Method of Diagnosing Tuberculosis

Abstract

The present invention relates to a method of diagnosing tuberculosis in a subject, said method comprising steps of: detecting anti-Mycobacterium tuberculosis (M.tb.) isocitrate dehydrogenase (ICDs) antibody in the subject, and diagnosing tuberculosis in the subject.

Description

LEGENDS

Table 1: PCR primers and thermal cycle parameters for amplification of M.tb ICD-1 and ICD-2

FIG. 1: Affinity purification of M.tb ICD-1 and M.tb ICD-2. Histidine-tagged recombinant protein was purified by nickel column chromatography under native condition and stained with Coomassie Blue following electrophoresis on 10% SDS polyacrylamide gels. The different lanes are: lanes 1 and 2: M.tb ICD-2; lane M: protein molecular size markers (200 kDa, 116 kDa, 97 kDa, 66 kDa, 45 kDa, 31 kDa and 21.5 kDa); lanes 3 and 4: M.tb ICD-1.

FIG. 2: M.tb ICD-1 and ICD-2 show high B-cell reactivity to sera from TB infected patients from different groups as opposed to BCG vaccinated healthy controls. The humoral immune responses directed against the recombinant proteins, M.tb ICD-1 (2A) and M.tb ICD-2 (2B) and control antigens HSP 60 (2C) and PPD (2D) were compared between different categories of patients and healthy controls. Group 1: fresh infections, Group 2: relapsed infection and Group 3: extrapulmonary TB. The respective sample numbers and p values are shown.

FIG. 3: M.tb ICDs are more immunogenic than HSP 60. The ELISA reactivity to M.tb ICD-1, M.tb ICD-2 and control antigen HSP 60 was compared in different patient groups. Horizontal bands represent the mean reactivity or average levels of humoral response in each category.

FIG. 4: M.tb ICDs could significantly distinguish TB-infected sera from NTMs and non-TB patient sera. Recombinant M.tb ICD-1 and M.tb ICD-2 as well as HSP 60 were tested against sera of NTM (4A) and non-tuberculosis patients (4B). The respective humoral responses were compared to TB-infected sera, the p values for which are given in the figures. HSP 60 could not distinguish TB-infected patient from either NTM or non-TB significantly. Horizontal bands represent the mean reactivity in each category.

TABLE 1
PCR primers and thermal cycle parameters for
amplification of M.tb ICD-1 and ICD-2
		PCR	Amplicon
Primers	Sequence	Parameters	Size
M.tb	ggatccATGTCCAACGCA	94° C. for 2′	~1.2	Kb
icd-1FP	CCCAAGATA
M.tb	aagcttCTAATTGGCCAG	(35 Cycles)
icd-1RP	CTCCTTTTC	94° C. for 30″
		50° C. for 1′
		72° C. for 3′
		72° C. for 7′
M.tb	AGCTTggatccATGAGCG	94° C. for 2′	~2.23	Kb
icd-2FP	CCGAACAGCC
M.tb	CATGGaagcttTCAGCCT	(10 Cycles)
icd-2RP	TGGACAGCCT	94° C. for 30″
		50° C. for 30″
		72° C. for 3.30′
		(25 cycles)
		94° C. for 30″
		58° C. for 30″
		72° C. for 3.30′
		72° C. for 7′

Claims

1. A method of diagnosing tuberculosis in a subject, said method comprising steps of: a. detecting anti-Mycobacterium tuberculosis (M.tb.) isocitrate dehydrogenase (ICDs) antibody in the subject, andb. diagnosing tuberculosis in the subject.

2. A method as claimed in claim 1, wherein the subject is mammal including human.

3. A method as claimed in claim 1, wherein the antibodies are detected using enzyme linked immunosorbent assays (ELISA).

4. A method as claimed in claim 1, wherein the ICDs are antigenic in two isoforms ICD-1 and ICD-2.

5. A method as claimed in claim 1, wherein the method distinguishes subject with tuberculosis from healthy BCG-vaccinated subject.

6. A method of developing drug against tuberculosis, said method comprising step of: a. targeting the proposed drug towards enzyme isocitrate dehydrogenase (ICD), andb. developing the drug against tuberculosis.