Vol.2, No.3, 171-176
Copyright © 2010 SciRes Openly accessible at http://www.scirp.org/journal/HEALTH/
(2010) Health
Rapid immunodiagnostic assays for Mycobacterium
Tuberculosis infection
Roba M. Talaat1*, Gamal S. Radwan2, Abdelaziz A. Mosaad3, Saleh A. Saleh4, Kalied Bassiouny1
1Department of Molecular Biology, Gen etic Eng i neering a nd Biotec hnology Research Institute (GEBRI), Minufiya University, Egypt;
2Department of Molecular Diagnosis, Genetic Engineering and Biotechnolo gy Research Institute (GEBRI), Minufiya University, Egypt
3Department of Microbiology, Faculty of Veterinary Me dicine, Minufiy a University, Egypt
4Diagsera /VACSERA , Ministry of Health, Egypt
Received 24 November 2009; revised 6 January 2010; accepted 8 January 2010.
Purpose: There is a need for a continued effort
to develop rapid immunodiagnostic assays for
tuberculosis (TB) infection with greater sensi-
tivity and specificity that can be used in the field
and in the laboratory and that can be formatted
for use with multiple species. This would help to
obtain definitive early diagnosis of TB. The
present study was developed to determine the
role of using early secreted antigenic target-6
(ESAT-6) in immunodiagnosis of Mycobacterium
tuberculosis. Methods: Serum samples were
obtained from TB infected patients and normal
healthy controls. Two rapid immunodiagnostic
assays (Enzyme-linked immunosorbent assay
(ELISA) and Immunoblotting) were performed.
Results: The sensitivity of immunoblotting assay
was 100%; however, ESA T-6 antigen was not able
to discriminate between patients and normal
controls. Application of direct ELISA using ESAT
-6 antigen yielded 97.6% sensitivity and 75%
specificity for the diagnosis of TB infection.
Conclusion: In conclusion, the detection of an-
tibodies against ESAT-6 antigen in the sera of
TB patients by direct ELISA could be used as a
preliminary assay for diagnosis of human M.
tuberculosis infection. A combination of the
ELISA with either radiological or microscopic
examination is required to overcome the low
specificity of the assay for negative results.
Keywords: Tuberculosis; Diagnosis; ESAT-6;
ELISA; Immunoblotting
Tuberculosis (TB) is a global health problem with one-
third of the world’s population latently infected with
Mycobacterium tuberculosis (MTB) and about 8 million
cases of active disease occurring each year [1,2]. Due to
the increasing numbers of persons with MTB in the last
years, the worldwide dissemination of HIV infection, the
strongest risk factor for MTB development, and the con-
tinual migration of people from areas with a high inci-
dence of TB to highly industrialized countries, MTB is
considered a global emergenc y [3-6]. In general, infection
by MTB is controlled initially by host defenses, and the
infection remains latent. However, latent TB infection has
the potential to develop into active TB at any time. Be-
cause active TB is infectious and leads to the spread of
MTB, rapid diagnosis and effective treatment of indi-
viduals with active TB are the m ost im portant com ponent
of TB control programs. Moreover, identification and
treatment of pe rsons with latent MTB infection who are at
high risk of progressing to active disease, also may con-
tribute to TB control [7].
Diagnosis of MTB based on clinical symptoms, chest
radiography and sputum microscopy is sensitive but not
specific [8]. Culture of bacteria is time-consuming, and
usually the bacillus is not cultured [9], whereas nucleic
acid-based methods such as polymerase chain reaction
(PCR) are not consistently accurate enough for the di-
agnosis of smear-negative pulmonary TB [10]. The tu-
berculin ski n te st (TS T), usin g puri fie d pr ot ein deri vati ve
(PPD), is largely used for both diagnosis and screening.
The greatest drawback of PPD is its broad cross-reactivity
with antigens derived from several mycobacterial species,
e.g., attenuate d M. bovis bacillus Calmette-Gue´rin (BCG)
used for vaccination, greatly decreasing the specificity of
the TST [1 1,12]. Moreover , 10-25% of TB patients have a
negative TST result, and this percentage increases up to
50% in patients with advanced disease or with immuno-
deficiency due to HIV coinfection [13,14]. MTB infec-
tion evokes a strong cel l -mediated immune response, and
detection of specific T cells might be a mean to detect
infection [15-17]. Because of their ease of performance
and cost effectiveness, serodiagnostic tests constitute a
R. M. Ta laat et al. / HEALTH 2 (2010) 171-176
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promise for early detection of TB. In fact, no serological
test is commonly used in the diagnosis of TB [18].
In an effort to develop more accurate diagnostic tools,
recent studies have led to the identification of the ge-
nomic segment BCG-region 1 (RD1), present in MTB but
absent from all strains as well as alm ost all environmental
mycobacteria [19-21]. Therefore, RD1 gene products
offer the potential for the development of new diagnostic
tests that may differentiate MTB infection from BCG
vaccination as well as exposure to environmental myco-
bacteria. Two secretory, low molecular mass proteins,
early secreted antigenic target 6 (ESAT-6) and culture
filtrate protein 10 (CFP-10) have been identified as a
product of this gene [22,23]. The antigens have already
shown great potential for tuberculosis diagnosis [24,25].
Thus, the purpose of this study was to evaluate the di-
agnostic potential of two rapid immunodiagnostic assays
(Immunoblotting and Enzyme-linked immunosorbent
assay (ELISA) for the detection of M. tuberculosis in-
fections using ESAT-6 antigen.
2.1. Human Sera
Forty-two serum samples from patients with pulmonary
tuberculosis were obtained from Mamora Chest Hospital
(Alexandria, Egypt). They we re diagnose d by a specialist
using smear and/or sputum culture (Ziehl-Neelsen stain)
[26] and confirmed by chest X ray. Sixteen control sera
were collected from laboratory staffs (who never suffer
from TB infection and did not get a vaccination). All
investigations were done in accordance with the Ministry
of Health, health and human Ethical Clearance Com-
mittee guidelines for clinical researches. Minufiya Uni-
versity local ethics committee approved the study pro-
to co l. All patients and controls agreed to be enrolled in this
2.2. Sodium Dodecyl
Sulphate-Polyacrylamide Gel
Electrophoresis (SDS-PAGE)
ESAT-6 antigen was kindly prepared and provided by Dr.
William C. Davis (College of Veterinary Medicine,
Washington State University, Pullman, WA, USA). Pro-
tein content was estimated by the Brad ford method [27].
Antigen was analyzed by 12% SDS-PAGE using a dis-
continuous SDS-PAGE system [28] and stained with
Coomassie brilliant blue (Sigma Chemical Co., St. Louis,
MO, USA). The protein molecular weight standard was
from invitrogen (Invitrogen Corporation Carlsbad, CA,
2.3. Immunoblotting
ESAT-6 was subjected to 12% SDS-PAGE, electro-
transferred onto 0.22µm nitrocellulose (NC) membrane
(Bio-Rad Lab oratories, Ri chmond, CA, USA) [ 29]. Elec-
trophoretic transfer was accom plished in 1h at 4°C with a
constant 200V. After electrotransfer, the sheet was cut
into 0.2cm wide strips. The resolved bands on NC mem-
brane were visualized with specific immunological de-
tection as described by [29-30]. Briefly, the antigen-
containing strips were incubated with sera of MTB in-
fected patients diluted 1:50 in blocking buffer (phos-
phate-buffered saline (PBS) containing 5% non-fat milk
and 0.3% pol yoxyethyle ne-20 (Twee n-20) (Sigm a) for 1h.
All incubations were carried out at room temperature on
an orbital shaker (Bellco, Vineland, NJ. USA). After
incubation, the NC strips were washed with hot (65C)
washing buffer (PBS/0.3%Tween-20) and incubated for
1h with peroxidase-labeled goat anti-human IgG (GAH-
GPOD) (kindl y suppli ed by Dr. Victor Tsang, Divisio n of
Parasitic Diseases, Centers for Disease Control and Pre-
vention, Atlanta, GA) diluted 1:1000. The strips were
then washed 3 times with washing buffer and once with
PBS only. Reactive bands were visualized by incubating
the NC strips with freshly prepared substrate solution
[50mg of 3, 3'-diaminobenzidine (DAB) and 5µl H2O2
(30%) dissolved in 50ml PBS, pH 7.2) (Sigma Chemical
Company, St. Louis, MO]. Positive reaction bands ap-
peared within 10 min. The reaction was then stopped
using distilled water.
2.4. Enzyme Linked Immunosorbent Assay
The ELISA was performed in 96 well flat bottomed mi-
croplates (Griener Labortechnik, Kremsmunste r, Austria).
The optimum antigen, serum and conjugate concentra-
tions and the incubation times were determined by check-
erboard titration. The wells were coated with 100 l of
ESAT-6 diluted in PBS (pH 7.2). The plates were incu-
bated 1h at 37C then overnight at 4C. After one wash
with PBS containing 0.05% Tween-20 (PBS/Tw), 200 l
of 5% non-fat milk diluted in PBS/Tw were added to each
well and incubated for 1.5 h at 37C. After one wash with
PBS/Tw, tested TB-infected and normal healthy control
sera (100 l/well) diluted in dilution buffer (PBS/Tw
containing 1% non fat m ilk) were a dded in duplicates an d
incubated at 37C for 1.5h. At the end of the incubation
period, plat es were washed 3 tim es wit h PBS/Tw and 100
l GAHG-POD were incorporated to each well at dilution
1:1000 in dilution buffer. After 4 washes with PBS/Tw,
100 l of TMB (3, 3’, 5, 5’-tetramethyl benzidine) and
H2O2 (0.02% in citric acid buffer) substrate (Kirkegrd
and Perry Lab, Caithersuburg, MY, USA) were added to
each well. The incubation time for the substrate was
15min. at room temperature. The optical density was
measured at 620nm using the UV-max ELISA plate
reader (Molecular Devices Corp.).
R. M. Talaat et al. / HEALTH 2 (2010) 171-176
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2.5. Statistical Analysis
Data are expressed as mean OD ± SD and were analyzed
using the statistical software package for social science
(SPSS). Suitable cutoff value for the ELISA was deter-
mined by receiver operating characteristics (ROC)
analysis. Comparisons of patients and normal control
groups were performed by the Student’s t-test. The dif-
ferences were considered significant if the probability
was associated with p<0.05.
3.1. Gel Electrophoresis
ESAT-6 was subjected to SDS-PAGE followed by Comassie
brilliant blue staining. As the results in Figu r e 1 showed, 2
bands were identified in ESAT-6 (7.1 and 6.5 kDa).
3.2. Immunoblotting
On screening of the TB infected sera, the im munoblotting
assay sensitivity was 100% as all TB infected patients
reacted with ESAT-6. However, the antigen was not able
to discriminate between patients and normal controls
(Figure 2).
Each serum sample was tested for its reactivities against
ESAT-6. Figure 3 showed the reactivity of TB infected
sera and uninfected controls against ESAT-6. The mean
absorbance value in the TB patients was 0.566 0.017,
significantly higher (p<0.01) than that in normal control
Figure 1. Coomassie blue stained 12% SDS-
PAGE gel of ESAT-6 antigen.
Controls TB Pat ien ts
Figure 2. Western blot profile of M. tuberculosis patients and
normal controls. ESAT (0.1 µg/mm) were separated by 12%
SDS-PAGE, electrotransferred onto NC sheets, cut into 0.2 cm
identical strips and reacted with patients and normal human
serum (last 8 strips) diluted 1:50 in PBS/0.3%Tween-20/5%
non-fat milk. Arrow pointed to the reactive band (6 kDa).
Opti cal Densi ty (OD650)
Figure 3. Reactivity of sera from M. tuberculosis infected
patients against ESAT-6 antigen (as shown by direct ELISA).
ROC Curve
1 - Specificity
Figure 4. ROC curves for ELISA with ESAT-6.
group (0.360 0.029). After ROC analysis (Figure 4),
the area under the curve and standard error of the area
under the c urve were 0. 961 (95% confi dence interval [C I],
0.918 to 1.005) and 0.022, respectively. Among different
cutoff values from the ROC analysis, a cutoff value of
0.351 was selected, as the sensitivity of 97.6 % and
specificity of 75% at the chosen cutoff was optimal for
our ELISA. Out of 42 TB infected sera, only 1 serum
sample showed a negative result at the cutoff value (1/42)
(false negative). On the other hand, out of 16 normal
R. M. Talaat et al. / HEALTH 2 (2010) 171-176
Copyright © 2010 SciRes Openly accessible at http://www.scirp.org/journal/HEALTH/
controls, 4 serum samples showed a positive resu lt at the
cutoff value (4/16) (false positive).
Along with HIV and malaria, MTB has been declared a
global enemy [31]. Delay in diagnosis is significant re-
garding not only disease prognosis at the individual level
but also transmission within the community and the
productive rate of TB epidemic [32,33]. An effective TB
control program requires early diagnosis and immediate
initiation of treatment. To control tub erculosis (TB), it is
still necessary to find diagnostic methods that are both
more rapid to carry out and more sensitive than traditional
methods (smear and culture) but which are simpler and
less expensive than the new molecular diagnostic tests
that are based on the amplification of nucleic acids. Se-
rological methods, which seem to be the ideal choice, are
usually based on the detection of free soluble circulating
antibodies against mycobacterial antigens. Thu s, there is
a need for a continued effort to develop rapid immuno-
diagnostic assays with greater sensitivity and specificity
that can be used in the field and in the laboratory and that
can be formatted for use with multiple species. Several
secreted antigens hav e been characterized, and their util-
ity for diagnostic testing as well as their attributes as
vaccine components have been exploited [34-36].
An antibody detection test can be developed into sev-
eral formats depending on the membrane, antigen(s) coat-
ing, and incubation technique. Common designs include
ELISA and immunoblotting formats (commercial sero-
logical). TST has many drawbacks, such as the need for
patients to return for test reading, as well as variability
and subjectivity in test application and reading. Most
importantly, TST has low specificity as PPD, the antig en
used for the test, is a mixture of mycobacterial antigens
also present in non tuberculous mycobacteria and in the
BCG vaccine strains [1 1]. Although both assays (TST and
ELISA) do not offer a good specificity in detection of M.
tuberculosis in fection, EL ISA has many other ad vantages
over the TST. Objective quantitative results can be ob-
tained the day after blood sampling, and time spent on
return visits to have the TST read is spared. Furthermore,
since no antigen is injected, the problem of a booster
affect on sequential skin tests is avoided. The test is
simple t o perform , and ca n be use d even i n count ries with
less-developed infrastructure.
ESAT-6 protein is secreted at an early or active phase
of mycobacterial infection. It is virtually specific for M.
tuberculosis complex representing a potential candidate
for use in early detection as substitute or as improved skin
test antigen [21-23,37-39]. Here in, we investigated the
human antibody respon se against ESAT-6 antigen for the
detection of M. tuberculosis infection using different
immunoassays (immunoblotting and ELISA). Serial
samples of sera collected from M. tuberculosis infected
patients were subjected to our developed im munoblotting
and ELISA to determine the serological response to
ESAT6. Western blot analysis showed a reactive band at
6 kDa with all serum samples. This may suggest that,
using immunoblotting format assay, ESAT-6 antigen is
not only recognized with individuals with TB infection
but also with normal healthy controls. This result merely
reflects a case of cross reaction that may returned to
shared epitope identification afforded by this assay . Thus,
our work showed that western blotting using ESAT an-
tigen is not the suitable choice for diagnosis of M. tu-
berculosis infection and just used to support its diagnosis.
On the ot her hand, applicati on of ELISA has i ncreased the
sensitivity and specificity for diagnosis of M. tuber c ulosis
infection to 97.6% and 75%, respectively.
ESAT-6, for comparison, is broadly recognized early
during disease in different species infected with M. tu-
berculosis or M. bovis [37,40,41] and this antigen is
generally reported to trigger the release of high levels of
IFN- by sensitized peripheral blood mononuclear cells
(PBMC) from TB patients [42,43]. This antigen dis-
criminates TB patients from both BCG-vacci nated and M.
avium patients and has therefore been suggested as a
candidate for in vitro TB diagnosis [42,43]. The diag-
nostic potential of low molecular weight culture filtrate
antigens (ESAT-6 and CFP-10) of M. tuberculosis has
been reported in earlier studies [24,25,44]. It has been
previously repo rted th at subjects with latent TB infection
may respond to ESAT-6 peptides and to the whole
ESAT-6 protein [45-49]. Vincenti et al. [50] dem onstrated
that, patients with active TB recognized the whole ESAT-
6 protein or PPD. The lack of response in some TB pa-
tients may be due to their particular state of immune
suppression [43-50].
Although serologic testing can detect specific anti-
bodies to mycobacteria in serum and is attractive diag-
nostic method due to its ease of application, they present
low sensitivity and specificity due to the great heteroge-
neity of the humoral response in patients with TB [51]. It
is noteworthy that, in patients suffering from AIDS, in
whom the number of T cells is decreased or even null,
determining humoral response can be an invaluable tool
in making an early diagno sis and gainin g epidemiological
control over TB [52]. Thus, even with 75% specificity,
getting 97.6% sensitivity with ESAT-6 in ELISA format
has improved the diagnostic potential of this antigen for
TB diagnosis. Imaz et al. [18] suggested the use of
multi-antigen cocktail to improve the diagnostic utility of
the ELISA. The design of antigen combinations would
achieve the high diagnostic accuracy of the assay.
In conclusion, this study offers the potential for de-
veloping relatively rapid assays (ELISA and western
blotting) for detection of M. tuberculosis infection. We
have tested the application of antibody reactivity against
ESAT-6 antigens as a serodiagnostic marker. The results
showed that the use of ESAT-6 improves the diagnosis's
R. M. Talaat et al. / HEALTH 2 (2010) 171-176
Copyright © 2010 SciRes Openly accessible at http://www.scirp.org/journal/HEALTH/
sensitivity and provide an ELISA as a simple method that
could be applied in TB diagnosis. To overcome the low
specificity of the assay, a combination of the ELISA with
either radiologi cal or mi croscopic examination is require-
ed for negative results.
The current study was supported by Minufiya University and
VACSERA. Authors would like to thank Dr. William C. Davis (Collage
of Veterinary Medicine, Washington State University, Pullman, WA,
USA) for his support by providing us with ESAT-6 antigen.
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