Diagnosis method of active tuberculosis
The present invention relates to a method for the in vitro diagnosis of active tuberculosis, comprising a step of contacting lymphocytes of a patient suspected to have active tuberculosis with at least one protein of mycobacteria, said protein being an enzyme having a lipolytic activity, and a step of detecting the presence of specific activated lymphocytes.
1. A method for the diagnosis of active tuberculosis, comprising:
a step of contacting a sample of purified B lymphocytes of a patient suspected to have an active tuberculosis with at least one protein of mycobacteria, said protein being an enzyme having a lipolytic activity and being able to hydrolyse lipids having a carbohydrate chain comprising at least 12 carbons selected from the group consisting of:
Rv3452 as set forth in SEQ ID NO: 1,
Rv1984c as set forth in SEQ ID NO: 2,
LipY, also called Rv3097c, as set forth in SEQ ID NO: 3,
Rv0183 as set forth in SEQ ID NO: 4,
PlcA, also called Rv2351c, as set forth in SEQ ID NO: 5,
PlcB, also called Rv2350c, as set forth in SEQ ID NO: 6,
PlcC, also called Rv2349c, as set forth in SEQ ID NO: 7, and
PlcD, as set forth in SEQ ID NO: 9 or SEQ ID NO: 8,
said sample being devoid of serum or of any fluid liable to contain soluble antibodies,
a step of incubation during a determined time to allow the purified B lymphocytes to secrete specific antibodies directed against said enzyme having a lipolytic activity, and
a step of detecting the presence of an immune complex comprising said antibodies and said enzyme having a lipolytic activity.
2. The method for the diagnosis of active tuberculosis according to claim 1 , wherein B lymphocytes are chosen among plasmablasts and plasmocytes.
3. The method for the diagnosis of active tuberculosis according to claim 1 , wherein the step of detecting the presence of activated B lymphocytes is carried out by an immunological detection.
4. The method for the diagnosis of active tuberculosis according to claim 1 , wherein said determined time is from 1 hour to 72 hours.
5. A kit for the detection of active tuberculosis comprising:
at least one protein of mycobacteria immobilised on a support, said protein being an enzyme having a lipolytic activity, selected from the group consisting of:
Rv3452 as set forth in SEQ ID NO: 1,
Rv1984c as set forth in SEQ ID NO: 2,
LipY, also called Rv3097c, as set forth in SEQ ID NO: 3,
Rv0183 as set forth in SEQ ID NO: 4,
PlcA, also called Rv2351c, as set forth in SEQ ID NO: 5,
PlcB, also called Rv2350c, as set forth in SEQ ID NO: 6,
PlcC, also called Rv2349c, as set forth in SEQ ID NO: 7, and
PlcD, as set forth in SEQ ID NO: 9 or SEQ ID NO: 8,
antibodies directed against constant chain of immunoglobulins, and
means for specifically purifying B lymphocytes.
6. The kit according to claim 5 , wherein said means specifically purifying B-lymphocytes are chosen among specific B cell specific antibodies, or density gradients, or a combination of the above.
7. The method for the diagnosis of active tuberculosis according to claim 1 , wherein said enzyme having a lipolytic activity is selected from the following enzymes:
Rv3452 as set forth in SEQ ID NO: 1,
Rv1984c as set forth in SEQ ID NO: 2, and
LipY as set forth in SEQ ID NO: 3.
8. The method for the diagnosis of active tuberculosis according to claim 1 , wherein said determined time is from 6 hours to 48 hours.
9. The method for the diagnosis of active tuberculosis according to claim 1 , wherein said determined time is from 16 h to 30 hours.
10. The kit according to claim 8 , wherein the antibodies are labelled.