Methods for detecting a Mycobacterium tuberculosis infection
AssigneesOregon Health & Science University, The United States of America, DBA The Department of Veteran Affairs
InventorsDavid M. Lewinsohn, Deborah A. Lewinsohn
Life of this patent
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Abstract
Methods for detecting an infection with Mycobacterium tuberculosis (Mtb) in a subject are disclosed. The methods include detecting the presence of CD8+ T cells that specifically recognize an Mtb polypeptide. The methods include in vitro assays for detecting the presence of CD8+ T cells in a biological sample, and in vivo assays that detect a delayed type hypersensitivity reaction. The methods can also include detecting Mtb polypeptides and polynucleotides. Reagents for the detection of an Mtb infection are also disclosed.
Claims19
A method for detecting Mycobacterium tuberculosis in a subject, comprising:
- contacting a biological sample from the subject, wherein the biological sample comprises T cells, with one or more-isolated Mycobacterium polypeptides in vitro, wherein one of the isolated Mycobacterium polypeptides comprises the amino acid sequence set forth as SEQ ID NO:
- 11;
- and
- performing an enzyme-linked immunospot assay that detects interferon-γ in order to determine if the T cells specifically recognize the Mycobacterium polypeptide, wherein a positive enzyme-linked immunospot assay that detects interferon-γ in the biological sample determines the presence of T cells that specifically recognize the Mycobacterium polypeptide and detects Mycobacterium tuberculosis in the subject.
A method for detecting a Mycobacterium tuberculosis infection in a subject, comprising:
- contacting ex vivo a biological sample from the subject, wherein the biological sample comprises T cells, with antigen presenting cells presenting one or more Mycobacterium polypeptides, wherein the amino acid sequence of one of the one or more Mycobacterium polypeptides comprises the amino acid sequence set forth as SEQ ID NO:
- 11;
- and
- contacting the sample with a capture monoclonal antibody that specifically binds interferon γ, wherein the capture monoclonal antibody is immobilized on a solid support, to form a complex;
- and
- contacting the sample with an additional labeled antibody that specifically binds interferon γ to detect the presence of the complex, wherein the presence of the labeled antibody bound to the complex indicates the presence of T cells that specifically recognize one of the Mycobacterium polypeptides and detects the Mycobacterium tuberculosis infection in the subject.
A method for detecting Mycobacterium tuberculosis in a subject, comprising:
- incubating a biological sample comprising 105 to 107 peripheral blood mononuclear cells isolated from the subject in vitro with one or more-isolated Mycobacterium polypeptides in a culture wherein one of the isolated Mycobacterium polypeptides comprises the amino acid sequence set forth as SEQ ID NO:
- 11 and is present at a concentration of 10−1 to 103 μg per ml of the culture, and wherein the peripheral blood mononuclear cells are incubated with the polypeptide for about 4 to about 24 hours;
- and
- performing an assay in order to determine if T cells in the biological sample specifically recognize the Mycobacterium polypeptide, wherein the presence of T cells that specifically recognize the Mycobacterium polypeptide and detects Mycobacterium tuberculosis in the subject, wherein the assay comprises one or more of a) determining if the polypeptide binds T cells;
- b) evaluating cytolytic activity of the T cells;
- c) evaluating proliferation of the T cells, d) detecting the secretion of interferon gamma, interleukin-2 or tumor necrosis factor alpha, or e) any combination of a)-d).
A method for detecting Mycobacterium tuberculosis in a subject, comprising:
- isolating CD8+ T cells from a biological sample from the subject;
- incubating the CD8+ T cells isolated from the subject in vitro with antigen presenting cells and with one or more-isolated Mycobacterium polypeptides in a culture, wherein one of the isolated Mycobacterium polypeptides comprises the amino acid sequence set forth as SEQ ID NO:
- 11 and is present at a concentration of 10−1 to 103 μg per ml of the culture, and wherein the CD8+ T cells are incubated with the polypeptide for about 4 to about 24 hours;
- and
- performing an assay in order to determine if T cells in the biological sample specifically recognize the Mycobacterium polypeptide, wherein the presence of T cells that specifically recognize the Mycobacterium polypeptide and detects Mycobacterium tuberculosis in the subject, wherein the assay comprises one or more of a) determining if the polypeptide binds T cells;
- b) evaluating cytolytic activity of the T cells;
- c) evaluating proliferation of the T cells, d) detecting the secretion of interferon gamma, interleukin-2 or tumor necrosis factor alpha, or e) any combination of a)-d).
Dependencies are inferred from the claim text.