2. Biochemical Studies on
Mycobacterium Tuberculosis
Antigen
BY
Mohamed Mostafa Omran
,Biotechnology Research Center
New Damietta, Egypt

3. Introduction

4. Mycobacterium Tuberculosis
• One third of the world’s population are
infected with M. Tuberculosis.
• M. Tuberculosis lead to death of one
person every 10 minutes.
• M. tuberculosis is the causative agent
of Tuberculosis (TB).

5. M. tuberculosis
• Curved rods.
• More resistant than
other bacteria to acids.
•Acid fast bacilli (AFB).

6. EPIDEMILOGY OF TB
Range of rate
per 100,000
0-9
10-24
25-49
50-99
%(Egypt (0.1-2.4
or more 100
No report

7. Transmission of TB
Patient with TB
.Inhalation of M. tuberculosis

8. Types of human TB
• Pulmonary TB (~ 80%).
• Extra-pulmonary TB (~ 20%).

9. Control of TB
1. Vaccination (BCG)
2. Effective therapy
3. Education
4. Early diagnosis

10. Diagnosis of TB
Traditional methods:
1. Ziehl- Neelsen (ZN) stain.
2. Fluorescence microscope.
X-ray. 3
.Tuberculin test. 4
Cultures. 5
New methods:
6. Serodiagnosis (Detection of TB antibody)
7. PCR (Detection of DNA of M. tuberculosis)
8. Detection of TB antigen.

11. Traditional methods
ZN stain
M tuberculosis
.
showing as red
rods against blue
background.
M. tuberculosis
showing as white
Yellow rods against
.the dark background

12. Traditional methods
X-ray
Tuberculin test

13. Traditional methods
Cultures
Culture is highly sensitive and specific test but
require 6 to 8 weeks to achieve m
axim sensitivity
um

14. New methods
Detection of TB antibody
TB antibody do not differentiated between
latent and active infection

16. Aim of the work
1. Identification of a TB antigen in different
body fluids of Infected TB patients using
monoclonal antibody.
2. Biochemical characterization of TB antigen.
3. Diagnosis of pulmonary and extra.pulmonary TB using TB antigen

18. Total number in the study (506
individuals)
Patients (n= 389) :
Pulmonary TB: 296 patients .
Extra-Pulmonary TB : 93 patients.
Controls (117):
Healthy volunteers: 48 individuals
Non-tuberculous diseases: 69
individuals

19. Patients with Pulmonary and
extra-pulmonary TB
100
90
% 76
80
70
) %(Percentag
60
50
% 24
40
30
20
10
0
Pulmonary TB
Extra-Pulmonary TB

20. All samples were obtained from the
Department of Chest Diseases at Sayd
Galal University Hospital, Al-Azhar
University and Abbasia fever Hospital,
Cairo, Egypt.
1. Serum
2. Cerebrospinal fluid )CSF(
3. Ascetic fluid

21. Monoclonal Antibody
()TB-55 mAb

22. Sodium Dodocyl Sulphate- polyacrylamide
Gel Electrophoresis )SDS-PAGE(

23. W
estern blotting
1-Separation by
electrophoresis
2-Blotting tank
Peptides transferred to
nitrocellulose sheet (blot)
+ve
3-Blocking of free
binding sites
Bovine serum albumin
-ve
+
+
+
+
5- Blot results
4- Immunostaining of the blot
Substrate
Antigen bands
visualized
Primary antibody
(TB-55 mAb)
Secondary antibody
Enzyme - labeled

24. Purification of TB antigen
1. Preparative gel electrophoresis.
2. Electroelution.

25. Capillary electrophoresis

26. TB Antigen detection using
dot-ELISA
Nitrocellulose membrane

27. Statistical analyses
Sensitivity, specificity, efficiency, and positive
predictive )PPV( and negative predictive )NPV(
values were calculated as following
Reference
Test
Evaluated test
Total
+ ve
- ve
+ ve
True + ve (a)
False –ve (c)
a+c
- ve
False + ve (b)
True -ve (d)
b+d
a+
= a / (ab+c) × 100. c + d
Specificity = d / (b + d) ×100.
Efficiency = (a + d)/(a + b + c + d)
×100.
PPV
= a / (a +b) ×100.
NPV
= d / (c +d) ×100.
Total
Sensitivity
a+b+c+d

28. Results

29. 1. Identification of TB antigen in
BCG, serum, CSF and ascites.

30. Identification of the TB-55 mAb target antigen in BCG
SDS-PAGE and western blotting for BCG.

31. Identification of TB antigen ( 55 kDa)
in serum samples of pulmonary TB
(Mr.) : Molecular weight markers
Lane (1-4): serum sample from non infected individuals.
Lanes 5-9: serum sample from pulmonary TB patients.

32. Identification of TB antigen (55 kDa) in
serum samples of extra- pulmonary TB
;Mr.) : Molecular weight markers(
.Lane (1): serum sample from non infected individual
Lane 2: peritonitis TB;
Lane 3: meningitis TB
Lane 4: lymph nodes TB; Lane 5: genitourinary tract TB ;
; Lane 6: potts disease TB; Lane 7: arthritis TB
Lane 8: sinusitis TB;
Lane 9: millary tuberculosis TB

33. Identification of TB antigen
(55 kDa) in CSF
(Mr.) : Molecular weight markers
Lanes (1-3): CSF from nontuberculous diseases patients.
Lanes (4-9): CSF from tuberculous meningitis patients.

34. Identification of TB antigen
(55 kDa) in ascetic fluid
(Mr.) : Molecular weight markers;
Lanes (1-2): non-tuberculous ascites fluids.
Lanes (3-9): tuberculous ascetic fluid from peritonitis TB
patients.

35. 2. Purification of TB antigen
from serum, CSF and ascites

36. SDS-PAGE of TB 55 kDa antigen from
serum of patients with TB
Pulmonary
(Mr.)
Extra-Pulmonary
: Molecular weight markers;
Lane 1: Crud serum sample.
.Lane 2:The TCA precipitate fraction of TB purified antigen
.Lane 3: The TCA supernatant fraction of TB purified antigen

37. SDS-PAGE of purified 55 kDa
antigens from CSF and ascites
CSF
(Mr.)
Ascites
: Molecular weight markers;
Lane 1: Crud sample (CSF or ascites).
.Lane 2:The TCA precipitate fraction of TB purified antigen
.Lane 3: The TCA supernatant fraction of TB purified antigen

38. OD at 200 nm
Capillary electrophoresis for purified TB antigens
Serum
.)Time (min
OD at 200 nm
.)Time (min
Serum
.)Time (min
.)Time (min

39. 3. Biochemical Characterization
of TB antigen from serum,
CSF and ascites.

40. Partial biochemical characterization of
purified TB antigen.
Reactivity of purified
antigen using dotELISA
Treatment
Concentration
Incubation
time
Treated
Untreated
Acid
0.2 M HCl
1 hour
-Ve
+ Ve
Base
0.2 M NaOH
1 hour
-Ve
+ Ve
Type of reagents
Trichloroacetic
acid
+ Ve
40%
15 min.
a. Precipitate
+ Ve
b. Supernatant
-Ve
20 mM
18 hours
+ Ve
+ Ve
Mercaptoethanol
180 M
1 hour
-Ve
+ Ve
Protease enzyme
1 mg/ml
45 min.
-Ve
+ Ve
Pepsin enzyme
1 mg/ml
45 min.
-Ve
+ Ve
Periodate

41. Amino acid analysis of TB antigen using HPLC
Name
Concentration
(nmol/mg protein)
Leucine
Isoleucine
Valine
Proline
Methionine
Tyrosine
Alanine
47.14
49.28
81.42
59.28
98
94.28
113.14
Hydrophilic
amino acids
Glycine
Serine
Therionine
755.71
214.28
55.71
46.4 %
Basic amino
acids
Lysine
Arginine
Histidine
162.85
85.71
111.42
% 16.3
Acidic amino
acids
Glutamic acid
Aspartic acid
135.71
142.85
Type
Hydrophobic
amino acids
%
24.6%
12.7

42. Amino acid analysis of 55-kDa using HPLC
46.4
50
(%)
40
30
24.6
20
16.3
12.7
10
0
Hydrophobic
Hydrophilic
Basic
Acidic
The 55- kDa antigen is a basic polypeptide
.chain with a hydrophilic nature

43. 4. Diagnostic potential of TB
antigen in pulmonary and
extra-pulmonary TB

44. Detection of circulating 55-kDa antigen
using Dot- ELISA
(+ve) control : Serum sample of infected individual.
(- ve) control : Serum sample of non-infected individual
: Strong positive test
+).Serum samples with high antigen level (3+, 4
: Weak positive test
+). Serum samples with low antigen level (1+,2

45. Advantages of detection TB 55-kDa antigen
using Dot-ELISA in serum samples of
pulmonary TB
TB-Ag detection
(Clinical diagnosis (gold standard
+
. Pulmonary tuberculosis
patients
257 (a)
Patients with respiratory diseases
other than TB and Healthy
controls
( b )4
Total
Sensitivity = 87 %
%,
%PPV
= 98 %,
%,Efficiency = 90
261
Total
39 (c)
296
( d )113
117
152
413
Specificity = 97
NPV
= 74

46. Advantages of detection TB 55-kDa antigen using
Dot-ELISA in serum samples of Extra-pulmonary TB
Clinical diagnosis (gold
(standard
TB-Ag detection
Total
+
-
.Extra-pulmonary tuberculosis (a )84
patients
Patients with respiratory
( b )4
diseases other than TB and
Healthy controls
Total
Sensitivity = 90 %,
PPV
= 95 %,
88
(c )9
93
113
( d)
117
122
210
Specificity = 97 %,
NPV
= 93 %
Efficiency = 94 %,

47. Overall Advantages of detection TB 55-kDa antigen
using Dot-ELISA in 506 serum samples
(Clinical diagnosis (gold standard
TB-Ag detection
Total
+
-
TB patients
(pulmonary and extra-pulmonary)
(a )341
(c )48
389
Patients with respiratory diseases
other than TB and Healthy controls
( b )4
( d )113
117
345
161
506
Total
%, Sensitivity = 88 %,
PPV
= 99 %,
%,Efficiency = 90
Specificity = 97
NPV
= 70%

49. Conclusion
1. 55–kDa TB antigen was identified in serum, CSF
and ascites fluid of TB infected patients using
westen blot.
2. The purified TB antigen showed a single band at
55–kDa in SDS-PAGE and one peak when analyzed
by Capillary electrophoresis at 11 minutes.
3. The reactive epitopes of the purified antigen from
serum, CSF and ascites have the same
biochemical characteristics.
The 55- kDa antigen is a basic polypeptide. 4
.chain with a hydrophilic nature

50. 5. The TB antigen was detected using a simple
and rapid dot-ELISA in 87% of individuals with
pulmonary TB and 90% of individuals with
extra-pulmonary TB.
6. TB antigen detection has sensitivity (88%),
specificity (97%), and efficiency (90%) in 506
individuals.

52. WHO Recommendations
To replace the “gold standard”, For TB diagnosis,
a serological test must has sensitivity of over 80%
and specificity of over 95%.
So, the detection of 55-kDa TB antigen using
dot-ELISA could replace the WHO gold standard
for TB diagnosis.