www.elsevier.com/locate/clinchim

Clinica Chimica Acta

Application of a circulating antigen detection immunoassay for

laboratory diagnosis of extra-pulmonary and

pulmonary tuberculosis

Abdelfattah M. Attallaha,*, Sanaa Osmanb, Amr Saadb, Mohamed Omrana,

Hisham Ismaila, Gellan Ibrahima, Ahmed Abo-Nagllac

aR&D Department, Biotechnology Research Center, P.O. Box 14, 23 July St., Industrial Zone, 34517 New Damietta City, EgyptbFaculty of Science, Cairo University, Giza, Egypt

cFaculty of Medicine, Al-Azhar University, Cairo, Egypt

Received 18 October 2004; received in revised form 29 November 2004; accepted 29 November 2004

Abstract

Background: Diagnosis of extra-pulmonary tuberculosis is often difficult to establish using standard methods. Recently, a 55-

kDa mycobacterial antigen was identified in sera of individuals with pulmonary TB using a simple and rapid dot-ELISA based

on monoclonal antibody (TB-55 mAb). Here, we have evaluated the application of the dot-ELISA for the detection of target

antigen in sera of individuals with extra-pulmonary TB.

Methods: The Western blot and indirect immunoperoxidase staining was used to identify the target TB antigen using the TB-55

mAb. The dot-ELISA was used to detect the target antigen in serum samples.

Results: The target antigen was identified at 55-kDa molecular weight in serum, ascitic fluid and CSF samples from individuals

with extra-pulmonary TB. The purified antigen from these samples showed similar biochemical properties to the previously

described antigen. The target antigen was localized in areas without caseous necrosis in lymph tissues. The dot-ELISA detected

the target antigen in 90% sera of individuals with extra-pulmonary TB and in 87% sera of individuals with pulmonary TB with a

specificity of 97% among control individuals.

Conclusion: The detection of the 55-kDa antigen using dot-ELISA can be routinely employed to support clinical diagnosis of

extra-pulmonary TB and pulmonary TB.

D 2005 Elsevier B.V. All rights reserved.

Keywords: Tuberculosis; Extra-pulmonary; Diagnosis; 55-kDa; Circulating antigen; Serum enzyme immunoassay; dot-ELISA

0009-8981/$ - see front matter D 2005 Elsevier B.V. All rights reserved.

doi:10.1016/j.cccn.2004.11.036

* Corresponding author. Tel.: +20 57 402889, 20 57 403889, 20

57 404889; fax: +20 57 401889.

E-mail address: amattallah@hotmail.com (A.M. Attallah).

1. Introduction

Tuberculosis (TB) remains a leading infectious

cause of death and a leading public health priority in

356 (2005) 58–66

A.M. Attallah et al. / Clinica Chimica Acta 356 (2005) 58–66 59

the world [1]. The World Health Organization (WHO)

estimates that there are more than 8 million new cases

of TB each year, and that one-third of the world

population is infected with the bacterium Mycobacte-

rium tuberculosis and at risk for active disease [2].

Although the lung is the primary site of disease in 80–

84% of TB cases, extra-pulmonary TB has become

more common with the advent of HIV infection [3,4].

The most commonly reported extra-pulmonary sites

of the disease are the lymph nodes [5], pleura [6] and

bones or joints [7,8]. Other sites include the genito-

urinary system [9], the central nervous system [10,11],

the abdomen [12,13] and in rare cases, virtually any

other organ [14]. Extra-pulmonary TB remains an

important diagnostic and therapeutic problem. Efforts

to control the disease are currently hampered by the

lack of effective tools for the detection of infected

individuals, although new diagnostic tests are being

developed [15]. Bacteria in extra-pulmonary TB cases

can be present in low numbers at inaccessible sites

[3]. Despite rapid advances in molecular genetics for

detection of M. tuberculosis, it is clear that interest in

serodiagnosis remains high, especially for those

situations in which a specimen may not contain the

infecting agent [16]. Extensive efforts to devise a

sensitive and specific immunodiagnostic test for the

detection of M. tuberculosis-circulating antigen have

been made by several investigators [17,18]. Recently,

Attallah et al. [19] developed a simple and rapid dot-

ELISA based on IgG monoclonal antibody (TB-55

mAb) specific for a 55-kDa mycobacterial antigen and

evaluated its performance in the diagnosis of pulmo-

nary TB. In the present study, we have investigated

the presence of the target TB antigen in serum as well

as ascitic fluid, cerebrospinal fluid and lymph node

tissues of patients with extra-pulmonary TB and

evaluated the application of the developed dot-ELISA

for the detection of the target antigen in sera of

patients with extra-pulmonary and pulmonary TB.

2. Materials and methods

2.1. Samples

2.1.1. Serum samples

Serum samples of 506 individuals (383 males, 123

females; age 14–58 year) screened by radiograph at

the Department of Chest Diseases at Sayd Galal

University Hospital, Al-Azhar University, Cairo,

Egypt that approved the study were recruited. Patients

with extra-pulmonary TB (n=93) were diagnosed by

clinical symptoms, radiographic evidence and ultra-

sound, or a combination of these techniques, depend-

ing on the location of the infection in each patient.

The sites of extra-pulmonary TB include peritonitis

(n=25), meningitis (n=22), lymphadenitis (n=14),

genitourinary tract (n=19), Pott’s disease (n=5),

arthritis (n=3), sinusitis (n=3) and miliary (n=2).

None of extra-pulmonary TB patients had clinical or

radiological evidence of concurrent active pulmonary

TB. Patients with pulmonary TB (n=296) were

diagnosed by sputum smear for acid-fast bacilli

(AFB) or by culture for M. tuberculosis and all had

no prior clinical history of TB. In addition, selected

patients admitted to the hospital for a defined acute or

chronic non-TB diseases (n=69) including asthma

(n=10), ischemic heart disease (n=10), chronic

obstructive pulmonary disease (n=30), pneumonia

(n=5), bronchitis (n=5), lung cancer (n=5) and lung

infection (n=4) as well as 48 healthy volunteers (age

10–56) with no signs of clinical impairment and

normal chest radiographs were included as controls.

2.1.2. Ascitic fluid

Ascites were obtained from the 25 tuberculous

peritonitis patients (20 males, 5 females; age 45–58)

and centrifuged at 4000 rpm for 10 min. The deposits

were examined microscopically for AFB by Ziehl-

Neelsen (ZN) staining. Supernatants were coded and

used for the detection of antigen. Five patients with

non-tuberculous ascites (4 transudative and 1 exuda-

tive) negative for M. tuberculosis by smear were used

as controls.

2.1.3. Cerebrospinal fluid (CSF)

CSF samples were obtained from 22 tuberculous

meningitis patients (15 males, 7 females; age 36–50)

before antibiotic therapy. They were considered likely

to have meningitis on the basis of clinical features,

such as neck rigidity, positive Kernig’s sign and

compatible CSF biochemical parameters, e.g., ele-

vated protein levels (60–400 mg%, mean 98 mg%),

low glucose concentration (8–30 mg%, mean 23

mg%) and pleocytosis (30–700 cells/cm3) in their

CSF specimens. These patients had neither manifes-

Fig. 2. Identification of the TB-55 mAb target antigen in ascitic

fluids of patients with peritonitis tuberculosis using Western blot

Lanes 1, 2: Two patients with transudative and exudative non-

tuberculous respectively as negative controls; lanes 3, 4: two

patients with peritonitis tuberculosis. The TB-55 mAb identified a

reactive band at 55-kDa molecular weight only in the ascites and

serum samples of the patients with peritonitis tuberculosis

Molecular weight markers were not shown but indicated by arrows

A.M. Attallah et al. / Clinica Chimica Acta 356 (2005) 58–6660

tations of pulmonary TB nor had received chemo-

therapy for TB in the recent past. The CSF specimens

were collected from all patients under aseptic con-

ditions and were centrifuged at 5000�g for 30 min.

The deposits were examined by ZN staining.

2.1.4. Lymph node tissues

Paraffin sections of lymph node tissues from

patients of tuberculous lymphadenitis (n=14) were

processed for routine histological and immunohisto-

chemical examinations. Hematoxylin and Eosin

(H&E) staining was done to evaluate morphological

features (granuloma formation and caseation) and ZN

staining was done to look for AFB.

2.2. SDS–PAGE and Western immunoblotting assays

Serum, CSF, ascitic fluid and BCG (Egyptian

Organization for Biological Products and Vaccines,

Dokki, Giza, Egypt), at 30 Ag/lane were separated by

sodium dodecyl sulfate–polyacrylamide gel electro-

Fig. 1. Identification of the TB-55 mAb target antigen in sera of

patients with different types of extra-pulmonary tuberculosis using

Western blot. Lanes 1, 2: Sera from 2 healthy individuals as

negative controls. Lanes 3, 4: BCG vaccine and serum sample from

a patient with pulmonary tuberculosis; respectively as positive

controls. Lanes 5–10: Sera of 6 patients with extra-pulmonary

tuberculosis including: lymphadenitis tuberculosis (lane 5), genito-

urinary tract tuberculosis (lane 6), Pott’s disease (lane 7), arthritis

tuberculosis (lane 8), sinusitis tuberculosis (lane 9), miliary tuber-

culosis (lane 10). The TB-55 mAb identified two reactive bands at

82-kDa and 55-kDa molecular weights in BCG and one band at 55-

kDa molecular weight in serum samples of infected individuals.

Molecular weight markers (not shown but indicated by arrows)

include Phosphorylase B (97.4-kDa), Bovine serum albumin (66.2-

kDa), Glutamate dehydrogenase (55.0-kDa), Ovalbumin (42.7-

kDa), Aldolase (40-kDa), Carbonic anhydrase (31-kDa), Soya bean

trypsin inhibitor (21.5-kDa).

Fig. 3. Identification of the TB-55 mAb target antigen in

cerebrospinal fluids of patients with tuberculous meningitis using

Western blot. Lanes 1, 2: Two patients with non-tuberculous

meningitis as negative controls; lanes 3, 4: two patients with

tuberculous meningitis. The TB-55 mAb identified a reactive band

at 55-kDa molecular weight only in the CSF and serum samples of

the patients with tuberculous meningitis. Molecular weight markers

were not shown but indicated by arrows.

.

.

.

phoresis (SDS–PAGE) and immunostained using the

TB-55 mAb as previously described by Attallah et al.

[19].

2.3. Isolation and partial characterization of the 55-

kDa antigen from different sources

The 55-kDa antigen was purified from ascitic

fluids, CSF and serum samples of patients with

A.M. Attallah et al. / Clinica Chimica Acta 356 (2005) 58–66 61

extra-pulmonary TB by preparative gel electroelution.

To determine some characteristics of purified anti-

gens, samples of antigen fractions were treated with

protease and several other chemical reagents and

tested in dot-ELISA according to Attallah et al. [19].

2.4. Immunohistochemical staining

Paraffin sections (4-Am) of lymph node tissue were

stained by indirect immunoperoxidase to demonstrate

the presence of Mycobacterium antigens. Sections

Fig. 4. SDS–PAGE and capillary zone electrophoresis (CZE) analyses of th

extra-pulmonary tuberculosis. A. SDS–PAGE analyses. Lane 1: Sample

resolved proteins were Coomassie blue stained. A single band was s

electropherogram of the purified antigen from CSF. The purified antigen (2

buffer, pH 8.3, on a 65-cm�75-Am capillary, 30 kV, at 20 8C and UV de

were washed several times with 0.05 M Tris-buffered

saline with Tween 20, pH 7.6, TBS-T. Smears were

then treated with 3% H2O2 for 5 min and washed, then

TB-55mAb antibody diluted in 0.01MTBSwas added

and incubated for 30 min. Subsequently, horseradish

peroxidase conjugated goat antibody to mouse IgG

diluted 1:500 in 1.5% BSA in TBS was added and

incubated for 60 min. After that, the sections were

washed then incubated for 10 min at room temperature

in a diaminobenzidine tetrachloride substrate. Finally,

the sections were counterstained with Harris hematox-

e 55-kDa antigen purified from different body fluids of patients with

from ascites, CSF or serum; lane 2: purified antigen fraction. The

hown at 55-kDa molecular weight in purified fraction. B. CZE

5 Ag per 1 ml of distilled water) was separated with 100-mM Borate

tection at 200 nm. A single peak was shown at 11 min.

A.M. Attallah et al. / Clinica Chimica Acta 356 (2005) 58–6662

ylin, dehydrated, cleared in xylene, mounted in

Permount (Sigma) and visualized under a microscope.

Specimens of tuberculous lymphadenitis with omission

of TB-55 mAb were used as negative controls.

2.5. Dot-enzyme linked immunosorbent assay

(dot-ELISA)

The dot-ELISA as a simple and rapid assay was

used for screening of TB antigen in serum using

specific IgG TB-55 mAb according to Attallah et al.

[19]. In brief, all the assay steps run out on the surface

of nitrocellulose membrane filter fixed in a plastic

cartilage. Tested samples (500 Ag/dot) were added

onto the membrane surface. After washing, the TB-55

mAb antibody diluted in 0.01M PBS was added. The

alkaline phosphatase conjugated goat antibody diluted

in 0.05 M Tris buffer was added onto the membrane

surface. The NBT/BCIP substrate solution was used

to develop the color within 2 min. The reaction was

then stopped and the result was evaluated.

3. Results

3.1. Identification of the TB-55 mAb target antigen in

body fluids

The target antigen of the TB-55 mAb was

identified by the Western blot at 55-kDa in serum

Fig. 5. Localization of the TB-55 mAb target antigen of lymph tissue usin

(indicated by arrows) lymph tissue stained with the TB-55 mAb (�400

necrosis, was seen as solid, beaded or fragmented rods, within phagocyte

seen on ZN staining. Diffuse staining in the form of antigenic dust was

immunostaining lymph tissue (�400).

samples (Fig. 1), ascites (Fig. 2) and CSF samples

(Fig. 3) from patients with extra-pulmonary TB. In

addition to 55-kDa, a high molecular weight reactive

epitope was identified at 82-kDa in BCG vaccine, Fig.

1. The 55-kDa target antigen was purified from these

body fluids using electroelution from preparative

polyacrylamide slab gels. The antigen fractions

purified either from serum, ascites and CSF of extra-

pulmonary TB patients showed a single band at 55-

kDa in Coomassie blue stained SDS–PAGE (Fig. 4A)

and one peak when analyzed by CZE at 11 min (Fig.

4B). The reactive epitope on the purified antigens was

sensitive (i.e. showing negative result using dot-

ELISA; colorless dot) to acid and base hydrolysis,

mercaptoethanol, protease and pepsin treatments. The

purified antigens were precipitated with TCA, and the

reconstituted precipitates showed high reactivity (i.e.

showing positive result using dot-ELISA; colored dot)

toward the TB-55 mAb. In contrast, the supernatants

of purified antigens showed no reactivity. Periodate

oxidation did not affect the reactivity of the target

epitope on the purified antigens.

3.2. Localization of the target antigen of TB-55 mAb

using indirect immunoperoxidase staining

The target antigen of the TB-55 mAb was seen in

areas without caseous necrosis as solid, beaded or

fragmented rods within phagocyte cytoplasm, some of

which were similar in size and shape to AFB as seen

g indirect immunoperoxidase staining. A. Positive immunostaining

). The target antigen of the TB-55 mAb, in areas without caseous

cytoplasm, some of which were similar in size and shape to AFB as

also seen in giant cells and epithelioid cell cytoplasm. B. Negative

Table 1

Validation of dot-ELISA test for diagnosis of extra-pulmonary

tuberculosis

Group No. dot-ELISAa % Positive

Positive Negative

Extra-pulmonary tuberculosis 93 84 9 90%

Controls 117 4 113 3%

Non-tuberculous disease 69 2 67 3%

Healthy individuals 48 2 46 4%

a Rapid detection of the 55-kDa circulating antigen in serum.

A.M. Attallah et al. / Clinica Chimica Acta 356 (2005) 58–66 63

on ZN staining. Their distribution in these areas was

microanatomically similar to the distribution of AFB

as seen on ZN staining. Diffuse staining in the form of

antigenic dust was also seen in giant cells and

epithelioid cell cytoplasm (Fig. 5A). Specimens of

tuberculous lymphadenitis with omission of the TB-

55 mAb were used as negative controls (Fig. 5B).

3.3. Rapid detection of the 55-kDa circulating antigen

in sera using dot-ELISA

The performance characteristics of dot-ELISA for

the detection of the 55-kDa antigen in sera of patients

with extra-pulmonary TB are shown in Table 1. The

55-kDa circulating antigen was detected in 90% sera

of extra-pulmonary TB. Only 3% of 69 non-TB

patients and 4% of 48 healthy controls had a false-

positive antigen showing a total specificity of 97%. In

a large number of individuals with pulmonary TB

(n=296), the target antigen was detected using dot-

ELISA in 87% of their sera. All samples showing

false-negative results using the dot-ELISAwere tested

using the more sensitive Western blot and the 55-kDa

antigen was detected in all (100%) false-negative

samples.

4. Discussion

Extra-pulmonary TB is often difficult to diagnose

because of its diverse clinical presentations [15,20].

The most affordable diagnostic methods for the

clinical setting are the immunoassays, since it is rapid,

easy to perform and require simple reagents. Many

serological assays have been developed for specific

antibody detection in TB patients [17,18,21]. How-

ever, people in the tropical areas are in contact with

various pathogens and developed cross-reacting anti-

bodies responsible for poor specificity [22]. Moreover,

the sensitivity of antibody detection tests is much

lower in HIV seropositive patients coinfected with

tuberculosis [23]. In addition, changes in antigen

conformation that may occur as a result of passive

coating of the antigens to solid supports may cause

technical artifacts resulting in false-positive and false-

negative reactions [24]. Recently, more efforts are

directed toward developing reliable, and less costly

immunoassays based on the detection of mycobacte-

rial antigens in different body fluids using specific

antibodies. Such tests could be useful for the diagnosis

and follow-up of TB patients [25]. Several M. tuber-

culosis antigens were detected in different body fluids

of infected individuals, e.g., 30-kDa antigen and 31-

kDa antigen in serum [26,27], 43-kDa antigen in

ascitic fluid [28], 43-kDa antigen, antigen 5, and 14-

kDa antigen in CSF [29–31]. It is possible that such

antigens could be shed directly into the infected area

or may arise from sequestered Mycobacterium in

tissues. Moreover, the diagnostic potential of Myco-

bacterium antigen detection has been evaluated in

serum [26,32,33], ascitic fluid [28] and CSF [34,35],

with sensitivity rates of 41–93% and specificity rate of

86–100%. However, none of these tests to detect

mycobacterial antigens has become available for

clinical utility nor achieved widespread use for the

diagnosis of TB. Recently, a 55-kDa circulating

Mycobacterium antigen was identified in sera of

individuals with pulmonary TB using specific mono-

clonal antibody designated TB-55 mAb [19]. In the

present work, the 55-kDa antigen was identified in

serum, ascitic fluid and CSF samples of patients with

extra-pulmonary TB. However, it is of interest that an

antigen with similar size; 55-kDa; has not been

previously reported in serum samples of patients with

extra-pulmonary TB. The 55-kDa target antigen was

purified from serum, ascites and CSF samples of

extra-pulmonary TB patients. The protein nature of

the reactive epitope was confirmed by several experi-

ments. These purified antigens showed the same

biochemical characteristics as the previously

described 55-kDa antigen purified from sera of

pulmonary TB patients [19]. In addition to the 55-

kDa reactive epitope, a higher molecular weight

epitope was identified at 82-kDa in BCG vaccine

suggesting that the 55-kDa serum antigen may be the

A.M. Attallah et al. / Clinica Chimica Acta 356 (2005) 58–6664

stable degradation product from the higher molecular

weight antigen. However, further molecular study is

required for confirmation. Earlier studies of immuno-

histochemical staining have shown the utility of

polyclonal and monoclonal antibodies to identify M.

tuberculosis antigens in the lung, brain and lymph

node and joint specimens of tuberculous patients [34].

The distribution of the 55-kDa target antigen in areas

without caseous necrosis of lymph node tissues was as

described for other mycobacterial antigens [36].

Regardless of the mechanism by which the antigen

appears in body fluids, the detection of the 55-kDa

circulating antigen in ascitic fluid, CSF and the

presence of this antigen in serum samples of extra-

pulmonary TB will make it easier for diagnosis.

Although the ELISA system is very practical and

sensitive, the testing equipment required is not always

available in areas where tuberculosis is endemic. An

alternative to ELISA could be the dot blot method,

which uses only a paper matrix onto which the antigen

is spotted, and the development of the antigen–

antibody reaction is done by an enzyme or the use

of a colloidal gold conjugate [37]. We previously

demonstrated the use of a simple and rapid dot-ELISA

based on the TB-55 mAb for the direct detection of a

circulating Mycobacterium antigen in sera of pulmo-

nary TB [19]. The technical aspects of the dot-ELISA

can be performed very simply and the staff of a single

laboratory can easily handle large number of serum

specimens. In addition, the reagents used in the assay

have shelf life of more than 2 years, and more

importantly, the assay is reproducible. The test can

be used for the initial diagnosis of extra-pulmonary

TB such as tuberculous peritonitis, meningitis, lym-

phadenitis, genitourinary tract, Pott’s disease, arthritis

and miliary TB. In the present study, we evaluated the

sensitivity and specificity of the dot-ELISA in patients

with extra-pulmonary TB and in a large number of

patients with pulmonary TB. The reported overall high

degree of sensitivity (88%) was similar to that

reported in our previous study [19]. Moreover, all

samples showing false-negative results using the dot-

ELISA were tested using the more sensitive Western

blot and the 55-kDa antigen was detected in all

(100%) false-negative samples. The dot-ELISA test

showed few false-positive results among healthy

individuals (4%) and the selected control group of

patients with non-tuberculous diseases (3%) resulting

in a high degree of specificity (97%). This degree of

specificity was slightly higher than that (93%)

previously reported of the test [19]. The false-positive

reactions in countries where TB is endemic will

diminish the utility of any assay. Any new test should

have a specificity of N90%, otherwise there will be

significant over-treatment [24]. It is clear that our

assay can be used in populations characterized by a

high prevalence of TB. As all selected patients with

non-tuberculous diseases in our study were sputum

negative, only radiographs and clinical symptoms

were used for their diagnosis. Therefore, it is possible

that a concurrent TB infection could also be present in

some of these patients, diagnosed with circulating

antigen detection, i.e., showing false positives. How-

ever, a large number of different controls still required

ascertaining the specificity of the test. Hopefully the

high degree of specificity of the test will not be

changed. In conclusion, we have identified of the TB-

55 mAb target antigen in ascitic fluid, CSF, serum and

lymph node tissue specimens. Mycobacterium antigen

detection using dot-ELISA is sensitive, specific, rapid

and cost effective, and could find practical application

for laboratory diagnosis of pulmonary and extra-

pulmonary TB, even in the laboratories with limited

resources and limited technical expertise. Hence, we

recommend this method as a routine test for the early

and rapid diagnosis of TB in populations characterized

by a high prevalence of TB. Further studies regarding

the detection of the TB circulating antigen before and

after treatment of extra-pulmonary TB infected

patients will be performed.

Acknowledgement

The authors would like to thank Dr. Khaled Zalata,

Pathology Department, Faculty of Medicine, Man-

soura University for his kind assistance.

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