB population mean that only the most highly reactive TB patients can be classified on the basis of their IgA or IgG reactivity. In contrast a smaller number of TB patients show any dlgA reactivity against this antigen, but the extremely low background reactivity among control non-TB patients means that the assay cutoff (mean plus 2 standard deviations) is a low OD, and the reactive samples are generally well separated from the assay cutoff.

J0207] In one embodiment, an ELISA is performed comparing dlgA and IgG reactivities against MTb antigen, lipoarabinomannan (LAM), BEI Resources, NIH. As shown in Figure 1 1, (A) Antigen-specific dlgA is readily detected in 11/60 patients with confirmed pulmonary TB but also in 2/50 matched controls, using serum samples from Vietnam (FIND). (B). IgG is readily detected but is also present in a large proportion of the controls, with a large degree of overlap between the two patient populations. Although the IgG responses show clear statistical differences between the TB and non-TB patient populations (Mann- Whitney test), the high degree of overlap between the populations and the high standard deviation among the non-TB population mean that only the most highly reactive TB patients can be classified on the basis of their IgG reactivity. In contrast a smaller number of TB patients show any dlgA reactivity against this antigen, but the low background reactivity and standard deviation among control non-TB patients means that the assay cutoff (mean plus 2 standard deviations) is a low OD, and the reactive samples are generally well separated from the assay cutoff.

[0208] In one embodiment, an ELISA is performed comparing dlgA and IgA reactivities against MTb antigen, culture filtrate protein (CFP), BEI Resources, NIH. As shown in Figure 12, (A) Antigen-specific dlgA is readily detected in 12/60 patients with confirmed pulmonary TB but also in 3/50 matched controls, using serum samples from Vietnam (FIND). (B). IgA is readily detected but is also present in a large proportion of the controls, with a large degree of overlap between the two patient populations. Although the IgA responses show clear statistical differences between the TB and non-TB patient populations (Mann- Whitney test), the high degree of overlap between the populations and the high standard deviation among the non-TB population mean that only the most highly reactive TB patients can be classified on the basis of their IgA reactivity. In contrast a smaller number of TB patients show any dlgA reactivity against this antigen, but the low background reactivity and standard deviation among control non-TB patients means that the assay cutoff (mean plus 2 standard deviations) is a low OD, and the reactive samples are generally well separated from the assay cutoff.

[0209] In one embodiment, an ELISA is performed comparing dlgA and IgA reactivities against MTb antigen, Anda A60 (commercial IgA ELISA kit plate), Anda Biologicals, France. As shown in Figure 13, (A) Antigen-specific dlgA is readily detected in 14/60 patients with confirmed pulmonary TB but also in 1/50 matched controls, using serum samples from Vietnam (FIND). (B). IgA is readily detected but is also present in a large proportion of the controls, with a significant degree of overlap between the two patient populations. Although the IgA responses show clear statistical differences between the TB and non-TB patient populations (Mann- Whitney test), the high degree of overlap between the populations and the high standard deviation among the non-TB population mean that only the most highly reactive TB patients can be classified on the basis of their IgA reactivity. In contrast a smaller number of TB patients show any dlgA reactivity against this antigen, but the low background reactivity and standard deviation among control non-TB patients means that the assay cutoff (mean plus 2 standard deviations) is a low OD, and the reactive samples are generally well separated from the assay cutoff.

[0210] In one embodiment, a correlation between IgA and dIgA reactivity is assessed using commercial Anda A60 antigen-coated plates. As shown in Figure 14, a proportion of the IgA reactive and dIgA reactive samples (with cutoff set at mean plus 2 standard deviations of control non-TB patients) show some correlation, and the 9 samples that are reactive for both IgA and dIgA demonstrate that the dIgA and IgA reactivity combined can provide confirmation of TB infection in this population. However a number of IgA reactive samples show no dIgA reactivity, and a number of dIgA reactive samples show no IgA reactivity, demonstrating that the dIgA and IgA responses are at least partly independent, and highlighting the additional value of using dIgA rather than only IgA for TB diagnosis.

[0211] In one embodiment, the antibody response to MTb antigens is shown to be heterogeneous among both TB patients and control non-TB patients. As shown in Figure 15, each colour column shows different antigen-isotypes: yellow dIgA, green IgA, and blue IgG. The shading of cells represents the strength of reactivity, as shown on the left, being greater than the mean plus either 2, 3 or 6 SD of the non-TB patient population. The rows represent individual patient samples. Some patients are very reactive to many antigens across multiple antibody types, while others show unique reactivity with only one or two antigens/isotypes. Strikingly, it can be seen that most of the dIgA reactive samples among TB patients are positive at the highest cutoff (mean + 6 SD), with a smaller proportion of IgA reactive samples positive at this cutoff, and the lowest proportion of IgG reactive samples positive at this cutoff.

[0212] As demonstrated herein, Figure 16 shows the very high reactivity of dIgA reactive samples which allows the use of the very high cutoff of mean plus 6 SD of control non-TB patients. Across the limited number of antigens tested, this provides a sensitivity of 27% and specificity of 98% for dIgA detection, but only 8% sensitivity for IgG detection and 15% sensitivity for IgA detection, with 98% and 100% specificity respectively. This demonstrates that dlgA is a useful component of serological testing strategies for diagnosis of active TB.

[0213] The heterogeneity of the antibody response to multiple MTb antigens has been observed previously in studies focused on the IgG response to multiple TB protein antigens. As illustrated in Figure 17, the heat map reproduced from Kunnath-Velayudhan et al, PNAS 107: 14703-14708, 2010 shows that when using the 13 antigens that were found to be most highly reactive out of the entire MTb proteome (rows), IgG responses between patients (columns) were very heterogeneous and it is necessary to combine a large number of antigens to achieve acceptable sensitivity using IgG antibody responses. The relative reactivity of antigens with dlgA or IgA rather than IgG has not been evaluated.

[0214] Figure 18 illustrates a correlation between IgA, dlgA and IgG reactivity on a subset of MTb antigens (pure antigens LAM, PstS l , LM, TDM, mAGP, SL1 , PIM6 and crude antigens or mixtures, CWF, CYT, CFP, AndaA60 and Omega commercial kits). dlgA reactivity is shown to have some correlation with IgA reactivity, and IgA to have some correlation with IgG reactivity, but dlgA shows no significant correlation with IgG reactivity. Most attempts at screening different MTb antigens for their potential use as diagnostic reagents has focused on IgG or to a lesser extent IgA. The independence of dlgA reactivity versus IgG (and to a lesser extent, IgA) highlights the utility of dlgA as an independent diagnostic marker for MTb infection.

[0215] Figure 19 illustrates results of screening 5 selected antigens for reactivity of dlgA, IgA or IgG among FIND patient samples, using a cutoff that gives 100% specificity among FIND non-TB control samples. Only the TB patient reactivities are shown because of this selected cutoff rendering all non-TB patients non-reactive. Using the antigens LAM, CWF, CFP, CYT, PstSl (all BEI Resources), IgA and IgG reactivity combined are able to detect 24/60 samples (40%) with 100% specificity. The inclusion of dlgA detects a further 7 samples that are uniquely reactive for dlgA (marked with red highlights; 3 of the samples reactive for dlgA against more than one antigen and 1 sample reactive against all five antigens), for a total of 31/60 (51.7%) sensitivity or a 29% increase over the 40% sensitivity seen with IgA and IgG alone. These results demonstrate that inclusion of dlgA detection using the R HpIgR reagent improves the sensitivity of antibody-based tests for active TB infection.

[0216] Combination screening (see Figure 20) provides very informative results using an expanded panel of MTb antigens for dIgA, IgA and IgG identifies unique reactivities, based on a cutoff of the mean plus 2 standard deviations of the non-TB sample population. Using 1 1 different antigens, 55/60 of the patients demonstrate reactivity for one or more of dIgA, IgA or IgG, with similar numbers being uniquely reactive only for dIgA (8), IgA (6) or IgG (7). Consistent with the results shown in Figure 15, this suggests that dIgA reactivity contributes as much as IgG or IgA to the sensitivity of improved serological tests for MTb-specific antigens in diagnosis of active TB. An optimal combination of assays from this set of 1 1 antigens would use dIgA with Anda A60, IgA with Anda A60, IgA with SCWP, IgG with 38kd-LPS (Omega) and IgG with SCWP, with a total 50/60 TB patients detected (82% sensitivity) and 6/50 non-TB patients (88% specificity). Notably, it is anticipated that the screening of additional MTb antigens will increase the sensitivity of a serological diagnostic algorithm when using dIgA alone or in combination with IgA and/or IgG, while the specificity of the assays may be improved by inclusion of a "rule-out" test in the diagnostic algorithm, such as C-reactive protein (CRP). CRP is always elevated in patients with active TB, and the absence of elevated CRP can be used to rule out active TB in at least that proportion of non-TB patients who do not have elevated CRP for other medical reasons.

[0217] The disclosure of every patent, patent application, and publication cited herein is hereby incorporated herein by reference in its entirety.

[0218] Many modifications will be apparent to those skilled in the art without departing from the scope of the present invention. TABLE 1

Summary of sequence identifiers

SEQUENCE ID NO: DESCRIPTION

1 Nucleotide sequence of human pIgR

2 Amino acid sequence of human pIgR

3 Nucleotide sequence of rabbit pIgR

4 Amino acid sequence of rabbit pIgR

5 Nucleotide sequence of chimeric human/rabbit pIgR

6 Amino acid sequence of chimeric human/rabbit pIgR

7 Nucleotide sequence of N-terminal rabbit domain 1 chimeric human/rabbit pIgR

8 Amino acid sequence of N-terminal rabbit domain 1 chimeric human/rabbit pIgR

9 Nucleotide sequence of C-terminal human domains 2-4 of chimeric human/rabbit pIgR

10 Amino acid sequence of C-terminal human domains 2-4 of chimeric human/rabbit pIgR

1 1 Nucleotide sequence of human pIgR with cytoplasmic domain of CD4

12 Amino acid sequence of human pIgR with cytoplasmic domain of CD4

13 Nucleotide sequence of rabbit pIgR cytoplasmic domain with cytoplasmic domain of CD4

14 Amino acid sequence of rabbit pIgR with cytoplasmic domain of CD4

15 Nucleotide sequence of chimeric human/rabbit pIgR with cytoplasmic domain of CD4

16 Amino acid sequence of chimeric human/rabbit pIgR with cytoplasmic domain of CD4

17 Nucleotide sequence of N-terminal rabbit domain 1 of chimeric human/rabbit pIgR with cytoplasmic domain of CD4

18 Amino acid sequence N-terminal rabbit domain 1 of chimeric human rabbit pIgR with cytoplasmic domain of CD4

19 Nucleotide sequence of C-terminal human domains 2-4 of chimeric human/rabbit pIgR with cytoplasmic domain of CD4

20 Amino acid sequence of C-terminal human domains 2-4 of chimeric human/rabbit pIgR with cytoplasmic domain of CD4 TABLE 2

Amino acid sub-classification

TABLE 3

Exemplary and Preferred Amino Acid Substitutions

Original Residue Exemplary Substitutions Preferred Substitutions

Ala Val, Leu, He Val

Arg Lys, Gin, Asn Lys

Asn Gin, His, Lys, Arg Gin

Asp Glu Glu

Cys Ser Ser

Gin Asn, His, Lys, Asn

Glu Asp, Lys Asp

Gly Pro Pro

His Asn, Gin, Lys, Arg Arg lie Leu, Val, Met, Ala, Phe, Norleu Leu

Leu Norleu, He, Val, Met, Ala, Phe He

Lys Arg, Gin, Asn Arg

Met Leu, He, Phe Leu

Phe Leu, Val, He, Ala Leu

Pro Gly Gly

Ser Thr Thr

Thr Ser Ser

Trp Tyr Tyr

Tyr Trp, Phe, Thr, Ser Phe

Val lie, Leu, Met, Phe, Ala, Norleu Leu BIBLIOGRAPHY

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Claims

1. A diagnostic process for detecting the TB status of a test subject, the process comprising obtaining a biological sample comprising antibodies from the subject and contacting same with a dlgA binding agent, to allow formation of a test complex between

TB antigen specific antibody and antibody binding agent, and measuring the level of the test complex and comparing the level of the test complex formed to a corresponding level formed in a control sample.

2. A diagnostic process for detecting the TB status of a test subject, the process comprising contacting a biological sample comprising antibodies from the subject with two or three specific antibody binding agents selected from IgA, IgG and dlgA-binding agents, to allow formation of test complexes between TB antigen specific antibody and antibody binding agent, and measuring the level of each test complex and comparing the level of the test complexes formed to corresponding levels formed in control samples, wherein the levels of at least two test complexes are substantially independent and contribute to enhancing the diagnostic power of the process.

3. The process of claim 2 wherein the at least two antibody binding agents are IgA and IgG-binding agents or IgA and dlgA binding agents or IgG and dlgA binding agents.

4. The process of claim 3 wherein the at least two antibody binding agents are IgG and dlgA binding agents.

5. The process of claim 2 wherein the subject antibody binding agents are IgA, IgG and dlgA-binding agents.

6. The process of any one of claims 1 to 5 wherein the TB status is active TB.

7. The process of claim 6 wherein the sensitivity of the process is at least 70%, or at least 80% or at least 88%.

8. The process of any one of claims 1 to 7 wherein the biological sample is a blood or serum sample.

9. The process of any one of claims 1 to 8 wherein one or more TB antigens is/are directly or indirectly bound to a solid surface and capture/s TB antigen specific antibody from the biological sample applied to the surface to form one or more immobilised TB antigen-TB antigen specific antibody complex/es.

10. The process of any one of claims 1 to 8 wherein the specific dlgA-binding agent is directly or indirectly bound to a solid surface, and the TB antigen specific dlgA-dlgA binding agent complex is probed with TB antigen.

1 1. The process of any one of claims 1 to 8 wherein the IgA and/or IgG-binding agent is directly or indirectly bound to a solid surface, and the TB antigen specific antibody-and IgA and/or IgG binding agent complex is probed with TB antigen.

12. The process of any one of claims 1 to 11 wherein the TB antigen is selected from one, two or three of the group comprising SCWP, LAM, CWF, CFP, CYT, PstSl, Anda A60, Omega, TDM, mAGP, PIM6, LM, 38KD-LPS and SL1.

13. The process of claim 12 wherein the TB antigen is one, two or three of Anda A60, SCWP and 38KD-LPS.

14. The process of any one of claims 1 to 13 wherein the dlgA-binding agent is plgR or a dlgA-binding variant thereof.

15. The process of any one of claims 1 to 14 wherein the following antigen-antibody combinations are included: two or three or four or five of Anda A60-dIgA, Anda A60-IgA, Anda A60, SCWP-IgA, 38KD-LPS-IgG and SCWP-IgG.

16. The process of any one of claims 1 to 15 which further employs an anti-CRP binding agent to assess the level of C-Reactive protein in the biological sample.

17. The process of any one of claims 1 to 16 which further employs an anti- procalcitonin binding agent to assess the level of procalcitonin in order to detect or rule out pneumonia.

18. The process of any one of claims 1 to 15 wherein active TB is diagnosed where IgA or IgG are positive at 2 standard deviations cut off or two out of three from IgA or IgG at 1 standard deviation cut off and CRP is more than 39 μg/ml and not CRP less than 6 μg/ml.

19. The process of any one of claims 1 to 18 which is an immunoassay, ELISA, or chromatographic process.

20. The process of any one of claims 1 to 19 comprising diagnosis and treatment, wherein the treatment comprises administering treatment to the subject if they are diagnosed with active TB.

21. A method of treating active TB in a subject, the method comprising requesting a test for diagnosis of active TB of any one of claims 1 to 20 and administering treatment to the diagnosed subject if the test is positive for active TB.

22. A kit for diagnosing active TB in a subject, comprising: i) an immunochromatographic device comprising a porous membrane operably connected to a sample portion, a test portion, and optionally a control portion; and further comprising a sucker portion, portion comprising a dlgA-binding agent, a portion comprising a TB antigen and optionally a conjugate portion; and (ii) instructions for using the immunochromatographic device to detect the presence of antigen specific dig A antibody.

23. A kit for diagnosing active TB in a subject, comprising: i) an immunochromatographic device comprising a porous membrane operably connected to a sample portion, a test portion, and optionally a control portion; and further comprising a sucker portion, portion comprising a portion or portions comprising two or three specific antibody binding agents selected from IgA, IgG and dlgA-binding agents, a portion or portions comprising two or three specific antibody binding agents selected from IgA, IgG and dlgA-binding agents, and optionally a conjugate portion; and (ii) instructions for using the immunochromatographic device to detect the presence of two or three of antigen specific IgA, IgG and dlgA in the sample.

24. The kit of claim 22 or 23 wherein the dlgA binding agent is a recombinant plgR or a dlgA-binding variant thereof.

25. The kit of claim 22 or 23, wherein the dlgA binding agent is an antibody or antigen binding fragment thereof or anti-J chain antibody.

26. The kit of any one of claims 22 to 25 wherein two or three or four or five of the Anda A60-dIgA, Anda A60-IgG, Anda A60-IgA, SCWP-IgA, 38kD-LPS-IgG, SCWP-IgG are assessed.

27. Use of a dlgA-binding agent in a screening method to identify dlgA-reactive TB antigens.

28. A dlgA-binding agent for use in diagnosing active TB in a subject.

29. The use of claim 27 or the agent of claim 28 wherein the dlgA binding agent is plgR or a dlgA-binding variant thereof.

30. The use or agent of claim 29 wherein the dlgA-binding agent is R/HpIgA.