Serodiagnostic efficacy of Mycobacterium tuberculosis 30/32-kDa mycolyl transferase complex, ESAT-6, and CFP-10 in patients with active tuberculosis.

Abstract

Elimination of tuberculosis (TB) largely depends upon definitive rapid diagnosis and treatment. Widely used diagnostic tests do not qualify for use in a developing country due to lack of either desired accuracy or their cost. In the present study an enzyme-linked immunosorbent assay was used to evaluate the diagnostic potential of an immuno-dominant 30/32-kDa mycolyl transferase complex (Ag85 complex) and Mycobacterium tuberculosis-specific proteins (ESAT-6 and CFP-10) of the RD1 region. Higher sensitivity (84.1%) with Ag85 complex was observed compared with ESAT-6 (64.9%) and CFP-10 (66%), with almost similar specificity (Ag85: 85.2%, ESAT-6: 88.9%, CFP-10: 85.2%), whereas the individual components of Ag85 complex, i.e. Ag85A, Ag85B, and Ag85C, showed sensitivities of 44.6, 34, and 80.9% and specificities of 55.6, 74.1, and 40.7% respectively. A cocktail of Ag85 complex, ESAT-6, CFP-10, Ag85A, Ag85B, and Ag85C antigens also could not help in increasing either sensitivity (51.1%) or specificity (85.2%). Furthermore, immunoblot analysis using clinical isolates as well as a standard strain (H37Rv) of M. tuberculosis also showed strong reactivity of sera from TB patients to Ag85 complex and, to a lesser extent, also to ESAT-6. To conclude, use of Ag85 complex along with ESAT-6 and CFP-10 seems to be promising in minimizing the heterogeneous sero-responses of adult TB cases.

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ORIGINAL ARTICLE
Serodiagnostic Efficacy of Mycobacterium tuberculosis 30/32-kDa
Mycolyl Transferase Complex, ESAT-6, and CFP-10 in Patients
with Active Tuberculosis
Gavish Kumar•Pradeep Kumar Dagur•Prashant Kumar Singh•
Hari Shankar•Virendra S. Yadav•Vishwa M. Katoch•Bharat Bajaj•
Rajesh Gupta•Utpal Sengupta•Beenu Joshi
Received: 29 January 2009/Accepted: 2 June 2009/Published online: 5 January 2010
? L. Hirszfeld Institute of Immunology and Experimental Therapy, Wroclaw, Poland 2010
Abstract
depends upon definitive rapid diagnosis and treatment.
Widely used diagnostic tests do not qualify for use in a
developing country due to lack of either desired accuracy
or their cost. In the present study an enzyme-linked
immunosorbent assay was used to evaluate the diagnostic
potential of an immuno-dominant 30/32-kDa mycolyl
transferase complex (Ag85 complex) and Mycobacterium
tuberculosis-specific proteins (ESAT-6 and CFP-10) of the
RD1 region. Higher sensitivity (84.1%) with Ag85 com-
plex was observed compared with ESAT-6 (64.9%) and
CFP-10 (66%), with almost similar specificity (Ag85:
85.2%, ESAT-6: 88.9%, CFP-10: 85.2%), whereas the
individual components of Ag85 complex, i.e. Ag85A,
Ag85B, and Ag85C, showed sensitivities of 44.6, 34, and
80.9% and specificities of 55.6, 74.1, and 40.7% respec-
tively. A cocktail of Ag85 complex, ESAT-6, CFP-10,
Ag85A, Ag85B, and Ag85C antigens also could not help in
increasing either sensitivity (51.1%) or specificity (85.2%).
Furthermore, immunoblot analysis using clinical isolates as
well as a standard strain (H37Rv) of M. tuberculosis also
showed strong reactivity of sera from TB patients to Ag85
complex and, to a lesser extent, also to ESAT-6. To
Eliminationoftuberculosis(TB)largely
conclude, use of Ag85 complex along with ESAT-6 and
CFP-10 seems to be promising in minimizing the hetero-
geneous sero-responses of adult TB cases.
Keywords
30/32-kDa Mycolyl transferase complex ?
ESAT-6/CFP-10 ? Serodiagnosis ? Tuberculosis
Secretory proteins ?
Introduction
Tuberculosis (TB) is a growing health problem in the
developing world. Nine million new cases of TB and three
million deaths are reported every year around the globe
(WHO 2007). In asymptomatic individuals, TB infection
can be diagnosed with the help of the intra-dermal skin test
using purified protein derivative (PPD). It is impossible to
distinguish between present and past infection on the basis
of a PPD test and this test is also not specific in BCG-
vaccinated individuals (Huebner et al. 1993). Furthermore,
conversion of the PPD reactivity from negative to positive
challenges the clinician (Fine et al. 1999).
The specificity of acid-fast bacilli (AFB) staining is
typically [99% and the sensitivity of this test varies
between 25 and 75% (American Thoracic Society 2000). A
positive AFB test and negative culture result may be caused
by nonviable bacilli, when a person is receiving anti-TB
drugs. The culture technique has an advantage in distin-
guishing the morphology of Mycobacterium tuberculosis
from those of some non-tuberculous mycobacteria. How-
ever, it requires more time for results (3–4 weeks)
(Thornton et al. 1998). Advanced molecular techniques
such as PCR and BACTEC are, although rapid, sensitive,
and specific (Hawkinds et al. 1991; Noordhoek et al. 1994),
are not available everywhere and are also not cost effective.
G. Kumar ? P. K. Dagur ? P. K. Singh ? H. Shankar ?
V. S. Yadav ? V. M. Katoch ? U. Sengupta ? B. Joshi (&)
National JALMA Institute for Leprosy and Other Mycobacterial
Diseases (ICMR), Taj Ganj, Agra 282001, India
e-mail: bjjalma@gmail.com; beenuj2002@yahoo.co.in
B. Bajaj
State TB Demonstration Centre,
S. N. Medical College, Agra 282002, India
R. Gupta
Department of TB and Chest Diseases,
S. N. Medical College, Agra 282002, India
Arch. Immunol. Ther. Exp. (2010) 58:57–65
DOI 10.1007/s00005-009-0055-4

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Active pulmonary TB is contagious and leads to the
spread of M. tuberculosis. Therefore, rapid and early
diagnosis is needed for a TB control program. Various
studies have been undertaken to develop a serodiagnostic
test using secretory proteins of M. tuberculosis as these are
known as immuno-dominant and early markers for TB
(Kanaujia et al. 2003; Malen et al. 2008; Samanich et al.
2000). Among these proteins, ESAT-6 (Rv3875) and CFP-
10 (Rv3874) are known as M. tuberculosis-specific anti-
gens as they are absent in BCG and in most of the
nonpathogenic mycobacteria (Arend et al. 2000; Harboe
et al. 1996); therefore these antigens could differentiate
infected and BCG-vaccinated individuals with TB. The
role of these antigens in the early diagnosis and latent TB
was previously reported (Silva et al. 2003). A well-defined
fraction of the secretory proteins is Ag85 complex (30/32-
kDa mycolyl transferases) abundantly found in culture
filtrate of M. tuberculosis (Wiker and Harboe 1992). This is
a family of three closely related proteins (Ag85A, Ag85B,
and Ag85C) found in all mycobacteria (Wiker et al. 1986).
Ag85 complex was recently reported to be an immuno-
dominant marker for TB (Malen et al. 2008).
In our recent report we showed the potential of Ag85C in
childhood TB cases (Kumar et al. 2008). The present study
wasconductedtofindoutthediagnosticpotentialofimmuno-
dominantAg85complexaswellasitsindividualcomponents
and highly specific (ESAT-6, CFP-10) secretory antigens
using enzyme-linked immunosorbent assay (ELISA) for the
serodiagnosis of adult TB in a tuberculosis and leprosy
endemic country, India. Furthermore, the study was also
extended using a cocktail of all these antigens. The reactivity
of sera was also evaluated using immunoblot analysis.
Materials and Methods
Study Subjects
Serum samples of 86 confirmed active TB patients were
obtained from the State Tuberculosis Demonstration
Centre, Agra, and the Department of Tuberculosis and
Chest Diseases, S. N. Medical College, Agra. The patients
were from the following well-defined categories.
Of the total pulmonary TB cases (n = 76), fresh TB
cases (n = 48) comprised patients who had infection with
M. tuberculosis bacilli for the first time and had no history
of TB treatment. Defaulter TB cases (n = 20) comprised
TB patients who had not taken the complete course of
antitubercular treatment and the symptoms reoccurred.
Relapse TB cases (n = 8) comprised TB patients who were
treated earlier for TB but symptoms reoccurred after
completion of the treatment (Table 1). All these patients
were examined microbiologically (Ziehl Nielsen staining/
culture) with clinical evaluations. All the pulmonary TB
patients were smear positive for AFB. Extra-pulmonary TB
patients (n = 10) had TB involvement in organs other than
the lungs in which the disease was confirmed by tuberculin
skin test and suggestive clinical findings (Table 1). The
history of tuberculosis contact was available for all the TB
patients. Bacteriologically negative, partially or fully
treated cases of TB (n = 10) were also included in this
study. All patients were categorized according to the
guideline of the Revised National Tuberculosis Control
Program, Central Tuberculosis Division, Government of
India.
Healthy controls (n = 29) included in this study were
staff members or temporary students working in our
Institute who had no disease involvement or any family
contact history with TB/leprosy patients. Eighteen well-
confirmed leprosy controls (7 lepromatous, 8 borderline
tuberculoid, 2 mid-borderline, and 1 tuberculoid leprosy)
were also included. These leprosy cases were taken from
the outpatient department (OPD) of the National JALMA
Institute for Leprosy and Other Mycobacterial Diseases
(ICMR), Agra. Two untreated active TB patients, two
healthy BCG-vaccinated individuals, and one contact of
TB were selected for Western blot analysis on the basis of
their antibody response shown in ELISA.
All the patients and controls included in the present
study were over 18 and under 65 years of age. Human
Table 1 Details of different
groups of patient with
tuberculosis and control
subjects studied in this study
Study subjects Total No.
SexMean age
MaleFemale Year Range
1. Tuberculosis 8653 33
i. Pulmonary cases 48 32 1632.6 ± 13.218–65
ii. Extra-pulmonary cases 103731.0 ± 14.0 18–65
iii. Defaulter TB cases 2010 1028.7 ± 8.8 18–50
iv. Relapse TB cases880 32.0 ± 10.824–55
2. Healthy controls 29218 25.9 ± 6.722–52
3. Leprosy controls18 126 33.3 ± 6.8 25–60
4. Anti tuberculosis treated cases 108235.3 ± 12.818–58
58Arch. Immunol. Ther. Exp. (2010) 58:57–65

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immunodeficiency virus (HIV) testing was performed for
all the cases and HIV-positive cases were excluded from
the study. The study was approved by the institutional
ethics committee and informed consent was obtained from
each patient.
Antigen and Antibody
Purified r-ESAT-6, r-CFP-10, native Ag85 complex, its
individual components (Ag85A, Ag85B, Ag85C), mono-
clonal antibody (CS-90) to Ag85 complex, and polyclonal
antibody to ESAT-6 and CFP-10 were obtained from
Colorado State University (Colorado, USA) through a TB
Research Materials and Vaccine Testing Contract (NIH
Contract HHSN266200400091C/ADB Contract NOI-AI-
40091).
Enzyme-Linked Immunosorbent Assay
One hundred ll of the antigens ESAT-6 (12.5 ng/ml), CFP-
10 (12.5 ng/ml), Ag85 complex (12.5 ng/ml), Ag85A
(50 ng/ml), Ag85B (6.25 ng/ml), Ag85C (25 ng/ml), and a
cocktail of all these antigens having the same concentration
as described above was coated on a 96-well ELISA plate
(flat bottom, Nunc Maxisorp, Roskilde, Denmark) in
0.05 M carbonate bicarbonate buffer, pH 9.6. The working
dilution of each antigen was standardized using checker-
board titration as described earlier (Kumar et al. 2008).
After overnight incubation at 37?C, the contents of all the
wells were aspirated and the plate was washed three times
with 150 mM PBS, pH 7.4. The plate was blocked with 2%
BSA (Sigma, St. Louis, MO, USA) in PBS (200 ll/well)
for 1 h. After washing with PBST (with 0.05% Tween-20),
serum diluted 1:200 in assay diluent (1% BSA in PBS with
0.05% Tween-20) was added in duplicate wells and incu-
bated at 37?C for 2 h. Anti-human IgG peroxidase-
conjugated antibody (Sigma, St. Louis, MO, USA) diluted
1:10,000 in assay diluent was added to each well. After
incubation for 1 h, 100 ll of substrate solution (orthophe-
nylene diamine (OPD) tablet of 5 mg dissolved in 10 ml of
distilled water and 50 ll H2O2) was added to each well and
kept at room temperature for 20 min in the dark. The
reaction was stopped by adding 50 ll of 7% H2SO4(stop
solution) to each well and the absorbance was measured at
492 nM using a Spectramax-M2 Reader (Molecular
Devices, Sunnyvale, CA, USA). All the experiments were
carried out at least two times.
Preparation of Whole-Cell Extracts
The growth of M. tuberculosis culture was harvested at the
late log phase (3 weeks) from Sauton’s medium and
washed twice with PBS (150 mM, pH 7.4). Bacterial
growth was then re-suspended (0.2 g growth/ml) in lysis
buffer (50 mM Tris, 10 mM MgCl2, 1 mM EGTA, 1 mM
PMSF, pH 7.4) and subjected to sub-cellular fractionation
(Brodie et al. 1979) using a Vibra-Cell probe ultrasonicator
fora totalof20 minusing
(100% = 475 W) and 50% duty cycle (on/off) at 4?C. The
extracts were then centrifuged at 23,0009g for 30 min to
remove debris and the supernatant was collected. The
protein concentration of each sample was determined using
Bradford’s method (Bradford 1976). These extracts were
stored at –20?C until used.
50% outputcontrol
Immunoblot Analysis
Polyacrylamide gel electrophoresis (PAGE) was done under
reducing conditions (Laemmli 1970) using 12% (w/v)
resolving gel in a Mini-Protean gel apparatus (Bio-Rad
Laboratories, Hercules, CA, USA) by loading 20 lg/lane
of protein extract. The molecular mass marker was
obtained from Bio-Rad. The resolved proteins were trans-
ferred (Towbin et al. 1979) onto a nitrocellulose membrane
(0.45-lm pore size; Sigma, St. Louis, MO, USA) using a
TransBlot apparatus (Bio-Rad Laboratories, Hercules, CA,
USA). The membrane was washed with PBS and blocked
with 1% BSA (Sigma, St. Louis, MO, USA). The mem-
brane was probed with sera diluted 1:400 in assay diluent
(1% BSA in PBS containing 0.05% Tween-20) overnight at
4?C. The blots were washed with PBS containing 0.05%
Tween-20 and incubated with anti-human IgG peroxidase-
conjugated antibody (Sigma, St. Louis, MO, USA) diluted
1:5,000 in assay diluent for 1 h. After final washing, the
color was developed with diamino benzedine (Sigma, St.
Louis, MO, USA) in citrate phosphate buffer (0.5 M, pH
5). When reactivity was observed, the reaction was stopped
by rinsing the membrane with distilled water. Data were
analyzed by the Gel Documentation system (Bio-Rad
Laboratories, Segrate (Milan), Italy) using Quantity One
Software.
Statistical Analysis
Receiver operating characteristic (ROC) curves were
constructed using Software Stata-7 (Strata Corporation,
College Station, Texas, USA) to determine the cutoff,
sensitivity, and specificity. Scattergrams were plotted using
GraphPad prism software version 3.02 (San Diego, CA,
USA).
Results
The focus of the present study was to identify the sero-
diagnostic potential of various secretory antigens of
Arch. Immunol. Ther. Exp. (2010) 58:57–6559

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M. tuberculosis (ESAT-6, CFP-10, Ag85 complex as well
as its individual components (A, B, C), and a cocktail of all
these antigens) using ELISA. The antibody response to
each antigen was analyzed in 76 well-characterized
patients with active pulmonary TB (48 fresh TB, 20
defaulters, 8 relapsed TB), 10 extra-pulmonary TB, 10
partially/fully treated smear-negative cases of TB, 29
healthy controls, and 18 leprosy controls. Furthermore, the
reactivity of sera against clinical isolates of M. tuberculosis
was also evaluated by immunoblotting.
Sensitivity and specificity for a particular antigen were
determined using ROC analysis. For all the antigens, the
same numbers of TB cases, healthy individuals, and leprosy
patients were taken. The cutoff was selected at the point
which showed the best accuracy (correctly classifying
individuals to their groups), sensitivity, and specificity by
ROC. A predictive value (PV) to define the probability of a
disease is very important as it provides the significance of a
disease to characterize a patient for the particular disease
from the patient’s population (?PV), and a high -PV is
also needed to exclude the disease (Table 2); hence these
parameters were also analyzed. Furthermore, concordance
of IgG response to antigens that show good results in terms
of sensitivity, specificity, and accuracy was determined to
assess the chance of agreement (Table 3).
Antibody Response of Different Groups of TB Patients
and Controls to the Various Secretory Antigens
We observed antibody reactivity of 64.9% and specificity
of 88.9% for ESAT-6 using the cutoff determined by ROC
(Table 2). Positivity at this cutoff was 68.8% for fresh TB,
70% for defaulters, 62.5% for relapse, and 70% for extra-
pulmonary TB. None of the leprosy patients were positive
for this antigen, whereas only 30% of the treated TB cases
were found to be positive for it (Fig. 1).
We noted slightly increased reactivity to CFP-10 antigen
in comparison with ESAT-6 when sera from fresh TB
(72.8%), relapse cases (75%), and treated cases (40%) were
measured. But this antigen was less reactive (55%) with the
sera of defaulter cases of TB. Reactivity of extra-pulmonary
TB was found to be similar (70%) to that of ESAT-6. We
did not note any reactivity of the sera of leprosy patients to
this antigen (Fig. 1). The overall sensitivity to this antigen
was 66.0% and specificity 85.2% (Table 2).
The reactivity for Ag85 complex was the highest
(84.1%) of all the antigens used in this study, with a
specificity of 85.2% (Table 2). We observed 88.9% reac-
tivity in the fresh TB group, 90% in defaulters, 75% in
relapse cases, 80% in extra-pulmonary TB, 60% in treated
cases of TB, and 6.89% in leprosy patients to this antigen
(Fig. 1).
The reactivities to the individual components Ag85A,
Ag85B, and Ag85C of Ag85 complex were 44.6, 34.0, and
80.9% and specificity 55.6, 74.1, and 40.7%, respectively
(Table 2). Furthermore, 36.6, 35.4, and 93.7% of fresh TB
cases were reactive to the Ag85A, B, and C components
respectively. The observed reactivities of sera from the
defaulter cases were 55, 45, and 80%, respectively. We
noted 62.5% reactivity with Ag85A, 62.5% with Ag85B,
and 87.5% with Ag85C in the sera of relapse cases. None
of the extra-pulmonary TB cases showed antibody response
to Ag85A or Ag85B, although 30% reactivity was noted
using Ag85C. The leprosy controls reacted more (44.4%)
with the Ag85C component than to Ag85A (33.3%) and
Ag85B (5.5%). Reactivities in the treated TB cases were
Table 2 Immunoglobulin G reactivity against various secretory antigens of M. tuberculosis at cut off decided by ROC analysis
AntigenCutoff Sensitivity (%)Specificity (%) ?PV (%) -PV (%) ?LR-LR ROC area
ESAT-6 0.33164.988.9 95.342.1 5.840.390.743
CFP-100.29366.0 85.293.941.84.45 0.40 0.746
Ag85 complex 0.15084.185.2 95.260.5 5.67 0.190.879
Ag85A0.119 44.655.677.423.2 1.010.10 0.496
Ag85B0.16334.074.182.8 24.4 1.310.890.545
Ag85C0.072 80.940.7 82.637.91.36 0.470.603
Cocktail 0.114 51.185.2 92.333.3 3.450.57 0.685
Table 3 Correlation of antibody reactivity of various secretory antigens
ESAT-6–CFP-
10?/Total
patients
CFP-10–ESAT-
6?/Total
patients
Ag85–ESAT-
6?/Total
patients
Ag85–CFP-
10?/Total
patients
Ag85–
Ag85A?/Total
patients
Ag85–
Ag85B?/Total
patients
Ag85–
Ag85C?/Total
patients
Ag85–
Cocktail?/Total
patients
7/76 7/761/76Nil/76 5/764/767/76 4/76
60 Arch. Immunol. Ther. Exp. (2010) 58:57–65

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only 40, 20, and 70% for Ag85A, Ag85B, and Ag85C,
respectively (Fig. 1).
We observed lower reactivity (51.1%) using the cocktail
of all these antigens, but the specificity was the same
(85.2%) as that reported for the Ag85 complex (85.2%)
(Table 2). However, antibody reactivity in fresh, relapse,
and defaulter TB cases, treated cases of TB, and leprosy
cases was observed to be 64.6, 75, 50, 40, and 16.6%,
respectively. No reactivity was noted with this antigen in
the extra-pulmonary TB patients (Fig. 1). Interestingly,
four of the healthy individuals were reactive to all the
antigens used in this study.
Correlation of Reactivity of Individual Patients
to Various Secretory Antigens
The antibody responses to the various secretory antigens
were also correlated. We noted antibody response to
ESAT-6 in seven patients who did not show antibody to
CFP-10 and, similarly, antibody reactivity to CFP-10 was
noted in seven patients who were negative for ESAT-6. All
the patients negative for Ag85 complex were also negative
for CFP-10 and ESAT-6, except one patient who was
positive for ESAT-6. However, five patients who showed
antibody reactivity to Ag85A were not positive for Ag85
complex, and 4 patients who were negative for Ag85
complex were positive for both Ag85B and the cocktail.
Using Ag85C, we were able to detect seven more positive
patients who were negative for Ag85 complex (Table 3).
Furthermore, the areas under the ROC curves were com-
pared for all these antigens (Table 2 and Fig. 2); it was
highest for Ag85 complex (0.879), followed by CFP-10
(0.746) and ESAT-6 (0.743).
Sero-reactivity of Ag85 Complex, ESAT-6,
and CFP-10 by Employing Western Blotting
with H37Rv and Clinical Isolates of M. tuberculosis
We further analyzed the antibody reactivity to 39 clinical
isolates from different regions of India using the pooled
Fig. 1 Antibody reactivity to
various secretory antigens of
M. tuberculosis with sera
derived from different clinical
groups (FTB: fresh untreated,
DTB: defaulters, RTB: relapsed,
EPTB: extra-pulmonary TB) of
tuberculosis patients and
controls (HC: healthy controls,
LP: leprosy patients). TtTB
denotes the treated cases of
tuberculosis. Each scatter
represents a tested sample of
serum, the dotted line denotes
the cutoff decided by ROC
Arch. Immunol. Ther. Exp. (2010) 58:57–65 61

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sera from fresh TB patients. Reactivity to the Ag85 com-
plex was observed in almost all the isolates as well as in
H37Rv (data not shown). Finally, five clinical isolates were
randomly selected from all these clinical isolates, taking
their reactivity and growth profile into consideration. Using
these isolates, the seroreactivities of the individual sera
were evaluated (Fig. 3).
Significant reactivity at the 30-kDa region (Ag85
complex) in H37Rv as well as in all five clinical isolates
was observed in both the TB patients used in the
study (Fig. 3c, d) compared with two BCG-vaccinated
individuals (Fig. 3a, b). However, only one patient was
reactive to the 6-kDa region (ESAT-6) in 4 clinical iso-
lates and to the 10-kDa region (CFP-10) in 3 isolates, but
the reactivity was low (Fig. 3c, d). Interestingly, one
person with contact with TB exhibited reactivity to the 30-
kDa region (Ag85 complex) in H37Rv and in 2 clinical
isolates and to the 6-kDa region (ESAT-6) in two clinical
isolates (Fig. 3e). The reactivity to Ag85 complex in the
clinical isolates was observed to be slightly greater than
that in H37Rv when all these strains were hybridized with
antibody against Ag85 complex (Fig. 3h). Reactivity to
ESAT-6 and CFP-10 in the clinical isolates was shown
after treating the transferred proteins with anti-ESAT-6
and -CFP-10 antibody (Fig. 3g).
Discussion
The management of tuberculosis in the regions of devel-
oping countries urgently needs an efficient diagnostic test
that should be simple to perform with limited cost.
Extensive studies have been done using various antigens of
M. tuberculosis (Abebe et al. 2007; Mishra et al. 2008).
The antigens selected for the current study have been
reported to be strong targets for humoral and cell-mediated
immune response (Barnes 2004; Kanaujia et al. 2003;
Kanaujia et al. 2004; Mori et al. 2004; Samanich et al.
2001; Van Vooren et al. 1992).
Fig. 2 Area under ROC of three significant antigens with sera from
tuberculosis patients
Fig. 3 Lanes 1–5 of whole-cell extracts (WCEs) of clinical isolates
of M. tuberculosis. a, b Blotting with BCG-vaccinated healthy
individual’s sera. c–d Reactivity with individual tuberculosis patient’s
sera. e Reactivity pattern of contact with tuberculosis. f 12% SDS–
PAGE gel pattern of WCEs of clinical isolates and H37Rv.
g Hybridization of polyclonal antibody against ESAT-6 and CFP-10
with whole-cell extracts. h Hybridization of monoclonal antibody
(CS-90) against Ag85 complex with WCEs of clinical isolates and
H37Rv. Rv: M. tuberculosis laboratory reference strain H37Rv; arrow
shows the reactivity pattern of Ag85 complex
62Arch. Immunol. Ther. Exp. (2010) 58:57–65

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Studies using ESAT-6 suggested its role as a marker of
risk (Davidow et al. 2005; Silva et al. 2003) and progres-
sive TB (Demissie et al. 2006). Berthet et al. reported a
highly specific antigen present in the RD1 region, and CFP-
10 is found in the same strongly stimulatory fraction of
culture filtrate as ESAT-6, with limited distribution outside
the TB complex (Berthet et al. 1998). In our study, very
little difference between ESAT-6 (64.9%) and CFP-10
(66.0%) reactivity was observed. This finding was in sup-
port of a study in which some relatedness between ESAT-6
and CFP-10 at the amino-acid level was reported (Skjot
et al. 2000). Interaction between ESAT-6 and CFP-10
genes and the co-transcription of these antigens were also
demonstrated (Berthet et al. 1998). However, in our study,
antibody to ESAT-6 was observed in seven TB patients but
not to CFP-10 and vice versa (Table 3). This finding sug-
gests a sequence difference between CFP-10 and ESAT-6.
India is a known endemic country for tuberculosis and
leprosy and a study documented the presence of ortho-
logues of ESAT-6 and CFP-10 in other mycobacteria,
including M. leprae (Gey Van Pittius et al. 2001). There-
fore, control sera from leprosy patients were also included
in the present study to determine the cross-reactivity of
antigens in leprosy and tuberculosis. To our surprise, none
of the leprosy patients were found to have antibody
response against either ESAT-6 or CFP-10. We noted
reactivity in healthy individuals, due to which a higher
cutoff was chosen for these two antigens, which could be
one of the reasons for the low positivity among the leprosy
patients. Genes of these proteins are reported to be present
in other pathogenic mycobacteria, for example M. africa-
num, M. kansasii, M. marinum, M. szulgai (Harboe et al.
1996; Sorensen et al. 1995), and M. bovis (Harboe et al.
1996), as well as the slow-growing nonpathogenic myco-
bacterium M. gastri (Colangeli et al. 2000) and the fast-
growing nonpathogenic environmental species M. flavesens
(Harboe et al. 1996). All the healthy individuals were either
staff or students working at our institute, hence higher
reactivity to ESAT-6 could be due to the presence of cross-
reactive antibodies to these mycobacteria. In our previous
study we noted overall sensitivity and specificity of 53.01
and 91%, respectively, for ESAT-6 in pediatric TB patients
(Dayal et al. 2006).
Although Ag85 complex is reported to be cross-reactive
with other mycobacteria as well (Rinke de Wit et al.
1993), we observed the highest sensitivity (84.0%) and
85.2% specificity (Table 1) using Ag85 complex. In an
earlier report, Ag85 complex was reported to be 72%
sensitive and 100% specific in Mexican Totonac Indians
with pulmonary tuberculosis (Sanchez-Rodriguez et al.
2002). Similar findings have been confirmed by others
(Abdelnour et al. 2000). Furthermore, we observed high
antibody (60%)against Ag85 complexinpatients
undergoing anti-TB treatment, which suggests persistence
of this antigen for longer duration. A recent report also
indicates the immuno-dominant nature of Ag85 complex
(Malen et al. 2008).
The seroreactivity of Ag85A in our study was 44.68%
in TB patients and it was only 55.66% specific. This
antigen was also noted to be highly reactive with the sera
of leprosy patients (33.33%). Antibody reactivity to
Ag85B was only 33.0% and specificity 74.1% (Table 2).
Antibody response to different components of the Ag85
complex was also reported earlier (Lim et al. 1999;
Samanich et al. 2000; Van Vooren et al. 1992). Van
Vooren et al. suggested that Ag85B was the most useful
component of the Ag85 complex for the serodiagnosis of
the active form of TB (Van Vooren et al. 1991). This was
further supported by Lim et al., who showed that the
antibody responses to Ag85B and Ag85A in patients were
significantly greater than those to the Ag85C protein (Lim
et al. 1999). Ag85B (Rv1886c) was reported to be 41–94%
sensitive in various studies (Raja et al. 2002, 2005;
Steingart et al. 2009; Uma Devi et al. 2003; Vikerfors
et al. 1993). In contrast, McDonough et al. failed to detect
antibodies against Ag85B (McDonough et al. 1992). We
noted antibody to Ag85B in four more patients compared
with the Ag85 complex (Table 2). However, antibodies to
Ag85A and Ag85B have been reported to appear in
extensive disease state (Sada et al. 1990). Ag85C has also
been identified as an early marker for TB diagnosis;
reactivity to this antigen has been reported in 36% of
patients and it was reported to be 100% specific in HIV-
positive smear-positive cases of TB (Samanich et al.
2000). We noted sensitivity of 80.9% and specificity of
only 40.7% using this antigen. In contrast, Samanich et al.,
in their study on HIV-negative smear-positive TB patients,
showed 80% reactivity to this antigen (Samanich et al.
2000). In our study, Ag85C was found to be highly
reactive in leprosy patients (44.4%) among all the anti-
gens. In a very recent study we reported strong reactivity
with high specificity to Ag85C in pediatric TB patients
(Kumar et al. 2008), which could be possible because of
less exposure of pediatric patients to environmental
mycobacteria. Homologues of Ag85 complex are also
present in nonpathogenic mycobacteria (Rinke de Wit
et al. 1993) and, this being an endemic country, it is
possible that the controls taken for our study were already
exposed and had antibody to these antigens. Despite the
presence of cross-reactive epitope sequences, the stronger
reactivity of the sera from TB patients to Ag85 complex
suggests that this antigen also has some specific immuno-
dominant sequences for the diagnosis of TB.
Further in our study, the antibody reactivity of two
patients to all the clinical isolates is suggestive of simi-
lar expression of Ag85 complex genes and a relative
Arch. Immunol. Ther. Exp. (2010) 58:57–6563

Page 8

immuno-dominant nature of this antigen complex. Reac-
tivity to ESAT-6 in H37Rv and four clinical isolates was
observed in one TB patient. Furthermore, a blot with a
contact of TB also showed strong reactivity against Ag85
complex in H37Rv as well as two clinical isolates and with
ESAT-6 in two clinical isolates (out of 5) (Fig. 3e). These
results highlight the importance of Ag85 complex and
ESAT-6inminimizingthe
response of TB patients (Fig. 3c, d). Although the reac-
tivity was analyzed with only one contact of TB, this
finding indicates that exposure status could be identified
using Ag85 complex and ESAT-6. Furthermore, studies
taking more contacts of tuberculosis would be helpful for a
strong conclusion. However, the reactivity with CFP-10
was low.
Several reports indicate that the pattern of antigen
reactivity to various antibodies in serum varied greatly
from patient to patient and no antigen alone can perform
significantly for the diagnosis of TB (Abebe et al. 2007;
Lyashchenko et al. 1998). Therefore, a cocktail of these
antigens was made to minimize the possible heterogeneity
of antigen recognition by patients. The lower sensitivity
reported for the cocktail in the present study might be due
to steric hindrance or masking of some of the dominant
epitopes in the cocktail, as observed in our previous study
of pediatric patients with TB (Kumar et al. 2008).
This is the first report from northern India to show a
response to highly specific (ESAT-6, CFP-10) and
immuno-dominant antigen 85 in complex form as well as
its individual components in adult patients with tubercu-
losis. The strong antibody responses against Ag85 complex
(despite the presence of cross-reacting epitopes in BCG
and other non-tuberculous mycobacteria), ESAT-6, and
CFP-10 in all the categories of active TB suggest the
potential of these antigens. These antigens could be more
useful in a high-prevalence area of TB to develop an array
for serodiagnosis. Therefore, detailed epitope-based studies
of these secretory antigens might be promising to provide
specific sequences for the serodiagnosis of TB. Collec-
tively, these results also indicate that an array of antigens
would be more useful for developing a simple and inex-
pensive serodiagnostic test for the diagnosis of adult cases
of tuberculosis than a mixture of various antigens.
heterogeneous immune
Acknowledgments
State University, (Colorado, USA) for providing the recombinant and
native antigens of M. tuberculosis used in the present study. We thank
Dr. B. K. Girdhar, NJIL & OMD, Agra, for providing well-diagnosed
serum samples of leprosy patients, Ms Rajni Upadhyay and
Ms Bhavyata Dua, NJIL & OMD, for their help in preparing this
manuscript. The authors acknowledge ICMR and DBT, New Delhi,
for financial support and UGC-CSIR and ICMR, New Delhi, for
providing fellowships to GK and PKD, respectively. Help given by
Mr. M. M. Alam, Lab Technician, Immunology Department, NJIL &
OMD, Agra, for sample collection and Mr. Navneet Kulshreshtha,
We are grateful to Dr. John T. Belisle, Colorado
Lab Technician, Microbiology & Molecular Biology Department,
NJIL & OMD, for mycobacterial culture is also acknowledged.
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