CLINICAL AND VACCINE IMMUNOLOGY, Mar. 2010, p. 372–375
Copyright © 2010, American Society for Microbiology. All Rights Reserved.
Vol. 17, No. 3
Humoral Immune Responses against the Mycobacterium tuberculosis
38-Kilodalton, MTB48, and CFP-10/ESAT-6 Antigens in Tuberculosis?
Xueqiong Wu,1†* Yourong Yang,1† Junxian Zhang,1Bangying Li,1Yan Liang,1Chuiying Zhang,1
Mei Dong,2Hongbing Cheng,1and Jufang He2
Institute for Tuberculosis Research,1and Department of Clinical Detection,2the 309th Hospital of
Chinese PLA, Beijing 100091, China
Received 13 July 2009/Returned for modification 3 August 2009/Accepted 28 December 2009
The diagnosis of smear-negative and culture-negative patients with active tuberculosis (TB) is challenging.
The detection of Mycobacterium tuberculosis-specific antibodies in human sera has been an important diagnostic
aid. However, detection of antibody responses to a single antigen usually has a low sensitivity for diagnosis of
TB. In this study, humoral immune responses against recombinant M. tuberculosis 38-kDa, MTB48, and
CFP-10/ESAT-6 (culture filtrate protein 10/6-kDa early secreted antigen target of M. tuberculosis) antigens in
250 Chinese TB patients and 260 healthy subjects were evaluated by an enzyme-linked immunosorbent assay
(ELISA). The levels of antibodies against those antigens in TB patients, even in bacterium-negative ones, were
significantly higher than those in healthy subjects (P < 0.001). The serodiagnostic sensitivities to detect
antibodies against individual antigens, i.e., recombinant M. tuberculosis 38-kDa, MTB48, and CFP-10/ESAT-6
antigens, in TB patients were 73.6%, 73.2%, and 60.4%, respectively, with specificities of 85.4%, 77.7%, and
73.8%, respectively. Importantly, the sensitivity to positively detect humoral responses to one of the antigens
increased further. Our data suggest that the humoral immune responses to M. tuberculosis antigens in TB
patients are heterogeneous. The 38-kDa, MTB48, and CFP-10/ESAT-6 antigens can be used as the cocktail
antigens in the serodiagnosis of active TB, especially for smear- or culture-negative TB cases.
The control of tuberculosis (TB) remains challenging in
China (18). Currently, the diagnosis of active TB mainly relies
on clinical symptoms, radiologic findings, and the detection of
Mycobacterium tuberculosis in clinical samples using smear
staining and mycobacterial culture. However, the diagnosis of
TB in smear- and culture-negative TB patients is difficult. The
detection of M. tuberculosis-specific antibodies in human sera
has been an important aid in diagnosis of TB. Notably, several
antigens have been demonstrated to have merit in TB diagno-
sis, including the 38-kDa protein, which is commonly used in
serodiagnostic tests (4, 5, 8, 13, 19, 22, 23). Previous studies
suggest that the antibody responses to M. tuberculosis antigens
are heterogeneous among individuals (17) so that the detection
of antibodies against a single antigen usually has a low sensi-
tivity for diagnosis of TB, especially for bacterium-negative
cases. Therefore, it may be valuable to evaluate antibodies
against the 38-kDa antigen and other major antigens for the
diagnosis of active TB (14, 15).
Notably, the MTB48, CFP-10 (culture filtrate protein 10),
and ESAT-6 (6-kDa early secreted antigen target of M. tuber-
culosis) genes are conserved in M. tuberculosis and Mycobacte-
rium bovis isolates but partially deleted or absent in M. bovis
BCG as well as in most nontuberculous mycobacteria (NTM)
(1–3, 10, 16). Importantly, the proteins encoded by these genes
are immunogenic (7, 9, 12, 16). In this study, we cloned the
38-kDa, MTB48, CFP-10, and ESAT-6 genes and generated
recombinant 38-kDa, MTB48, and CFP-10/ESAT-6 fusion
proteins in Escherichia coli. Subsequently, we developed an
enzyme-linked immunosorbent assay (ELISA) for the charac-
terization of serum antibodies against 38-kDa, MTB48, and
CFP-10/ESAT-6 antigens in a population of 250 active TB
patients and 260 healthy subjects. We found that characteriza-
tion of antibodies against multiple M. tuberculosis antigens
were valuable for the diagnosis of active TB.
MATERIALS AND METHODS
Bacterial strains. Escherichia coli strain BL21(DE3) (Invitrogen, Carlsbad,
CA) was cultured in Luria-Bertani (LB) medium. Mycobacterium tuberculosis
reference strain (H37Rv) was obtained from the National Institute for the Con-
trol of Pharmaceutical and Biological Products, Beijing, China, and cultured on
Lowenstein-Jensen slants at 37°C for 4 weeks (6).
Generation of recombinant M. tuberculosis antigens. The procedures for the
cloning, expression, and purification of M. tuberculosis 38-kDa, MTB48, and
CFP-10/ESAT-6 antigens were described previously (15, 21, 24). Briefly, the
genes encoding the M. tuberculosis 38-kDa, MTB48, CFP-10, and ESAT-6 pro-
teins were amplified by PCR using specific primers. The sequences of these
primers were as follows. For the 38-kDa antigen, the forward primer was 5?-
GGTATTCCATATGTGTGGCTCGAAACCACCGAGC-3?, and the reverse
NheI and EcoRI restriction enzyme sites, respectively, are underlined. For MTB48,
the forward primer was 5?-GCTAGCCAGTCGCAGACCGTGACG-3?, and the
reverse primer was 5?-CCCAAGCTTCTTCGACTCCTTACTGTCCT-3?. The
NheI and HindIII restriction enzyme sites, respectively, are underlined. For CFP-10,
the forward primer was 5?-CCGGATCCATGGCAGAGATGAAGAC-3?, where
the BamHI restriction enzyme site is underlined, and the reverse primer was 5?-G
GCCCATTTGCGAGGACAGCGCCT-3?. For ESAT-6, the forward primer was
GCATGACAGAGCAGCAGTGGAATTTCGCGG-3?, and the reverse primer
was 5?-CCAAGCTTTGCGAACATCCCAGTGA-3?, where the HindIII restriction
enzyme site is underlined. The PCR products of the M. tuberculosis 38-kDa and
MTB48 DNA fragments were digested with NdeI/EcoRI or NheI/HindIII en-
zymes and cloned into pET24b (Novagen, San Diego, CA), followed by trans-
* Corresponding author. Mailing address: Institute for Tuberculosis
Research, the 309th Hospital of Chinese PLA, Beijing 100091, China.
Phone: (86) 13671334568. Fax: (8610) 62582972. E-mail: wu-xueqiong
† These two authors made equal contributions to this study.
?Published ahead of print on 6 January 2010.
formation into E. coli BL21(DE3), respectively. The CFP-10 and ESAT-6 DNA
fragments were used as the templates, together with a DNA strand linker en-
ine-glycine-glycine-glycine-glycine-serine, for generating the CFP-10–ESAT-6
fused gene by PCR using the following primers: forward (5?-CCGGATCCATG
GCAGAGATGAAGAC-3?) and reverse (5?-CCAAGCTTTGCGAACATCCC
AGTGA-3?). The PCR products were digested with BamHI and HindIII, gel
purified, and cloned into pET-28a (Novagen, San Diego, CA), followed by
transformation into E. coli BL21(DE3). After DNA sequencing, individual trans-
formants were cultured in LB medium containing kanamycin (50 ?g/ml) over-
night and treated with 1 mM isopropyl-?-D-thiogalactopyranoside (IPTG) for 4 h
to induce the expression of recombinant proteins. The recombinant proteins
were purified by metal chelate column chromatography using Ni-nitrilotriacetic
acid (Ni-NTA) resin, according to the manufacturer’s protocol (Qiagen). The
concentrations of the recombinant proteins were determined by spectrometry,
and their molecular weights and purities were estimated by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie brilliant
blue, as described previously (21). Individual preparations with a purity of ? 90%
were further aliquoted at 1 mg/tube, lyophilized, and then stored at ?80°C.
Collection of blood samples. Blood samples were collected from individual TB
patients in the hospital and control subjects at the clinical laboratory of the 309th
Hospital of Chinese PLA from January to April 2007, and their sera were
prepared by centrifugation and then stored at ?80°C. The clinical data were
surveyed retrospectively. A total of 250 serum samples were selected from the
patients with pulmonary TB, including 119 sera from smear- or culture-positive
patients and 131 from smear- or culture-negative patients confirmed retrospec-
tively. The acid-fast staining of bacillus smears and mycobacterial culture of
sputa were performed in our laboratory according to the Chinese Laboratory
Science Procedure of Diagnostic Bacteriology in Tuberculosis (6). An additional
260 serum samples from healthy volunteers were selected and used as controls.
All patients and healthy controls in this study were negative for HIV antibodies.
This study was performed in accordance with the guidelines of the Research
Ethics Committee of the 309th Hospital of Chinese PLA.
PPD skin tests. The purified protein derivative (PPD) from M. tuberculosis (50
IU/ml) was purchased from Beijing Gaoke Life and Technology, China. Healthy
individuals or subjects were injected intradermally with 0.1 ml of 5 IU PPD in the
left forearm (Mantoux technique). The diameters of both axes of skin induration
were measured and recorded by a certified doctor 72 h postinjection. A positive
response to PPD was defined as an induration greater than 5 mm in diameter.
ELISA. The humoral responses of individual subjects to M. tuberculosis anti-
gens were determined by an ELISA as described previously (10). In brief,
individual recombinant proteins (10 ?g/well) were used to coat the wells in
96-well microtiter plates (Nunclon; Nunc, Roskilde, Denmark) overnight at 4°C.
After the wells were washed, they were blocked with 1% bovine serum albumin
(BSA) in phosphate-buffered saline (PBS) for 1 h at room temperature. Subse-
quently, individual serum samples (1:100) were added in triplicate to the wells
and incubated for 1 h at 37°C. After the wells were washed, the bound antibodies
were detected by horseradish peroxidase (HRP)-conjugated goat-anti-human
IgG (1:10,000) for 1 h at 37°C and developed with o-phenylenediamine and
hydrogen peroxide, followed by the addition of 2 mol/liter sulfuric acid and
measurement at an optical density of 492 nm (OD492) in an ELISA plate reader.
Data management and statistical analysis. All data were entered into a Mi-
crosoft Office Excel file. The mean and standard deviation of the optical density
of individual groups for antibody responses to each of the M. tuberculosis anti-
gens were calculated. The differences between the values for the groups were
analyzed by Student t test. Furthermore, the receiver operating characteristic
(ROC) curves of the optical density values for antibody responses to each M.
tuberculosis antigen were plotted using SPSS 9.06 software and the area under
the curves and 95% confidence intervals (95% CIs) for responses to each M.
tuberculosis antigen were calculated. In addition, the optimal cutoff values were
chosen when Youden’s index (sensitivity ? specificity ? 1) was maximum. Sub-
sequently, individuals were scored as positive for the specific antibody response
when his/her optical density value was greater than or equal to the cutoff value,
and the positive rates and 95% CIs of individual groups for antibody responses
to each M. tuberculosis antigen were calculated and differences for the values of
the groups were analyzed by ?2test.
M. tuberculosis antigens. After the DNA fragments were
cloned by PCR, the individual positive colonies, which ex-
pressed each recombinant protein with six consecutive histi-
dine residues at the carboxyl terminus, were confirmed by
DNA sequencing. Expression of the recombinant 38-kDa, re-
combinant MTB48 (rMTB48), and recombinant CFP-10/
ESAT-6 (rCFP-10/ESAT-6) antigens were induced in E. coli
BL21(DE3), and the recombinant proteins were purified using
metal-chelate affinity chromatography. Individual preparations
of recombinant protein antigens with a purity of ?90% were
used for subsequent experiments. The successful generation of
recombinant 38-kDa, rMTB48, and rCFP-10/ESAT-6 proteins
provided a basis for the characterization of antibody responses
in different groups of subjects by an ELISA.
Serum antibody reactivities to M. tuberculosis antigens. The
levels of antibodies against the recombinant 38-kDa, rMTB48,
and rCFP-10/ESAT-6 antigens in individual sera were mea-
sured by an ELISA. The levels of antibodies against the re-
combinant 38-kDa, rMTB48, and rCFP-10/ESAT-6 antigens in
TB patients were significantly higher than those in healthy
controls (P ? 0.001) (Table 1). Furthermore, antibody re-
sponses to those antigens in the bacterium-positive TB patients
were stronger than those in the bacterium-negative TB pa-
tients (P ? 0.05). Importantly, the levels of antibodies against
these antigens in bacterium-negative TB patients were signif-
icantly higher than those in healthy controls (P ? 0.001). In
addition, the levels of antibodies against the recombinant 38-
kDa antigen and rMTB48, but not rCFP-10/ESAT-6, in the
PPD-positive subjects were significantly higher than those in
the PPD-negative ones (P ? 0.05). Apparently, characteriza-
tion of antibodies against those M. tuberculosis antigens can
effectively distinguish between TB patients and healthy con-
Furthermore, individual values of antibodies against recom-
binant 38-kDa, rMTB48, and rCFP-10/ESAT-6 antigens were
used for the generation of ROC curves (data not shown). The
areas under the curves and 95% CIs were calculated in Table
2. Accordingly, the optimal cutoff values of the optical densi-
ties of antibody responses to the 38-kDa, MTB48, and CFP-
10/ESAT-6 antigens for diagnosing TB patients were 0.0852,
0.0718, and 0.0690, respectively. As a result, the sensitivities of
detecting antibody responses to the 38-kDa, MTB48, and CFP-
TABLE 1. Serum antibody responses to recombinant M. tuberculosis
38-kDa, rMTB48, and rCFP-10/ESAT-6 antigens
Serum antibody response to antigena
0.064 ? 0.04
0.082 ? 0.07c
0.073 ? 0.06
0.052 ? 0.03
0.077 ? 0.06c
0.064 ? 0.05
0.040 ? 0.03
0.049 ? 0.05
0.044 ? 0.04
0.177 ? 0.13d
0.254 ? 0.26e
0.213 ? 0.20f
0.150 ? 0.16d
0.241 ? 0.29e
0.193 ? 0.23f
0.119 ? 0.16d
0.226 ? 0.39e
0.170 ? 0.30f
aSerum antibody responses to recombinant M. tuberculosis 38-kDa, rMTB48,
and rCFP-10/ESAT-6 antigens were measured by determining the optical density
at 492 nm (OD492).
br38kD, recombinant 38-kDa antigen.
cP ? 0.05 versus the PPD?healthy subjects.
dP ? 0.001 versus healthy controls.
eP ? 0.05 versus bacterium-negative TB patients.
fP ? 0.001 versus healthy controls.
VOL. 17, 2010HUMORAL IMMUNE RESPONSES TO M. TUBERCULOSIS ANTIGENS 373
10/ESAT-6 antigens were 61.2%, 74.4%, and 73.2%, respec-
tively, and the specificities were 85.4%, 77.7%, and 73.8%,
respectively. The overall positive rates of the detection of an-
tibodies against recombinant 38-kDa, rMTB48, and rCFP-10/
ESAT-6 antigens by an ELISA were calculated in Table 3.
Clearly, the positive rates in active TB patients were signifi-
cantly higher than those in healthy controls (P ? 0.001), and
the values in the PPD-positive subjects were significantly
higher than those in the PPD-negative subjects (P ? 0.05).
Importantly, the positive rates for antibody responses to two or
more antigens were significantly higher than those to single
antigen (P ? 0.05), respectively. Therefore, characterization of
serum antibody responses to multiple antigens may be valuable
for the diagnosis of M. tuberculosis infection and TB in pa-
Although the role of humoral immunity in host defense
against M. tuberculosis is poorly understood, the M. tuberculo-
sis-specific antibodies were present in TB patients, suggesting
that serological characterization of M. tuberculosis-specific an-
tibodies may be valuable in the diagnosis of TB in humans. In
this study, we successfully generated the recombinant 38-kDa,
rMTB48, and rCFP-10/ESAT-6 proteins and evaluated their
diagnostic potential by ELISA in detecting antibodies against
M. tuberculosis antigens in sera from confirmed TB cases and
healthy controls. We found higher levels of antibodies against
individual recombinant 38-kDa, rMTB48, and rCFP-10/
ESAT-6 antigens in patients with active TB than in healthy
controls, consistent with a previous report (17). The levels of
antibodies against these antigens in bacterium-positive TB pa-
tients were significantly higher than those in bacterium-nega-
tive patients. Importantly, the levels of antibodies against these
antigens in bacterium-negative TB patients were significantly
higher than those in healthy controls, indicating their values in
the diagnosis of smear/culture-negative patients (11, 19, 20,
22). Therefore, characterization of serum antibodies against
recombinant 38-kDa, rMTB48, and rCFP-10/ESAT-6 antigens
are effective in the diagnosis of TB in M. tuberculosis smear-
and culture-negative and -positive patients in the clinic.
Further analysis revealed that the sensitivities of the ELISA
for detecting humoral immune responses to the recombinant
38-kDa, rMTB48, and rCFP-10/ESAT-6 antigens were 61.2%,
74.4%, and 73.2%, respectively, with specificities of 85.4%,
77.7% and 73.8%, respectively. Importantly, the positive rates
of the ELISA in active TB patients were significantly higher
than those in healthy controls (P ? 0.001), and the positive
rates for antibody responses to two or more antigens were
significantly higher than those to single antigen (P ? 0.05).
Therefore, characterization of antibody responses to two or
more M. tuberculosis antigens can sensitively differentiate pa-
tients with active TB from healthy controls. Notably, the sen-
sitivities for detection of antibodies against these antigens in
our assays were higher than those reported previously (5, 9, 10,
12, 13, 16). The high sensitivity may stem from different genetic
backgrounds of the individuals studied or different antigens
used in the assays. We are interested in further directly com-
paring the commercial antigens if available and the antigens we
generated in characterizing humoral immune responses.
We found that the levels of antibodies against recombinant
38-kDa or rMTB48 antigen and the positive rate in PPD-
positive healthy controls were significantly higher than those in
PPD-negative ones. These results indicated that some subjects
might be at a latent stage of M. tuberculosis infection or recov-
ering from previously unrecognized TB. Alternatively, the hu-
moral immune responses to those antigens in the PPD-positive
subjects may result from previous BCG vaccination, as infec-
tion with mycobacteria can induce long-term memory re-
sponses in humans.
A serological test with appropriate sensitivity and specificity
can have significant advantages over currently available tests.
TABLE 2. Areas under the curve of antibody responses to
M. tuberculosis antigensa
aThe P values for TB patients versus healthy controls were ?0.001.
bAUC, area under the curve.
cr38kD, recombinant 38-kDa antigen.
TABLE 3. Positive rates of antibody responses to M. tuberculosis antigens
Group and status
Positive rate of antibody response to M. tuberculosis antigen (95% CI)
Single antigen Combinations of antigens
r38kD, rMTB48, and
ar38kD, recombinant 38-kDa antigen.
bP ? 0.05 versus PPD?controls determined by ?2test.
cP ? 0.001 versus healthy controls determined by ?2test.
374WU ET AL.CLIN. VACCINE IMMUNOL.
Unlike the tuberculin skin test (TST) and Quantiferon test, Download full-text
characterization of antibody responses to M. tuberculosis anti-
gens is less invasive and dangerous (adverse effects) and saves
time. Furthermore, it does not require the patient to return to
the clinic for evaluation. In addition, the serological assay can
be developed into a user-friendly and field-ready format. No-
tably, T-cell and macrophage-related inflammation is critical
for the pathogenesis and control of TB in humans. Accord-
ingly, the results from TST and the Quantiferon test can reflect
T-cell immunity and the status of inflammation in M. tubercu-
losis-infected individuals. The Quantiferon test measures sys-
temic T-cell immunity to the purified protein derivative of M.
tuberculosis, which can help in diagnosis of latent TB infection
and evaluation of TB progression. Our findings indicated that
simultaneous characterization of antibody responses to multi-
ple M. tuberculosis antigens effectively distinguished patients
with active TB from healthy controls. However, besides the
value of TB diagnosis, the implication of positive antibody
responses to M. tuberculosis antigens in the pathogenesis and
progression of TB in patients with active TB is unclear. Given
that antibody responses to M. tuberculosis antigens usually de-
pend on T cells, the results from our experimental system may
partially reflect systemic immune responses to M. tuberculosis.
Therefore, characterization of antibody responses to multiple
M. tuberculosis antigens should aid in the diagnosis of active
TB in clinic. We are interested in further simultaneous evalu-
ation of the serological assay, TST, and the Quantiferon test in
a larger population.
In summary, our data suggest that characterization of anti-
bodies against 38-kDa, MTB48, and CFP-10/ESAT-6 antigens
can increase the sensitivity and specificity for the diagnosis of
active TB. Therefore, these M. tuberculosis antigens can be
used as cocktail antigens in the serodiagnosis of active TB,
especially for smear- or culture-negative TB cases. We are
interested in further screening and evaluating the serological
responses to other antigens to identify the optimal combina-
tions of antigens and/or polyproteins for the sensitive and
specific diagnosis of active TB.
This study was supported by the Serious Infectious Diseases Foun-
dation of China (2008ZX-10003-001) and the Beijing Science and
Technology Foundation of China (D08050700640802).
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