Mol Biotechnol (2011) 47:18–25
DOI 10.1007/s12033-010-9307-0
RESEARCH
Clinical Evaluation of a Homemade Enzyme-Linked Immunospot
Assay for the Diagnosis of Active Tuberculosis in China
Xueqiong Wu • Qiaoke Li • Yan Liang •
Yourong Yang • Junxian Zhang • Jianqin Liang •
Lan Li • Fakuan Tang • Ansheng Wang
Published online: 2 July 2010
Ó Springer Science+Business Media, LLC 2010
Abstract The rapid diagnosis of smear-negative pul-
monary tuberculosis (TB) and extrapulmonary TB is a
significant problem in clinical practice. We evaluated the
usefulness of a homemade enzyme-linked immunospot
(ELISPOT) assay for the diagnosis of active TB in China.
Seventy-eight healthy volunteers, 60 patients with active
TB, and 32 patients with non-TB diseases were evaluated
by tuberculin skin test (TST), an ELISPOT assay using a
recombinant CFP-10/ESAT-6 fusion protein (rCFP-10/
ESAT-6) as a stimulant, and T-SPOT-TB assay. The spot-
forming cells (SFC) from 78 healthy subjects containing
both PPD-positive and -negative persons was 3.7 ± 6.5.
Among 31 diagnosed TB patients, the ELISPOT assay had
a sensitivity of 67.7%, compared to a sensitivity of 77.4%
for the T-SPOT-TB assay. The ELISPOT assay was more
sensitive in smear-positive TB cases (76.9%) than in
smear-negative TB cases (61.1%), while T-SPOT-TB had
roughly similar sensitivities in smear-positive (76.9%) and
smear-negative TB cases (77.8%). The specificity was
X. Wu (&)
Q. Li
Y. Liang
Y. Yang
J. Zhang
J. Liang

A. Wang
Institute for Tuberculosis Research, The 309th Hospital of
Chinese PLA, Beijing 100091, China
e-mail: wu-xueqiong@263.net
L. Li
Department of Digestion, The 309th Hospital of Chinese PLA,
Beijing 100091, China
F. Tang
Department of Cardiovascular Internal Medicine, The 309th
Hospital of Chinese PLA, Beijing 100091, China
Q. Li
Department of Internal Medicine, Army Police Sichuan General
Hospital, Sichuan 614000, China
90.6% for ELISPOT and 78.1% for T-SPOT-TB among 32
subjects with non-TB diseases. The SFC of TB cases was
significantly higher than that of non-TB disease cases, and
the SFC of smear-positive TB cases was significantly
higher than that of smear-negative TB cases (P \ 0.01).
We confirmed that the homemade ELISPOT assay appears
more specific for the diagnosis of active TB than T-SPOT-
TB. ELISPOT assay may be a useful method for the rapid
diagnosis of active TB, especially for cases of smear-
negative TB.
Keywords ELISPOT assay
T-SPOT-TB assay

Diagnosis
Active tuberculosis
Introduction
In China, it is estimated that there are 550 million people
infected with M. tuberculosis, 4.5 million patients with
pulmonary tuberculosis (TB), and 2 million patients with
smear-positive or culture-positive pulmonary TB [1]. At
present, the rapid diagnosis of TB relies primarily on
clinical symptoms and radiologic findings, as well as the
smear and culture of clinical samples. However, 55.6% of
patients with active TB are smear- and culture-negative,
and pathological samples for detection can be difficult or
impossible to obtain in these cases. Therefore, a confirmed
diagnosis of bacterium-negative TB can be difficult.
Immunological diagnosis using blood samples has some
promise for confirming diagnosis in these cases. However,
although tuberculin skin test (TST) is widely used to detect
TB infection and aid in the diagnosis of TB, it has broad
cross-reactivity with nontuberculous mycobacteria (NTM)
and the vaccine strains of M. bovis BCG. Further, the PPD
skin test may produce a false-negative result in patients
Mol Biotechnol (2011) 47:18–25
with advanced TB or who have decreased immune function
[2]. Therefore, there is an urgent need for a more sensitive
and specific immunological tool for the diagnosis of active
bacterium-negative TB.
Recently, an in vitro T-SPOT-TB assay using pools of
early secretory antigenic target 6 (ESAT-6) and culture
filtrate protein 10 (CFP-10) peptides was manufactured and
commercialized (Oxford Immunotec, Oxford, UK) [3, 4]. It
is based on the detection of interferon-gamma (IFN-c)
released by activated T lymphocytes. The stimulants used,
ESAT-6 and CFP-10 peptides, are located within region of
difference 1 (RD1) of the M. tuberculosis and M. bovis
genomes, but is absent from all strains of M. bovis BCG, as
well as from most NTM [5–7]. This assay is highly specific
in its ability to elicit a strong T-cell response in humans
with active TB or latent TB infection (LTBI) [3, 4, 8, 9],
and holds particular promise in countries with extensive
BCG vaccination, such as China. However, they cannot be
used to distinguish between active TB and LTBI [10]. In
addition, the use of peptides increases the cost of the kit,
making it difficult to afford in developing countries.
Results from Hill et al. suggest that a fusion protein of
ESAT-6 and CFP-10 was equivalent to overlapping pep-
tides for the diagnosis of LTBI [11]. A fusion protein as the
stimulant would offer a cheaper and more realistic alter-
native for large-scale production and clinical use in the
developing countries. Therefore, the primary objective
of this study was to evaluate the clinical value of a
homemade rCFP-10/ESAT-6-based enzyme-linked immu-
nospot (ELISPOT) assay for the diagnosis of active TB in
China. To our knowledge, this is the first clinical evalua-
tion of rCFP-10/ESAT-6 as the stimulant in an ELISPOT
assay in the diagnosis of active pulmonary TB and extra-
pulmonary TB.
Materials and Methods
Participants
Two hundred and twenty participants were recruited in the
309th Hospital of Chinese PLA, Beijing 100091, China
from October 2005 through December 2007. Ethical
approval for the study was granted by the 309th Hospital of
the Chinese PLA Research Ethics Committees. Written
informed consent was obtained from the participants. The
acid-fast bacilli in the smear of sputa, bronchoalveolar
lavage fluid (BALF), urinal sediment and biopsy tissue
slices from the patients were examined by Ziehl–Neelsen
staining in our laboratory according to the protocols in the
Chinese Laboratory Science Procedure of Diagnostic
Bacteriology in Tuberculosis [12]. Patients were untreated
or had received \2 weeks of therapy at the time of
19
venipuncture for the ELISPOT assay. The clinical data
were surveyed retrospectively. Ninety-two cases for which
a final diagnosis had been determined according to the
criteria as follows: (1) TB symptoms and signs: for
example, cough, cough producing phlegm, coughing up
blood, fever, night sweats, fatigue, loss of appetite, weight
loss, chest pain, breathing difficulty, etc.; (2) TB lesion on
chest X-ray or CT; (3) positive smear or positive culture;
(4) strongly positive PPD skin test; (5) bronchoscopy; (6)
biopsy of the affected tissue and pathological examination;
and (7) the anti-TB treatment was effective [13]. They
were selected as the subjects of our research, and divided
into two groups. The TB group contained 60 TB cases,
mean age 32.3 ± 16.6, 41 males and 19 females, in which
22 patients were retrospectively confirmed smear-positive
(19 sputa, 1 BALF, 1 biopsy tissue, 1 urine sample sedi-
mented for 24 h) and 38 patients were retrospectively
confirmed smear-negative. The non-TB disease group
contained 32 subjects, mean age 52.9 ± 18.6, 19 male and
13 female. These non-TB disease controls were older than
patients with confirmed TB (P \ 0.001). Seventy-eight
new soldiers from the Chinese army in Beijing were
interviewed, routinely examined, and selected as the
healthy controls, mean age 18.8 ± 1.9; all were male.
Details of patients and controls selected in the study are
shown in Table 1.
Preparation of rCFP-10/ESAT-6 Fusion Protein
The fusion protein of CFP10 (Rv3874) and ESAT-6
(Rv3875) was engineered as described previously [14, 15].
In brief, the individual genes were amplified from
M. tuberculosis H37Rv genomic DNA by PCR. During the
amplification steps the genes were fused with a linker
encoding glycine–glycine–glycine–glycine–serine–glycine–
glycine–glycine–glycine–serine–glycine–glycine–glycine–
glycine–serine. The product was subsequently cloned into
plasmid vector pET-28a (Novagen, San Diego, CA) con-
taining a C-terminal hexahistidine tag. Sequencing was
performed to confirm the identity of the cloned DNA
fragment. The recombinant fusion protein was overex-
pressed in Escherichia coli BL21 (DE3) (Invitrogen,
Carlsbad, CA, USA) and was purified by metal chelate
column chromatography using Ni–NTA resin according to
the manufacturer’s protocol (Qiagen). Recombinant protein
batches were analyzed by 15% sodium dodecyl sulfate-
polyacrylamide gel electrophoresis (SDS-PAGE), followed
by Coomassie brilliant blue staining and Western blotting
with a murine anti-His tag monoclonal antibody (Novagen,
San Diego, CA), to confirm the size and purity of the
protein. The fusion protein was used at a concentration of
40 lg/ml in the ELISPOT assays (see below).
20 Mol Biotechnol (2011) 47:18–25

Table 1 Study participants 37°C, followed by the addition of streptavidin–alkaline

Participants Number
phosphatase conjugate (Promega, USA) for 2 h at room
temperature. After a washing step, the nitroblue tetrazolium
1. TB patients 60 (NBT) and 5-bromo-4-chloro-3-indolylphosphate (BCIP;
Pulmonary TB 51 Promega, USA) chromogenic substrate was added. The
Tuberculous meningitis 3 number of spot-forming cells (SFC) in each well was auto-
Tuberculous pleurisy 2 matically counted with a CTL-ImmunoSpotÒ S5 Versa
Tuberculous peritonitis 1 Analyzer (Cellular Technology Ltd., USA). The responses
Cervical tuberculous lymphadenitis 1 were scored as positive if the test well contained at least 17
Left renal TB 1 more SFC than the negative control well or had at least twice
Epididymis TB 1 as many as SFC as the negative control well. PHA-positive
2. Non-TB disease 32 control wells were set to at least 100 SFC/well/250,000 cells.
Gastritis or colonitis 7 Negative control wells were required to have\17 SFC.
Gastric or colonic polyps 4
Gastric or colonic cancer 3 T-SPOT-TB Assay
Connective tissue disease 3
Cholecystitis 2 The T-SPOT-TB assay (Oxford Immunotec, Oxford, UK)
Gastrorrhagia and gastric ulcer 2 for human IFN-c was performed according to the manu-
Cerebral infarction 2 facturer’s recommendations. Briefly, 250,000 PBMCs were
Thrombocytopenic purpura 1 plated for 18 h on 96-well plates, which had been pre-
Mediastinal tumor 1 coated with a mouse anti-human IFN-c antibody per well.
Upper respiratory tract infection 1 The cells were left unstimulated (negative control) or were
Pneumonia 1 stimulated with 50 ll PHA (positive control), with 50 ll of
Acute lymphocytic leukemia 1
ESAT-6 and CFP-10 peptides in separate wells. The
and cytomegalovirus infection response of stimulated cultures was considered positive
Kidney transplant operation 1 when one or both test wells contained at least six more
Multiple myeloma 1 spots than the negative control wells or had at least twice as
Rheumatoid arthritis 1 many as SFC as the negative control wells.
Hypertension and coronary 1
heart disease PPD Skin Tests
3. Healthy controls 78
TST-negative 45 PPD produced from M. tuberculosis (50 IU/ml) was pur-
TST-positive 33 chased from Beijing Gaoke Life and Technology Inc,
Total 170 China. Seventy-eight new soldiers were injected intrader-
mally in the left forearm with 0.1 ml of 5 IU PPD (Man-
toux technique) after extraction of blood for the ELISPOT.
ELISPOT Assay The diameters of both axes of skin induration were mea-
sured and recorded by a certified doctor at 72 h after
A homemade ELISPOT assay for IFN-c was performed antigen injection. Results were expressed as the mean of
according to an improved method [16, 17]. 4–5 ml of venous the diameter of induration in millimeters. A positive result
blood samples from the volunteers were taken in heparinized was defined as an induration [5 mm in diameter according
glass tubes, and peripheral blood mononuclear cells (PBMC) to the national TB guideline.
were isolated and quantified at second day after the patients
went into hospital. 250,000 cells/well of PBMC were seeded Data Management and Statistical Analysis
in 96-well plates (Millipore, USA) pre-coated with anti-IFN-
c capture monoclonal antibody (R&D corporation, USA) and All data were entered into a Microsoft Office Excel file.
stimulated with the different antigens for 20 h at 37°C in air The mean and SD of SFC of the test well minus the neg-
plus 5% CO2. 40 lg/ml of rCFP-10/ESAT-6 fusion protein ative control well in individual groups were calculated. The
was used as a specific antigenic stimulant. PBMC in medium difference between groups was analyzed by student t tests.
alone and phytohemagglutinin (PHA, Sigma, USA) at All of the significance tests were two sided, and a P value
10 lg/ml were used as negative and positive controls, of \0.05 was considered to be statistically significant. The
respectively. Biotinylated anti-IFN-c detection monoclonal sensitivity and specificity were calculated. The sensitiv-
antibody (R&D corporation, USA) was added for 2 h at ity = correct number judged in TB group/total number in
Mol Biotechnol (2011) 47:18–25
TB group, which shows the ability of the assay in correctly
diagnosing TB; the specificity = correct number judged in
the control group/total number in the control group, which
shows the ability of the assay in correctly diagnosing non-
TB disease.
Results
Development of a Homemade ELISPOT Assay
In order to optimize the homemade ELISPOT assay, we
evaluated a number of experimental conditions. First, we
determined the optimal number of cells/well by comparing
the numbers of SFC in each well using 100,000, 200,000,
and 300,000 cells/well of PBMC from a TB patient; the
results demonstrated that 200,000 cells/well produced
the ideal number of SFC (Fig. 1a). We then determined the
optimal incubation time by comparing incubation times of
10, 20, and 30 h using 200,000 cells/well of PBMC stim-
ulated by rCFP-10/ESAT-6 protein; the results showed that
20 and 30 h of incubation both produced a satisfying
number of SFC (Fig. 1b). We then determined the optimal
concentration of stimulating antigen by comparing 10, 20,
and 40 lg/ml of rCFP-10/ESAT-6 protein, using 200,000
cells/well of PBMC; the results showed that the SFC pro-
duced using 20 and 40 lg/ml of the antigens had no sig-
nificant difference (Fig. 1c). These optimization studies led
us to our final set of conditions; we used 200,000 cells/well
of PBMC, an incubation time of 20 h, and 40 lg/ml of
rCFP-10/ESAT-6 protein in all subsequent experiments.
Determination of the Cutoff Value for Positivity
Seventy-eight new soldiers were enrolled as healthy controls
and tested with the PPD skin test and the homemade ELI-
SPOT assay. The numbers of SFC were obtained by sub-
tracting the SFC in the negative control wells from the SFC in
the stimulated wells, and expressed as the mean num-
ber ± SD of SFC. The results are shown in Table 2. The
numbers of SFC from 45 PPD-negative and 33 PPD-positive
healthy subjects were 3.2 ± 6.5 and 4.3 ± 6.6, respectively,
and were not significantly different (P [ 0.05). The number
of SFC from 78 healthy subjects was 3.7 ± 6.5, and this
mean plus two SDs was 16.7 SFC. Therefore, the homemade
ELISPOT assay was scored as positive when the number of
SFC in the stimulated wells minus the number in the negative
control wells was C17, and scored as negative when the
number of SFC in the stimulated wells minus the number in
the negative control wells was \17. There were three posi-
tive ELISPOT results in 45 PPD-negative healthy subjects
and two positive ELISPOT results in 33 PPD-positive
healthy subjects. The false positive rate was 6.4% (5/78).
21
1 2 3 4
A
Control 1×105 2×105 3×105
B
Control 10h 20h 30h
C
0µg/ml 10µg/ml 20µg/ml 40µg/ml
Fig. 1 The development of optimize experimental conditions of the
homemade ELISPOT assay. Figure 1a shows the comparison of
different PBMC levels from a TB patient in the homemade ELISPOT
assay. Lane 1 is a negative control, in which 3 9 105 PBMCs were
unstimulated. Lanes 2–4 are test wells, in which 1 9 105, 2 9 105
and 3 9 105 of PBMCs were stimulated by 20 lg/ml rCFP10-ESAT6
antigen for 20 h, respectively. Figure 1b shows the comparison of
different incubation times when 2 9 105 cells/well of PBMC were
stimulated by rCFP-10/ESAT-6 protein in the homemade ELISPOT
assay. Lane 1 is a negative control, in which 2 9 105 PBMCs were
unstimulated. Lanes 2–4 are test wells, in which 2 9 105 PBMCs
were stimulated by 20 lg/ml rCFP10-ESAT6 antigen for 10, 20, and
30 h, respectively. Figure 1c shows the comparison of different rCFP-
10/ESAT-6 protein concentrations when 2 9 105 cells/well of PBMC
were stimulated in the homemade ELISPOT assay. Lane 1 is a
negative control, in which 2 9 105 PBMCs were unstimulated. Lanes
2–4 are test wells, in which 2 9 105 PBMCs were stimulated by 10,
20, and 40 lg/ml of rCFP10-ESAT6 antigen for 20 h, respectively
Comparison of the Homemade ELISPOT Assay
with the T-SPOT-TB Assay
Using clinical data from our retrospective survey, we selected
31 confirmed TB cases and 32 non-TB disease cases and
compared available data from the homemade ELISPOT assay
and T-SPOT-TB assay at the same time. The results are shown
in Tables 3 and 4, respectively. 31 TB cases (30 cases of
pulmonary TB and one case of tuberculous pleurisy) were
classified as having either smear-positive (41.9%, 13/31) or
smear-negative (58.1%, 18/31) TB. For these TB cases, the
diagnostic sensitivity of the ELISPOT assay was 67.7%
overall, and it was more sensitive in smear-positive TB cases
(76.9%) than in smear-negative TB cases (61.1%). The
diagnostic sensitivity of the commercial T-SPOT-TB assay
22 Mol Biotechnol (2011) 47:18–25

Table 2 Comparison of homemade ELISPOT assay and PPD skin test in 78 healthy subjects
Diameter (mm) Number No. of positive Mean number
(mean ± SD) of cases (rate of positivity) of SFC ± SD

PPD skin test \5 (0.49 ± 1.12) 45 3 (6.7%) 3.2 ± 6.5

C5 (11.5 ± 3.6) 33 2 (6.1%) 4.3 ± 6.6a
Total 5.17 ± 6.03 78 5 (6.4%) 3.7 ± 6.5
a -
P [ 0.05 versus the PPD healthy subjects

Table 3 Comparison of the homemade ELISPOT assay with T-SPOT-TB assay in distinguishing TB cases from cases of non-TB disease
Diagnosed Smear No. of Homemade ELISPOT assay T-SPOT-TB assay
cases cases
No. of SFC ESAT-6 peptides CFP-10 peptides ESAT-6
positive peptides ? CFP-
cases 10 peptides
(positive No. of SFC No. of SFC No. of positive
rate) positive positive (positive rate)
(positive (positive
rate) rate)

Non-TB 32 3 (9.4%) 4.3 ± 7.6 6 (18.8%) 7.1 ± 12.7 2 (6.3%) 5.5 ± 19.1 7 (21.9%)
disease
TB ? 13 10 (76.9%) 88.8 ± 131.1a 9 (69.2%) 67.8 ± 103.5a 7 (53.8%) 103.5 ± 163.5a 10 (76.9%)
(41.9%)
- 18 11 (61.1%) 39.3 ± 47.6a,b 13 (72.2%) 31.4 ± 45.2a,b 13 (72.2%) 44.5 ± 69.8a,b 14 (77.8%)
a a
Total 31 21 (67.7%) 60.1 ± 93.7 22 (71.0%) 46.7 ± 78.5 20 (64.5%) 69.2 ± 119.7a 24 (77.4%)
a
P \ 0.001 versus the subjects with non-TB disease
b
P \ 0.001 versus the culture-positive TB patients

Table 4 Relativity between the homemade ELISPOT assay and T-SPOT-TB assay used in 31 TB cases
Homemade ELISPOT assay T-SPOT-TB assay Total
ESAT-6 peptides ? ESAT-6 peptides ? ESAT-6 peptides - ESAT-6 peptides -
CFP-10 peptides ? CFP-10 peptides - CFP-10 peptides ? CFP-10 peptides -

CFP-10/ESAT-6 protein ? 15 2 1 3 21
CFP-10/ESAT-6 protein - 3 2 1 4 10
Total 18 4 2 7 31
Note: Data are number of subjects

was 77.4%, with little difference in sensitivity between
the smear-positive (76.9%) and smear-negative TB cases
(77.8%). For non-TB disease subjects, the specificities of the
ELISPOT assay and T-SPOT-TB assay were 90.6 and 78.1%,
respectively. The SFC of TB cases was significantly higher
than that of non-TB disease cases (P \ 0.001). Likewise, the
SFC of smear-positive TB cases was significantly higher than
that of smear-negative TB cases (P \ 0.001).
Clinical Diagnostic Value of Homemade ELISPOT
Assay for TB
Using the clinical data from the retrospective survey, 78
healthy controls, 32 non-TB disease cases, and 60 TB cases
with available data from the homemade ELISPOT assay
were evaluated. The results are shown in Fig. 2 and
Table 5. 60 TB cases (51 pulmonary TB and nine extra-
pulmonary TB cases) were classified as having either
smear-positive (36.7%, 22/60) or smear-negative (63.3%,
38/60) TB. Of 51 pulmonary TB cases, the sensitivity of
smear for AFB was 41.2%. Total diagnostic sensitivity of
ELISPOT assay was 56.7%. For pulmonary TB cases, the
diagnostic sensitivity was 60.8%. It identified more smear-
positive TB cases (66.7%) than smear-negative TB cases
(56.7%). The diagnostic sensitivity in pulmonary TB was
significantly higher than that in extrapulmonary TB
(33.3%). The sensitivity of the combined smear with
ELISPOT in pulmonary TB was 74.5% (38/51). Although
the SFC of smear-positive cases with pulmonary TB was
higher than that of smear-negative cases with pulmonary
TB and extrapulmonary TB cases, there was no significant
difference between them (P [ 0.05).
Mol Biotechnol (2011) 47:18–25 23

1 2 3 that immunological diagnostic methods may have utility in

A The T-SPOT-TB assay has a higher specificity for the
B discriminate between active TB and LTBI [20]. T-SPOT-TB
C In this study, a homemade ELISPOT assay was devel-
Fig. 2 This figure shows representative results of the homemade
ELISPOT assay from three patients. Figure 2a shows a negative
result from a non-TB disease patient. Figure 2b shows a negative
result from a TB patient. Figure 2c shows a positive result from a TB
patient. Lane 1: negative control, in which 2 9 105 PBMCs were
unstimulated; lane 2: positive control, in which 2 9 105 PBMCs
were stimulated by PHA; lane 3: test well, in which PBMCs were
stimulated by rCFP10/ESAT6 antigen
Discussion
The rapid diagnosis of smear-negative pulmonary TB and
extrapulmonary TB is a significant problem in clinical
practice. Cell-mediated immunity plays a key role in the host
defense against M. tuberculosis infection [18], suggesting
the diagnosis of these difficult cases. At present, the TST and
the commercial T-SPOT-TB kit are used to diagnose LTBI.
diagnosis of LTBI than the TST [19]. The M. tuberculosis-
specific IFN-c release assays are not influenced by prior BCG
vaccination and have lower cross-reactivity to NTM [5–7].
However, China is a country with a high TB incidence and
high TB infection rate, and has a policy of BCG vaccination.
Neither the TST nor T-SPOT-TB can clearly and rapidly
for the diagnosis of active TB has a higher sensitivity (85–
95%) but lower specificity (68–80%) [20, 21]. However, a
negative T-SPOT-TB result can assist in excluding TB and is
helpful for differential diagnosis of non-TB diseases.
oped. We used a CFP-10/ESAT-6 fusion protein as the
stimulating antigen. It is important to note that this fusion
protein was different from the ESAT-6/CFP-10 fusion
protein used by Hill et al. [11] and is much cheaper to
prepare and detect than bulk peptides. We evaluated the
sensitivity and specificity of this ELISPOT assay using
confirmed cases of TB and controls. The SFC in the TB
patients was significantly higher than that in the healthy
control group, which contained both subjects with and
without LTBI (Table 2). Therefore, we selected the mean
number plus two SDs as the diagnostic cutoff value for
active TB. The specificity of the ELISPOT assay in 110
controls was 95.5%. Its sensitivity in 51 subjects with
pulmonary TB was 60.8%, which was higher than the
sensitivity in those with extrapulmonary TB (33.3%). This
sensitivity was higher than that of the AFB smear, which
only identified 41.2% of the pulmonary TB cases and
11.1% of the extrapulmonary TB cases. The results suggest

Table 5 Detection of TB cases with homemade ELISPOT assay

Diagnosed cases Smear No. of cases Homemade ELISPOT assay
No. of positive cases SFC (stimulated well minus
(positive rate) negative control well)

TB cases 60 34 (56.7%) 54.4 ± 84.4

Pulmonary TB 51 31 (60.8%) 57.1 ± 87.5
? 21 (41.2%) 14 (66.7%) 79.1 ± 117.0a
- 30 17 (56.7%) 41.7 ± 56.2
Extrapulmonary TB 9 3 (33.3%) 38.7 ± 65.6
Tuberculous meningitis - 3 1 (33.3%) 65.3 ± 111.4
Tuberculous pleurisy - 2 0 (0.0%) 8.0 ± 7.1
Cervical tuberculous lymphadenitis - 1 1 (100.0%) 88.0
Epididymis TB - 1 1 (100.0%) 88.0
Left renal TB ? 1 0 (0.0%) 0.0
Tuberculous peritonitis - 1 0 (0.0%) 0.0
a
P [ 0.05 versus the culture-negative TB patients or extrapulmonary TB patients
24
that our ELISPOT assay has potential utility not only in the
diagnosis of pulmonary TB, but also in the diagnosis of
extrapulmonary TB. Losi et al. [21] used T-SPOT-TB to
diagnose active tuberculous pleurisy, and found that the
sensitivity was very high (90 and 95% when performed on
PBMCs and pleural effusion mononuclear cells, respec-
tively), though the specificity was low (67 and 76% on
PBMCs and pleural effusion mononuclear cells, respec-
tively). We plan to further evaluate the diagnostic value of
our ELISPOT assay in extrapulmonary TB.
As far as we are aware, Hill et al. [11] were the first to
use an ESAT-6/CFP-10 fusion protein as a reagent to
monitor natural M. tuberculosis infection in humans. Our
study is the first formal assessment of the use of a CFP-10/
ESAT-6 fusion protein as the stimulating antigen in an
ELISPOT assay to diagnose active TB. Compared with the
T-SPOT-TB assay, the sensitivity of our ELISPOT assay
was lower (67.7 vs. 77.4%), but its specificity was higher
(90.6 vs. 78.1%) (Table 3). This is because the T-SPOT-
TB assay has a lower cutoff value of SFC such that it
includes the diagnosis of LTBI, while we chose a higher
cutoff value for our ELISPOT assay in order to specifically
diagnose active TB disease. Several studies have reported
that the specific IFN-c released from effector T-cells
detects a current or more active infection, and may wane
considerably with time after infection or anti-TB treatment
in TB patients and LTBI cases [22–24]. The persons with
higher SFC produced by more TB-specific effector T-cells
may present active TB or ‘‘recent’’ or ‘‘active’’ TB infec-
tion. Our results suggest that our ELISPOT assay can
separate active TB from LTBI and other non-TB diseases
with a high diagnostic sensitivity and specificity. The
sensitivities of our ELISPOT assay and the T-SPOT-TB
assay were similar in the smear-positive TB cases, while
the sensitivity of ELISPOT assay was lower in the smear-
negative cases. Of 31 TB cases, CFP-10 peptides had a
lower rate of positivity than ESAT-6 peptides (Table 4),
suggesting that ESAT-6 may be the immunodominant
antigen in China. These results are consistent with results
from a study in Gambia [11]. Both the CFP-10 and the
ESAT-6 antigens are encoded within a single operon, and
are expressed simultaneously at similar ratios [25].
Therefore, the differences in immune responses to indi-
vidual CFP-10 and ESAT-6 antigens may be explained by
polymorphisms in the HLA type in the host population, or
by differences in M. tuberculosis strains in different
countries. Six subjects were peptide positive and fusion
protein negative, and two were fusion protein positive and
peptide negative (Table 4). The differences in immune
responses to peptide antigens and the fusion protein might
result from differences in structure between peptides and
the fusion protein, leading to the presentation of different
epitopes.
Mol Biotechnol (2011) 47:18–25
The mean SFC of the fusion protein was significantly
higher than the count for ESAT-6 peptides, but lower than
CFP-10 peptides (Table 3). These findings suggest that the
antigenicity of CFP-10 peptides is stronger than the fusion
protein, and that of the ESAT-6 peptides is the weakest.
Jafari et al. found that the number of CFP-10-specific cells
in PBMC or BALF (24.5 and 49.5) was higher than the
number of ESAT-6-specific cells (17 and 37.5) [9]. Simi-
larly, Losi et al. found that the number of CFP-10-specific
cells in PBMC and pleural effusion mononuclear cells (25
and 98) was higher than the number of ESAT-6-specific
cells (22 and 61) [21]. The SFCs in the smear-positive TB
cases were higher than that in the smear-negative TB cases
using both the ELISPOT and T-SPOT-TB assays (Table 3).
Therefore, it appears that a higher number of SFCs may
correlate with a greater likelihood of smear positivity.
However, some smear-positive TB patients had negative
ELISPOT results, which could indicate lower cell-medi-
ated immune function in these patients. Combining the
smear with the ELISPOT assay in the diagnosis of pul-
monary TB has the potential to increase the sensitivity of
diagnosis. However, of 110 controls, five healthy subjects
and three non-TB patients (one multiple myeloma, one
cerebral infarction, and one gastric polyps) had positive
ELISPOT results for PBMCs (range 17–31 SFC). We
hypothesize that these subjects may have LTBI, and are
developing another cutoff value of SFC for the diagnosis of
LTBI.
Conclusion
Improved sensitivity for pulmonary TB was gained by
combining the ELISPOT assay with the smear. Therefore,
it may be used as a diagnostic aide for active TB, especially
smear-negative TB, and could be routinely used in
unconfirmed cases in clinical laboratories. We plan to
perform further studies to ascertain the potential utility of
this tool in the diagnosis of extrapulmonary TB, as well as
in patients with immunodeficiencies.
Acknowledgments This work was supported by the Serious
Infectious Diseases Special Foundation of China (2008ZX-10003-
001), and Chinese PLA Medical and Science Research Foundation
(No. 08G130).
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