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Ip-10 based immunological monitoring

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Title: Ip-10 based immunological monitoring.
Abstract: The present invention relates to an immunological method and, more particularly, a method for measuring cell-mediated immune reactivity (CMI) in mammals based on the production of IP-10. The invention further discloses an assay and a kit for measuring CMI to an antigen using whole blood or other suitable biological samples. The methods of the present invention are useful in therapeutic and diagnostic protocols for human, livestock and veterinary and wild life applications, thus the invention further relates to a method for diagnosing an infection in a mammal. ...

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Inventors: Morten Ruhwald, Pernille Ravn, Jesper Eugen-Olsen
USPTO Applicaton #: #20120015386 - Class: 435 792 (USPTO) - 01/19/12 - Class 435 
Chemistry: Molecular Biology And Microbiology > Measuring Or Testing Process Involving Enzymes Or Micro-organisms; Composition Or Test Strip Therefore; Processes Of Forming Such Composition Or Test Strip >Involving Antigen-antibody Binding, Specific Binding Protein Assay Or Specific Ligand-receptor Binding Assay >Assay In Which An Enzyme Present Is A Label >Heterogeneous Or Solid Phase Assay System (e.g., Elisa, Etc.)

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The Patent Description & Claims data below is from USPTO Patent Application 20120015386, Ip-10 based immunological monitoring.

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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.



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).

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Application #
US 20120015386 A1
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Other USPTO Classes
435 34
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