Ip-10 based immunological monitoring   

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of and claims the benefit of priority to U.S. patent application Ser. No. 12/438,515, filed Oct. 14, 2009, which is a National Phase application of and claims the benefit of priority to International Application No. PCT/DK2007/000399, filed on Sep. 5, 2007, designating the United States of America and published in the English language, which is an International Application of and claims the benefit of priority to Danish Patent Application No. PA200601145, filed Sep. 5, 2006, and Danish Patent Application No. PA200700262, filed Feb. 20, 2007. The disclosures of the above-referenced applications are hereby expressly incorporated by reference in their entireties.

The present invention relates generally to an immunological assay and, more particularly, an assay for measuring cell-mediated immune reactivity (CMI). Even more particularly, the present invention provides an assay and a kit for measuring a cell-mediated response to an antigen using whole blood or other suitable biological samples. The assay is useful in therapeutic and diagnostic protocols for human, livestock and veterinary and wild life applications.

Measurement of cell-mediated immune responses is important for immune diagnosis of many infectious and autoimmune diseases, as a marker for immunocompetence, and for detection of T-cell responses to endogenous and exogenous antigens (i.e. infections and vaccines).

The present invention provides a method for measuring CMI in a mammal by incubating a sample from the mammal which comprises T-cells or other cells of the immune system with an antigen. Production of IP-10 is then detected. The presence or level of immune effecter is then indicative of the level of cell mediated responsiveness of the subject.

BACKGROUND OF THE INVENTION

The discovery of mycobacterium tuberculosis (MTB)-specific immunodominant antigens has led to a significant new avenue for the diagnosis of tuberculosis (TB). Early work had shown the potential to replace the Tuberculin Skin Test (TST) by a test that assayed the in vitro production of interferon gamma (IFN-γ) by T cells in response to defined MTB antigens. Around the same time, a major advance was the discovery of the highly immunogenic antigens, early secreted antigenic target 6 (ESAT-6) and culture filtrate protein 10 (CFP-10) and TB7.7 that improved specificity significantly. These antigens are encoded within the region of difference 1 (RD1) of the pathogen and are consequently absent from all Bacille Calmette Guerin (BCG) vaccine strains and most non-tuberculous mycobacteria (exceptions include Mycobacterium kansasii, Mycobacterium marinum Mycobacterium szulgai). IFN-γ responses to overlapping peptides of the RD1 encoded antigens ESAT-6, CFP-10, TB7.7 form the basis for the detection of MTB infection in two licensed and commercially available tests.

QuantiFERON-TB Gold (Cellestis Limited, Carnegie, Victoria, Australia), a whole blood enzyme-linked immunoassay (ELISA) has European CE mark approval and recently received American Food and Drug Administration (FDA) approval for the detection of both latent TB infection and disease.

T-SPOT.TB (Oxford Immunotec, Oxford, UK), an enzyme-linked immunospot assay (ELISPOT) that uses peripheral blood mononuclear cells has European CE mark approval and was approved for use in Canada in 2005. T-SPOT.TB only uses ESAT-6 and CFP10.

However, the limitations of the currently available tests are: 1) The sensitivity may be impaired in immunosuppressed individuals (such as HIV positive or patients receiving immunosupressing medication), 2) In some situations a relatively large volume of blood is necessary (3 ml per QuantiFERON test and 8 ml for the T-SPOT.TB), which may limit its use in infants and severely ill and anaemic children, 3) The tests do not discriminate between active, latent and recent infection 4) The tests have not been demonstrated to be able to predict who will progress from recent or latent TB to active TB.

Most of the test limitations are due to measurement of the effect parameter IFN-γ at very low levels, close to the limit of even the most sensitive method (in the QuantiFERON test down to 0.35 IU/ml (17.5 pg/ml) and in the T-SPOT.TB 5 spots/field). Decreasing cut-off to enhance sensitivity will eventually result in impaired specificity of the tests. A recent publication based on the QuantiFERON test has shown that that repeated testing of people with test results in the lower range of IFN-γ varies around the cut-off level which underlines the potential risk of false positive and false negative results of the QuantiFERON (QFT) test (Pai, M. et al).

To overcome the evident fragility of the tests, sensitivity could be improved by using additional M. tuberculosis specific antigens and this has been done in the third generation of the QuantiFERON tests (QFT), the QuantiFERON In tube test (QFT-IT) which now comprises an additional antigen named TB7.7(p4) and potentially sensitivity is improved, but it still depends on measurements at very low IFN-γ levels.

This approach has been tried by others, i.e. recently is was shown that the Monokine Induced by IFN-γ (MIG/CXCL9) was specifically expressed in vitro after stimulation with M. tuberculosis specific antigens (ESAT-6/CFP10) and PPD. The sensitivity of CXCL9, however was very low and lower than that of IFN-γ. Another smaller study based on intracellular cytokine cytometry in CD4+ T cells following ESAT-6 stimulation, tested if expression of IFN-γ, IL-2, IL-4, IL-10 or the activation marker CD40L could distinguish TB from non-TB disease. None of these markers were found to be comparable or superior to IFN-γ (Abramo C, et al) (Hughes, A. et al).

Yet no sensitive and specific markers to replace IFN-γ for diagnosis of TB infection have been identified in the presently published literature. Various have disclosed IP-10 in connection to infections, but not as a marker in the diagnosis of infection with a prior antigen stimulation.

The diagnosis of genital chlamydial infections evolved rapidly from the 1990s. Nucleic acid amplification tests (NAAT), such as polymerase chain reaction (PCR), transcription mediated amplification (TMA), and the DNA strand displacement assay (SDA) now are the mainstays. The most commonly used and widely studied chlamydia NAATs in the US and many other industrialized countries are Aptima (Gen-Probe), Probe-Tec (Becton-Dickinson), and Amplicor (Roche). The Aptima Combo II assay tests simltaneously for C. trachomatis and Neisseria gonorrhoeae, the cause of gonorrhea. NAAT for chlamydia may be performed on swab specimens collected from the cervix (women) or urethra (men)

At present, the NAATs have regulatory approval only for testing urogenital specimens. The NAATs have largely replaced culture, the historic gold standard for chlamydia diagnosis, and the non-amplified probe tests, such as Pace II (Gen-Probe). The latter test is relatively insensitive, successfully detecting only 60-80% of infections in asymptomatic women, and occasionally giving falsely positive results. Culture remains useful in selected circumstances and is currently the only assay approved for testing non-genital specimens.

Chlamydia diagnosis is thus based on complicated and resource demanding technology such as PCR, not readily available in the developing world. A fast and easy technology would improve diagnostic measures for this important disease,

CA 2,478,138 discloses that elevated blood levels of the chemokine CXCL10 polypeptide are associated with respiratory illnesses (e.g. SARS, influenza and community-acquired pneumonia) and are useful in diagnosis of patients. Methods are provided for diagnosis and treatment of patients suffering from respiratory illnesses.

WO 05/091969 discloses markers for TSE (Transmissible Spongiform Encephalopaties) that are present prior to formation of detectable pathological prion protein are useful to detect this infection prior to clinical signs. IP-10 is just one of several markers disclose and this application does not disclose any antigen stimulation.

US2004-038201 discloses distinct gene expression programs activated in response to different pathogens in macrophages. IP-10 is again just one in many markers mentioned and this application does not disclose any antigen stimulation.

Annalisa Azzurri et al. discloses IFN-γ-inducible protein 10 and pentraxin 3 plasma levels are tools for monitoring inflammation and disease activity in Mycobacterium Tuberculosis infection. The article shows that IP-10 plasma level is spontaneously increased in patients with TB, again this reference does not disclose any antigen stimulation.

WO 03/063759 discloses a method of identifying a heat shock protein (Hsp) derived peptide useful for diagnosis or therapy. The effect of the compounds of this invention was tested on pheripheral blood monocytes by measuring the IP-10 level in response to subsequent stimulation with LPS. Direct stimulation with the test compounds without subsequent stimulation with LPS, did not give rise to an increase in the IP-10 level.

WO 07/039,400 discloses a method and kit for the diagnosis of Immune Restoration Syndrome which is associated with tuberculosis (TB-IRS) in patients infected with tuberculosis as well as HIV/TB co-infected patients. In order to diagnose TB-IRS the inventors detected the level of Th1 response against PPD and/or a 16 KDA protein in comparison to the Th1 response against ESAT-6, CFP-10, 85B (negative control) and used a rise in the Th1 response as an indicative of TB-IRS.

To overcome the problems of impaired sensitivity and low levels of detectable IFN-γ by the currently available tests using antigen stimulation, the present inventors suggest the use of an alternative biomarker than IFN-γ.

SUMMARY

The present invention proposes a novel diagnostic principle. A test system that can detect infection with e.g. tuberculosis eishmania or chlamydia, based on measuring the chemokine IP-10 following stimulation of immune cells with antigenic proteins/peptides.

The described test system IP-10 is more sensitive than tests based on IFN-γ as effect parameter, it improves testing and diagnosing. The test can be performed using lower amounts of blood because the samples can be diluted at time of incubation or before analysis. The test can be performed at shorter incubation times. Furthermore, the test system may allow discriminating between various stages of infection such as e.g. active, recent and latent TB infection.

In short the present invention can be described as an immunological method comprising the steps of incubating a sample obtained from a mammal with at least one antigen, determining the IP-10 level in said sample and comparing said determined IP-10 level with a reference-level, thereby determining whether mammal has previously encountered the antigen generating immunological reactivity to the antigen, or previously encountered other antigens generating immunological cross reactivity to the antigen.

DETAILED DESCRIPTION

The present invention provides an assay of the potential or capacity of a subject to mount a CMI response. The assay is based on measuring immune effecter molecule production by cells of the immune system in response to antigenic stimulation. The immune effecters may be detected using ligands such as antibodies specific for the effecters or by measuring the level of expression of genes encoding the effecters.

The present invention provides, therefore, means to determine the responsiveness of CMI in a subject and, in turn, provides means for the diagnosis of infectious diseases, pathological conditions, level of immunocompetence and a marker of T-cell responsiveness to endogenous or exogenous antigens

One aspect of the invention relates to an assay of the potential or capacity of a subject to mount an IP-10 response. The assay is based on measuring IP-10 production by cells of the immune system in response to antigenic stimulation. The IP-10 production may be detected using ligands such as antibodies specific for IP-10 or by measuring the level of expression of genes encoding IP-10. The present inventors have demonstrated the test principle using two different types of infection: Tuberculosis and Chlamydia. In the case of tuberculosis a test based on M. Tuberculosis specific stimulation and subsequent determination of IP-10, can identify persons infected with M. tuberculosis. In the case of Chlamydia a test based on stimulation with C.Trachomatis extract can identify persons infected with Chlamydia.

The described test system measures higher levels of the biomarker IP-10, than the levels of IFN-γ, the marker the currently available assays are based on. The test system based on IP-10 is as specific as and more sensitive than tests based on e.g. IFN-γ as effect parameter, it improves testing and diagnosing of immunocompromised individuals (Example 10), the test can be performed using lower amounts of blood because the samples can be diluted at time of incubation or before analysis (Examples 9 and 13). It also improves the speed of diagnosis, as IP-10 is produced in significant amounts after few hours of incubation as shown in example 11. In the case of tuberculosis, the test system may allow discriminating between active, recent and latent TB infection and, in addition the test system is potentially capable of identifying persons at risk of progressing to active TB. The test system is based on IP-10 detection using an immunoassay (i.e. ELISA or Luminex) and the test system can potentially be developed into a field friendly immunochromatographic test applicable in low resource settings, where the test result is presented by a colour reaction detectable to the naked eye.

The assay described in the present invention solves a series of problems. The currently available assays measure the effect parameter IFN-γ at very low levels, close to the limit of even the most sensitive detection method (in the case of tuberculosis tests, the QuantiFERON test has a cut-off level for positive test at 0.35 international units/ml (17.5 pg/ml) and in the T-SPOT.TB test 5 spot forming units/field). Decreasing cut-off to enhance sensitivity will eventually result in impaired specificity of the tests. Publications based on the Quantiferon test have shown that that repeated testing of people with test results in the lower range of IFN-γ varies around the cut-off level which underlines the potential risk of false positive and false negative results of the Quantiferon (QFT) test. In addition the current test may give false negative results in immunosuppressed individuals who are unable to mount an IFN-γ response above the cut-off level. As the amount of IP-10 release is much higher after antigen stimulation when compared to IFN-γ: the sensitivity is higher, fewer tests are deemed false negative, test results are more reproducible, and, less indeterminate test results are expected in immunosuppressed individuals.

Furthermore, because antigen induced IP-10 is secreted in such high concentrations it is possible to dilute the sample before or after the incubation step. This means that the amount of sample material (e.g. whole blood) needed to perform the test can be reduced e.g. in the case of whole blood down to or even at or below 0.25 ml, such as 0.20 ml, e.g. 0.15 ml, such as 0.1 ml, e.g 0.05 ml. In a preferred embodiment the test can be performed down to or even below 0.1 ml. Thus a “mini assay” suitable for patients with low blood volume (e.g. children/infants or anaemics) can be developed. Furthermore, using e.g. blood from e.g. a finger-prick, the mini assay can be made even more user friendly as vein puncture is avoided.

Furthermore, in the case of tuberculosis none of the currently available tests can discriminate between Active and Latent infection. Surprisingly the inventors found the concentration of IP-10 in the un-stimulated, but incubated sample material i.e. the nil sample (e.g. whole blood), is higher among patients with active disease compared to healthy individuals with latent disease. The present inventors propose that the IP-10 concentration in sample material incubated with an inactive solution (Nil) in combination with an antigen-specific test can be used as marker for active infection (e.g. tuberculosis) versus latent infection (e.g. tuberculosis).

Thus, one aspect of the present invention relates to an immunological method comprising the steps of a) incubating a sample obtained from a mammal with at least one test-antigen b) determining the IP-10 level in said sample c) comparing said determined IP-10 level with a reference-level, thereby determining whether mammal has previously encountered the test-antigen generating immunological reactivity to the test-antigen or previously encountered other antigens generating immunological cross reactivity to the test-antigen.

It is to be understood that any of the methods described in the present invention is platform independent. Accordingly, any immunological method such as but not limited to ELISA, Luminex, Multiplex, Immunoblotting, TRF-assays, immunochromatographic lateral flow assays, Enzyme Multiplied Immunoassay Techniques, RAST test, Radioimmunoassays, immunofluorescence and various immunological dry stick assays (e.g. chromatographic stick test) may be applicable to the present invention.

In a second aspect, the present invention relates to a method for diagnosing an infection, said method comprising the steps of a) incubating a sample obtained from a mammal with at least one test-antigen under the proviso that said test-antigen is not PPD or LPS b) determining the IP-10 level in said sample c) comparing the determined IP-10 level with a reference-level, thereby determining whether said mammal is infected with a micro organism, if the determined IP-10 level is above the reference-level.

The present inventors have demonstrated that direct stimulation with the selected test-antigen(s) or antigen(s) for evaluation is sufficient to obtain a readout that enables the skilled addressee to conclude whether the sample has encountered the test-antigen(s) generating immunological reactivity to the test-antigen(s) or previously encountered other test-antigens generating immunological cross reactivity to the chosen test-antigen(s).

Thus, in one embodiment, the present invention also relates to immunological methods as described herein, wherein any further or subsequent stimulation is excluded.

Subsequent or further stimulation may cover any type of stimulation e.g. priming or stimulating a sample with a biological inactive substance or a substance with a biological effect related to the inflammatory response, such as but not limited to mitogens, bacterial products or biological active proteins.

Thus, in one embodiment, the present invention relates to immunological methods as described herein, wherein any further or subsequent stimulation with LPS is excluded.

Accordingly, said method is applicable for the detection of an infection in e.g. people who is in high risk of developing infectious diseases such as but not limited to Tuberculosis, Chlamydia, Leishmania, Trypanosoma and Schistosoma.

The present invention also relates to a method according to the present invention, wherein the sample is divided into at least 2 fractions and a) incubating the first fraction of the sample with the test-antigen(s) to generate a response sample b) incubating the second fraction of the sample with an inactive solution to generate a nil sample c) determining the IP-10 level in the two fractions d) determining the antigen-dependent IP-10 response of the sample by subtracting the IP-10 level determined in the nil sample from the IP-10 determined in the response sample e) comparing the test-antigen-dependent IP-10 response or a value derived thereof with the reference-level or a value derived thereof, thereby determining whether mammal has previously encountered the test-antigen(s) and thus generate immunological reactivity to the test-antigen(s) or previously encountered other antigens generating immunological cross reactivity to the test-antigen(s) and/or is going to develop infection.

In one embodiment the assay performance can be potentiated by concomitant addition of an immunostimulatory molecule with the antigen selected for evaluation, said immunostimulatory molecule being a cytokine selected from the non-limiting group consisting of IL-2, IL-12, TNF-α, and IFN-γ.

In another embodiment the immunostimulatory molecule is a soluble receptor (e.g. di- or polymere of the B7 molecules (CD80/CD86)) or an antibody (e.g. an CD28-binding antibody).

In another embodiment the immunostimulatory molecule has the property of providing T cells a co-stimulatory signal (known to those skilled in the art as the signal 2), said co-stimulatory signal is unable to induce an IP-10 response alone but will increase the IP-10 response if the cells generate an CMI response to the antigen selected for evaluation.

In another embodiment potentiating the assay can be achieved by inhibiting anti-inflammatory processes occurring during the incubation step. In one embodiment the assay can be potentiated with inhibiting antibodies or soluble receptors that bind anti-inflammatory molecules such as but not limited to IL-4, IL-10 and TGF-β.

In another embodiment potentiating the assay can be achieved by inhibition of anti-inflammation mediated through inhibition or elimination of cell populations acting inhibitory to the CMI response such as regulatory T-cells.

Specifically as used herein the term Purified Protein Derivative (PPD or tuberculin) is a precipitate of non-species-specific molecules. PPD or tuberculin is obtained by extracting proteins from a mixture of M. tuberculosis or other mycobacteria such as M. avium. PPD is commonly employed in testing for the presence of cellular immunity or Th1 response generated either against BCG or against M. tuberculosis. For example, it can be obtained from the TubersolB of Connaught Laboratories Limited prepared from a large Master Batch, Connaught Tuberculin (CT68), or in the form of RT23 obtained from the Statens Serum Institute (SSI, Copenhagen Denmark).

The ESAT-6 protein (early secreted antigenic target 6) is a major secreted antigen which has been purified from M. tuberculosis short-term culture filtrates. As referred herein ESAT-6, CFP-10 (culture filtrate protein 10) and 85B can be obtained from cell lysate and purification, by recombinant techniques or produced as synthetic peptides. For example ESAT6 can be obtained as a recombinant protein from Statens Serum Institute.

Tuberculin or PPD (purified protein derivative) differs from ESAT-6, (early secreted antigenic target 6) CFP-10 (culture filtrate protein 10), and TB7.7 which are encoded by genes (in the RD-1 region) located only within the M. tuberculosis genome and are not contained in BCG (the Bacille of Calmette et Guérin). It differs from PPD because PPD is also contains other antigens that are shared with e.g. BCG sub-strains and with several non-tuberculous mycobacterial species with low or no pathogenicity.

Optionally, the method may further comprise dividing the sample into 3 fractions and incubating the third fraction of the sample with a T cell activator to generate a positive control. Here immune cells may be incubated in e.g. three separate populations: nil control (e.g. saline), Ag stimulated (e.g. Chlamydia or tuberculosis specific proteins or derivates hereof) and positive control (e.g. Phytohemagglutinin). Immune cells can be in the form of whole blood, diluted whole blood or various purifications of cell population like peripheral blood mononuclear cells, monocytes or T cells. The cells can be obtained from blood, urine, pleural fluid, bronchial fluid, oral washings, tissue biopsies, ascites, pus, cerebrospinal fluid, aspirate, and/or follicular fluid. Immune cells incubate for e.g. 4-24 h at 37° C.

In one embodiment, the sample is divided into at least 2 fractions and a) incubating the first fraction of the sample with the antigen to generate a response sample b) incubating the second fraction of the sample with an inactive solution to generate a nil sample c) determining the IP-10 level in the two fractions d) determining the antigen-dependent IP-10 response of the sample by subtracting the IP-10 level determined in the nil sample from the IP-10 determined in the response sample e) comparing the antigen-dependent IP-10 response or a value derived thereof with the reference-level or a value derived thereof, f) comparing the antigen spontaneous IP-10 response or a value derived thereof with the reference-level or a value derived thereof, thereby determining whether mammal has previously encountered the antigen and thus generate immunological reactivity to the antigen or previously encountered other antigens generating immunological cross reactivity to the antigen and thereby determining whether the mammal has an active, a recent, or a latent infection if mammal is responding to treatment, or is going to develop infection.

More specifically, the sample is divided into at least 3 fractions and a) incubating the first fraction of the sample with the antigen to generate a response sample b) incubating the second fraction of the sample with an inactive solution to generate a nil sample c) incubating the third fraction of the sample with a stimulatory solution (e.g. PHA) to generate a mitogen sample c) determining the IP-10 level in the three fractions d) determining the antigen-dependent IP-10 response of the sample by subtracting the IP-10 level determined in the nil sample from the IP-10 determined in the response sample e) comparing the antigen-dependent IP-10 response or a value derived thereof with the reference-level or a value derived thereof, f) determining the mitogen dependent IP-10 response of the sample by subtracting the IP-10 level determined in the nil sample from the IP-10 determined in the mitogen sample g) comparing the mitogen dependent IP-10 response or a value derived thereof with the reference-level or a value derived thereof, h) comparing the antigen spontaneous IP-10 response or a value derived thereof with the reference-level or a value derived thereof, thereby determining whether mammal has previously encountered the antigen and thus generate immunological reactivity to the antigen or previously encountered other antigens generating immunological cross reactivity to the antigen and thereby determining whether the mammal has an active, a recent, or a latent infection, or if mammal is responding to treatment, is going to develop infection, or is immune-suppressed.

The term “mitogen” refers to any chemical or chemical composition promoting cell division. Mitogens may act upon both T cells and B cells either separately or simultaneously. Accordingly, the term mitogen also covers the terms T cell-activator and B cell-activator, and is thus used herein interchangeably. The mitogens of the present invention covers all mitogens known by the skilled person such as but not limited to phytohaemagglutinin (PHA), concanavalin A (conA) lipopolysaccharide (LPS) and pokeweed mitogen (PWM). In a preferred embodiment of the present invention the mitogen is a T-cell activator and even more preferably the mitogen is PHA. In another preferred embodiment of the present invention the mitogen is a monocyte/macrophage activator. IP-10 production is then determinate by any cytokine or chemokine detection method known to the skilled addressee such as but not limited to antibody-based technologies e.g. xMAP, multiplexing, Luminex, ELISA, ELISPOT, lateral stick assay or mRNA based techniques like Real time polymerase chain reaction (RT-PCR) or Intracellular flow cytometri (IC-FACS).

The quantity of IP-10 in response to antigens (e.g. Chlamydia extract antigens or tuberculosis specific proteins or derivatives hereof) may be determined by subtracting the background production of IP-10 and the probable infection with e.g. M. tuberculosis is interpreted on the basis of this antigen-specific IP-10 response.

The tuberculosis data in the present application is developed using existing technology: QuantiFERON® TB-Gold In-Tube test (Cellestis, Carnegie, Australia), in which whole blood is drawn directly into vacutainer tubes pre-coated with either saline (nil), TB specific peptide antigens (Ag) or mitogen (PHA). The tubes were incubated in a presently preferred embodiment at 18 h at 37° C., where after cytokine concentration was measured by xMAP technology on the Luminex platform (Luminex Corporation, USA), using Biosource reagents (Biosource Camarillo USA), and was thus able to switch the effect parameter from the traditional parameter IFN-γ to IP-10, which is expressed in higher concentrations and performs better.

The high sensitivity of the present invention makes this method an excellent tool for differentiating between active infection, latent infection, recent infection, infection in a child/newborn and/or long term latent infection. Thus in one embodiment, the present invention relates to a method wherein an antigen-dependent IP-10 response above the reference-level together with a nil indicates that the mammal has an active infection, a latent infection, a recent infection, and/or a long term latent infection.

In another embodiment the present invention relates to a method where the amount of sample material (e.g. whole blood) used in the test is reduced. In the case of whole blood down to the range of 3 to 0.1 ml, and in the case of PBMCs the cell number is in the range of 1×106 to 0.05×106. This “mini assay” suitable for patients with low blood volume (especially children/infants or patients with anaemia) is innovative because it can diagnose a disease (e.g. tuberculosis) without haemodynamic consequences for the donor or be performed on a very scarce sample material e.g. cells from spinal fluid, pleural fluid or blood from the umbilical cord.

Combination with Other Markers

Measuring IP-10 in combination with one or more of the following markers may reduce the number of false positive and increase the discriminatory power. Thus, in one embodiment, the method further comprises, a) determining the level of IP-10 and MCP-1 in response to the antigenic stimulation, b) combining the determined level of IP-10 and MCP-1, and c) comparing said combined level with a combined reference-level.

As understood by the skilled addressee the combined reference-level is determined by measuring the IP-10 level and any of the suggested combinatorial markers such as but not limited to MCP-1 level in a healthy population and combining said determined IP-10 and MCP-1 level by means of arithmetic such as but not limited to addition. The combined reference-level is determined at a selected cut-off point related to the distribution of the combined reference-level e.g. mean+2 standard deviations in a healthy population or by other means known to the skilled addressee.

Further combinatorial markers comprises IL-2 and INF-γ.

In one embodiment, the present invention discloses a method which further comprises a) determining the level of INF-γ and optionally MCP-1 and/or IL-2 in response to the antigenic stimulation, b) combining the determined level of IP-10 and INF-γ and optionally MCP-1 and/or IL-2, and c) comparing said combined level with a combined reference-level.

In one embodiment, the method comprises, a) determining the level of IP-10 in response to the antigenic stimulation, b) comparing the level of IP-10 to a reference level or a value derived thereof c) determining whether said mammal has previously encountered said antigen and thus generated IP-10 reactivity to the antigen d) determining the level of MCP-1 in response to the antigenic stimulation, e) comparing the level of MCP-1 to a reference level or a value derived thereof f) determining whether said mammal has previously encountered said antigen and thus generated MCP-1 reactivity to the antigen g) combining the determined IP-10 reactivity and MCP-1 reactivity thereby determining whether the mammal has previously encountered the antigen and thus generated immunological reactivity to the antigen with at least one biomarker.

In one embodiment, the method comprises, a) determining the level of IP-10 in response to the antigenic stimulation, b) comparing the level of IP-10 to a reference level or a value derived thereof c) determining whether said mammal has previously encountered said antigen and thus generate IP-10 reactivity to the antigen d) determining the level of IL-2 in response to the antigenic stimulation, e) comparing the level of IL-2 to a reference level or a value derived thereof f) determining whether said mammal has previously encountered said antigen and thus generate IL-2 reactivity to the antigen g) combining the determined IP-10 reactivity and IL-2 reactivity thereby determining whether the mammal has previously encountered the antigen and thus generate immunological reactivity to the antigen with at least one biomarker

In one embodiment, the method comprises, a) determining the level of IP-10 in response to the antigenic stimulation, b) comparing the level of IP-10 to a reference level or a value derived thereof c) determining whether said mammal has previously encountered said antigen and thus generate IP-10 reactivity to the antigen d) determining the level of IFN-γ in response to the antigenic stimulation, e) comparing the level of IFN-γ to a reference level or a value derived thereof f) determining whether said mammal has previously encountered said antigen and thus generate IFN-γ reactivity to the antigen g) combining the determined IP-10 reactivity and IFN-γ reactivity thereby determining whether the mammal has previously encountered the antigen and thus generate immunological reactivity to the antigen with at least one biomarker

In one embodiment, the method comprises, a) determining the level of IP-10 in response to the antigenic stimulation, b) comparing the level of IP-10 to a reference level or a value derived thereof c) determining whether said mammal has previously encountered said antigen and thus generate IP-10 reactivity to the antigen d) determining the level of INF-γ and/or MCP-1 and/or IL-2 in response to the antigenic stimulation, e) comparing the level of INF-γ and/or MCP-1 and/or IL-2 to reference levels for each biomarker or values derived thereof f) determining whether said mammal has previously encountered said antigen and thus generate INF-γ and/or MCP-1 and/or IL-2 reactivity to the antigen d) combining the determined IP-10 reactivity and/or INF-γ and/or MCP-1 and/or IL-2 reactivity thereby determining whether the mammal has previously encountered the antigen and thus generate immunological reactivity to the antigen with at least one of the examined biomarkers

In one embodiment, and as stated previously, IP-10 may be used for diagnosis of subjects suspected of various immunological states, such as infections. When used in diagnosis the method according to the present invention may help to determine the presence of immunological states, such as infections, usually accomplished by evaluating clinical symptoms and further laboratory tests. The test may diagnose various stages of infection i.e. a recently encountered infection in an individual without any symptoms, an infection encountered many years back in an individual with no symptoms of that infection, an active infection where the patients has symptoms due to the infection.

In another embodiment IP-10 may be used for diagnosis of subjects suspected of tuberculosis (e.g. active, latent or recent TB infection) and in particular patients at increased risk for progression from latent to active tuberculosis i.e. patients receiving immunosuppressing medication (i.e. monoclonal antibody treatment (anti-CD20 antibodies (e.g. Rituximab©) or TNF-α blocking treatment (e.g. Remicade©, Enbrel©, Humira©))) or steroids or cancer-chemotherapy; or, patients suffering from immunosuppressing conditions (e.g. HIV infection, cancer, IDDM or non-insulin dependent diabetes mellitus (NIDDM), autoimmune conditions, malnutrition, old age, intravenous drug use (IVDU) or inherited immune disorders), and in individuals who have recently been infected. In fact following standard guidelines these patients should be screened for active, latent or recent TB before initiation of medical treatment.