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A newer approach for the laboratory diagnosis of tuberculous meningitis
Diagnostic Microbiology and Infectious Disease 39 (2001) 225–228
www.elsevier.com/locate/diagmicrobio
Mycobacteriology
A newer approach for the laboratory diagnosis of tuberculous meningitis A. Mathaia, V. V. Radhakrishnana,*, S. M. Georgea, C. Saradab a b
Department of Pathology, Sree Chithra Tirunal Institute for Medical Sciences &Technology, Thiruvananthapuram – 695 011, Kerala State, India Department of Neurology, Sree Chithra Tirunal Institute for Medical Sciences &Technology, Thiruvananthapuram – 695 011, Kerala State, India Received 11 September 2000; accepted 15 March 2001
Abstract In this prospective study, a simple method was standardized for measuring circulating mycobacterial antigen in the cerebrospinal fluid (CSF) for the laboratory diagnosis of tuberculous meningitis (TBM). The heat-inactivated CSF specimens from tuberculous and nontuberculous patients were subjected to sodium dodecyl sulfate (SDS) - polyacrylamide gel electrophoresis (PAGE) (SDS-PAGE) and they were subsequently transferred onto nitrocellulose membrane (NCM) Using a rabbit polyvalent antibody to M tuberculosis, a heat stable 82 kDa mycobacterial antigen was demonstrated in the CSFs of patients with TBM. This antigen was conspicuous by its absence in the CSFs of non-tuberculous subjects. Due to inactivation of CSF specimens, there is a minimal risk of handling of infectious material in the laboratory. Besides, this newer approach is simple, inexpensive and can be readily applied in any routine clinical laboratory and it is particularly suited to developing countries. © 2001 Elsevier Science Inc. All rights reserved. Keywords: Tuberculous meningitis; Mycobacterium tuberculosis; Immunodiagnosis; 82 k Da antigen; Cerebrospinal fluid; Heat inactivation
1. Introduction TBM is one of the common clinical and morphologic manifestations of extrapulmonary tuberculosis in developing countries. Currently the global prevalence of the disease has increased following HIV epidemic (Berenguer et al., 1992). Rapid confirmatory laboratory diagnosis of TBM and initiation of appropriate anti tuberculosis chemotherapy (ATT) is essential in reducing the mortality and morbidity rates in patients with TBM. Direct demonstration of acid fast - bacilli in CSF specimens by Zeihl- Neelsen stain and isolation of M tuberculosis by the conventional culture method are still considered as ‘Gold standard’ for the laboratory diagnosis of TBM. However these direct microbiologic techniques in CSF specimens are less sensitive for the diagnosis of TBM. Even short-term radiometric methods i.e., BACTEC culture, is less sensitive for the isolation of M * Corresponding author. Tel.: ⫹91-0471-524508; fax: ⫹91-0471446433. E-mail address: [email protected] (V.V. Radhakrishnan).
tuberculosis in CSF (Bonington et al., 1998). As an adjunct for the laboratory diagnosis of TBM, several immunologic and molecular biologic techniques have been described earlier (Krambovitis et al., 1984; Sada et al., 1983; Kox et al., 1995). In this report, we describe an immunoblot method to detect 82 kDa mycobacterial antigen in the heat-inactivated CSF of patients with TBM. The utility of this approach is that, this is simple, rapid and it has practical application for the laboratory diagnosis of TBM in developing countries where the laboratory resources and technical expertise are limited.
2. Materials and methods 2.1. Patients and collection of clinical specimens CSF specimens were obtained from 30 patients admitted to the Sree Chitra Tirunal Institute for Medical Sciences, Thiruvananthapuram, Kerala State, India, with a clinical
0732-8893/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved. PII: S 0 7 3 2 - 8 8 9 3 ( 0 1 ) 0 0 2 3 0 - 9
226
A. Mathai et al. / Diagnostic Microbiology and Infectious Disease 39 (2001) 225–228
diagnosis of TBM. As this Institute is one of the tertiary referral center for neurologic diseases, the time interval between the collections of CSF specimens from the onset of disease varied considerably in most of the patients. A provisional diagnosis of TBM was made on the basis of relevant clinical manifestations, elevated protein levels and pleocytosis in CSF specimens. In five patients, the diagnosis of TBM was confirmed by the isolation of M tuberculosis in CSF specimens by the conventional bacteriologic cultures (Lowenstein–Jensen medium). In the remaining 25 patients, a repeat CSF cultures for M tuberculosis, fungi, and pyogenic bacteria were reported to be negative on two occasions. PCR was also simultaneously performed in the CSFs of patients with TBM. Although there was no microbiologic confirmation, these 25 patients were clinically regarded as ‘probable’ TBM. Patients with ‘confirmed’ and ‘probable’ TBM received (ATT) for 2– 4 weeks during their hospital study. CSF specimens from 40 patients with non- tuberculous neurologic diseases formed the control group [Partially treated pyogenic meningitis (n ⫽ 14), viral encephalitis (n ⫽ 8); CSF collected at myelography (n ⫽ 8); demyelinating disorder (n ⫽ 10)]. Cisternal CSF, collected at an autopsy from a CSF culture positive case of TBM was used as the positive control in this study. 2.2. Heat inactivation of CSF samples 2 mL of CSF from TBM and control groups were autoclaved at 15 lbs/sq inches for 30 min. The protein in CSF specimen was precipitated by 50% ammonium sulfate and centrifuged at 3500 rpm for 30 min. The precipitate was reconstituted to 200L in 0.15M PBS after removing the ammonium sulfate by continuous dialysis for 24 h.
3. Tuberculin purified protein derivative antigen (PPD) Tuberculin PPD was prepared from the culture filtrate of the H37Ra strain of M tuberculosis and polyvalent antibody to M. tuberculosis was raised as described earlier (Mathai et al., 1989; Radhakrishnan et al., 1990).
4. SDS- PAGE and electrotransfer SDS- PAGE (Laemmli, 1970) was performed using vertical electrophoretic unit (Mighty small, Hoefer). Tuberculin PPD antigen 2g/slot, 200L of heat inactivated CSFs from TBM and control group were subjected to discontinuous SDS-PAGE and subsequently transferred onto nitrocellulose membrane (NCM) (Towbin et al., 1979). Follow-
ing the transfer, the NCM strips were cut at 4 mm width and the strips were stored at 4°C until used for immunostaining.
5. Immunostaining NCM strips containing tuberculin PPD antigen, heat inactivated CSF from ‘confirmed’, ‘probable’ TBM and control groups were thoroughly washed with 0.15M PBS and then quenched with 2.5% skimmed milk for 1 h. All the incubations in the assay were carried at 40°C. The NCM strips were washed with 0.15 M PBS and incubated with (1: 500) polyvalent rabbit IgG to M tuberculosis for 2 h, followed by (1:600) anti rabbit IgG - biotin conjugate (Sigma) for 2 h. The NCM strips were then washed and incubated with (1: 400) extr-avidin HRP (Sigma) for 2 h. Subsequently the strips were treated with a substrate, containing 3 mg of 4-chloro-1-naphthol in 1 mL of cold methanol and 3l of 30% hydrogen peroxide in 4 mL of PBS. A positive reaction was indicated by development of a clean blue band in the NCM strip (Fig. 1).
6. Result Fig. 1 depicts the results of this study. Tuberculin PPD antigen demonstrated four mycobacterial antigens possessing molecular weights of 35, 45, 66 and 82 kDa respectively. The CSF from ‘confirmed’ TBM patients contained 45, 66 and 82 kDa antigens, of which 82 kDa antigen appeared conspicuous. In 16 out of 25 patients with ‘probable’ TBM, 82 kDa antigen was also demonstrated in the NCM strips. In the remaining 9 patients in the ‘probable’ TBM, 82 kDa antigen was absent. The CSFs from control group showed both 38 and 66 kDa antigens but 82 kDa was absent in all of them. Based on this observation in NCM, we regard that; the presence of 82 kDa antigen in the heat inactivated CSF in a patient will distinguish tuberculous from non-tuberculous etiology. All those TBM patients with 82kDa antigen in their heat-inactivated CSFs were regarded as positive for tuberculous etiology. Accordingly five ‘confirmed’ patients with TBM and 16/25 patients with ‘probable TBM were treated with ATT during their hospital stay and they showed optimal neurologic recovery at the time of discharge from the hospital. Nine out of the 25 ‘probable’ TBM did not show 82 kDa antigen in their CSfs. Hence they were not regarded as TBM and ATT was withdrawn. These nine patients were extensively investigated to establish the cause of meningitis. Despite of that the etiology of meningitis remained undetermined in them.
7. Discussion The data of this study highlight three relevant observations. Firstly, heat stable 82 kDa mycobacterial antigen was
A. Mathai et al. / Diagnostic Microbiology and Infectious Disease 39 (2001) 225–228
227
Fig. 1. Showing the immunoblot in PPD, TBM and non TBM groups. Note the presence of 82 kDa antigen in only CSFs of TBM patients
seen in the CSF of 5/5 ‘confirmed’ and 16/25 ‘probable’ TBM patients and was absent in the CSF of control group. Thus assay carried high degree of specificity for tuberculous etiology. Secondly, the assay can easily be performed in any routine clinical laboratory and does not require any expensive equipment. Thirdly, heat inactivation of CSF specimens considerably reduced the risk of handling of infectious material in the laboratory. This becomes relevant, because of the increasing prevalence of tuberculosis among HIV patients. The rabbit antibody to M tuberculosis can easily be stored at -20°C for at least 6 months without any decrease in their titers. The applications of the recent techniques like PCR, for the routine laboratory diagnosis of TBM, still remain speculative. In this be study PCR was performed simultaneously in all 30 CSFs from patients with TBM and showed an overall sensitivity of 33.3% and also required 72 h to perform in the laboratory. By contrast, the technique as described in this study required only 48 h to perform and
Table 1 Showing the results of Immunoblot and PCR Immunoblot Confirmed TBM (n ⫽ 5) Probable TBM (n ⫽ 25) Control Group Sensitivity (%) Confirmed Probable Specificity (%) Duration of the assay
PCR
5 16 0
3 7 3
100 64 100 48 h
60 28 66 72 h
more importantly the assay could be done in any routine clinical laboratory (Table 1) and is best suited to laboratories in developing countries. Hence we advocate this new method for rapid laboratory diagnosis of TBM. Acknowledgments The authors are grateful to the Director, Sree Chithra Tirunal institute for Medical Sciences and Technology, Thiruvananthapuram, India for providing all the facilities to undertake this study and also for the kind permission to publish this paper. The authors are also indebted to Miss Susha Kumari for her excellent secretarial assistance. References Berenguer, J., Moreno, S., Laguna, F. (1992). Tuberculous meningitis in patients infected with the human immuno deficiency virus. N Engl J Med 36, 668 – 672. Bonington, A., Strang, J. I. G., Klapper, P. E., Hodd, S. V., Rubombora, W., Penny, M., Willers, R., & Wilkins, E. G. L. (1998). Use of Roche Amplicor Mycobacterium tuberculosis PCR in early diagnosis of Tuberculous meningitis. J Clin Microbiol 36, 1251–1254. Krambovitis, E., Mc Illmurray, M. B., Lock, P. E., Hendrickse, W., & Hozel, H. (1984). Rapid diagnosis of tuberculous meningitis by latex particle agglutination. Lancet, ii1229 –1231. Sada, E., Riuz Palacio, G. M., Lopex- Videl, Y., & Ponce De Leon, S. (1983). Detection of mycobacterial antigens in cerebrospinal fluid of patients with tuberculous meningitis by enzyme –linked immunosorbent assay. Lancet ii:651– 652. Kox, F. F., Kuijper, S., & Kolk, H. J. (1995). Early diagnosis of tuberculous meningitis by polymerase chain reaction. Neurology, 45, 2228 – 2232. Mathai, A., Radhakrishnan, V. V., Sehgal, S., & Mohan, P. K. (1989). Enzyme linked immunosorbent assay for IgG antibody to tuberculin
228
A. Mathai et al. / Diagnostic Microbiology and Infectious Disease 39 (2001) 225–228
purified protein derivative in cerebrospinal fluid specimens of patient with tuberculous meningitis. Indian J Med Microbiol, 7, 98 –104. Radhakrishnan, V. V., Mathai, A., Rao, B. S., & Sehgal, S. (1990). Immunoelectrophoresis of mycobacterial antigens. Indian J Exp Biol, 28, 812– 815.
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head bacteriophage T4. Nature (London), 227, 680 – 685. Towbin, H., Stanelin, J., & Gorden, J. (1979). Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc Natl Acad Sci (USA), 76, 4350 – 4354.
www.elsevier.com/locate/diagmicrobio
Mycobacteriology
A newer approach for the laboratory diagnosis of tuberculous meningitis A. Mathaia, V. V. Radhakrishnana,*, S. M. Georgea, C. Saradab a b
Department of Pathology, Sree Chithra Tirunal Institute for Medical Sciences &Technology, Thiruvananthapuram – 695 011, Kerala State, India Department of Neurology, Sree Chithra Tirunal Institute for Medical Sciences &Technology, Thiruvananthapuram – 695 011, Kerala State, India Received 11 September 2000; accepted 15 March 2001
Abstract In this prospective study, a simple method was standardized for measuring circulating mycobacterial antigen in the cerebrospinal fluid (CSF) for the laboratory diagnosis of tuberculous meningitis (TBM). The heat-inactivated CSF specimens from tuberculous and nontuberculous patients were subjected to sodium dodecyl sulfate (SDS) - polyacrylamide gel electrophoresis (PAGE) (SDS-PAGE) and they were subsequently transferred onto nitrocellulose membrane (NCM) Using a rabbit polyvalent antibody to M tuberculosis, a heat stable 82 kDa mycobacterial antigen was demonstrated in the CSFs of patients with TBM. This antigen was conspicuous by its absence in the CSFs of non-tuberculous subjects. Due to inactivation of CSF specimens, there is a minimal risk of handling of infectious material in the laboratory. Besides, this newer approach is simple, inexpensive and can be readily applied in any routine clinical laboratory and it is particularly suited to developing countries. © 2001 Elsevier Science Inc. All rights reserved. Keywords: Tuberculous meningitis; Mycobacterium tuberculosis; Immunodiagnosis; 82 k Da antigen; Cerebrospinal fluid; Heat inactivation
1. Introduction TBM is one of the common clinical and morphologic manifestations of extrapulmonary tuberculosis in developing countries. Currently the global prevalence of the disease has increased following HIV epidemic (Berenguer et al., 1992). Rapid confirmatory laboratory diagnosis of TBM and initiation of appropriate anti tuberculosis chemotherapy (ATT) is essential in reducing the mortality and morbidity rates in patients with TBM. Direct demonstration of acid fast - bacilli in CSF specimens by Zeihl- Neelsen stain and isolation of M tuberculosis by the conventional culture method are still considered as ‘Gold standard’ for the laboratory diagnosis of TBM. However these direct microbiologic techniques in CSF specimens are less sensitive for the diagnosis of TBM. Even short-term radiometric methods i.e., BACTEC culture, is less sensitive for the isolation of M * Corresponding author. Tel.: ⫹91-0471-524508; fax: ⫹91-0471446433. E-mail address: [email protected] (V.V. Radhakrishnan).
tuberculosis in CSF (Bonington et al., 1998). As an adjunct for the laboratory diagnosis of TBM, several immunologic and molecular biologic techniques have been described earlier (Krambovitis et al., 1984; Sada et al., 1983; Kox et al., 1995). In this report, we describe an immunoblot method to detect 82 kDa mycobacterial antigen in the heat-inactivated CSF of patients with TBM. The utility of this approach is that, this is simple, rapid and it has practical application for the laboratory diagnosis of TBM in developing countries where the laboratory resources and technical expertise are limited.
2. Materials and methods 2.1. Patients and collection of clinical specimens CSF specimens were obtained from 30 patients admitted to the Sree Chitra Tirunal Institute for Medical Sciences, Thiruvananthapuram, Kerala State, India, with a clinical
0732-8893/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved. PII: S 0 7 3 2 - 8 8 9 3 ( 0 1 ) 0 0 2 3 0 - 9
226
A. Mathai et al. / Diagnostic Microbiology and Infectious Disease 39 (2001) 225–228
diagnosis of TBM. As this Institute is one of the tertiary referral center for neurologic diseases, the time interval between the collections of CSF specimens from the onset of disease varied considerably in most of the patients. A provisional diagnosis of TBM was made on the basis of relevant clinical manifestations, elevated protein levels and pleocytosis in CSF specimens. In five patients, the diagnosis of TBM was confirmed by the isolation of M tuberculosis in CSF specimens by the conventional bacteriologic cultures (Lowenstein–Jensen medium). In the remaining 25 patients, a repeat CSF cultures for M tuberculosis, fungi, and pyogenic bacteria were reported to be negative on two occasions. PCR was also simultaneously performed in the CSFs of patients with TBM. Although there was no microbiologic confirmation, these 25 patients were clinically regarded as ‘probable’ TBM. Patients with ‘confirmed’ and ‘probable’ TBM received (ATT) for 2– 4 weeks during their hospital study. CSF specimens from 40 patients with non- tuberculous neurologic diseases formed the control group [Partially treated pyogenic meningitis (n ⫽ 14), viral encephalitis (n ⫽ 8); CSF collected at myelography (n ⫽ 8); demyelinating disorder (n ⫽ 10)]. Cisternal CSF, collected at an autopsy from a CSF culture positive case of TBM was used as the positive control in this study. 2.2. Heat inactivation of CSF samples 2 mL of CSF from TBM and control groups were autoclaved at 15 lbs/sq inches for 30 min. The protein in CSF specimen was precipitated by 50% ammonium sulfate and centrifuged at 3500 rpm for 30 min. The precipitate was reconstituted to 200L in 0.15M PBS after removing the ammonium sulfate by continuous dialysis for 24 h.
3. Tuberculin purified protein derivative antigen (PPD) Tuberculin PPD was prepared from the culture filtrate of the H37Ra strain of M tuberculosis and polyvalent antibody to M. tuberculosis was raised as described earlier (Mathai et al., 1989; Radhakrishnan et al., 1990).
4. SDS- PAGE and electrotransfer SDS- PAGE (Laemmli, 1970) was performed using vertical electrophoretic unit (Mighty small, Hoefer). Tuberculin PPD antigen 2g/slot, 200L of heat inactivated CSFs from TBM and control group were subjected to discontinuous SDS-PAGE and subsequently transferred onto nitrocellulose membrane (NCM) (Towbin et al., 1979). Follow-
ing the transfer, the NCM strips were cut at 4 mm width and the strips were stored at 4°C until used for immunostaining.
5. Immunostaining NCM strips containing tuberculin PPD antigen, heat inactivated CSF from ‘confirmed’, ‘probable’ TBM and control groups were thoroughly washed with 0.15M PBS and then quenched with 2.5% skimmed milk for 1 h. All the incubations in the assay were carried at 40°C. The NCM strips were washed with 0.15 M PBS and incubated with (1: 500) polyvalent rabbit IgG to M tuberculosis for 2 h, followed by (1:600) anti rabbit IgG - biotin conjugate (Sigma) for 2 h. The NCM strips were then washed and incubated with (1: 400) extr-avidin HRP (Sigma) for 2 h. Subsequently the strips were treated with a substrate, containing 3 mg of 4-chloro-1-naphthol in 1 mL of cold methanol and 3l of 30% hydrogen peroxide in 4 mL of PBS. A positive reaction was indicated by development of a clean blue band in the NCM strip (Fig. 1).
6. Result Fig. 1 depicts the results of this study. Tuberculin PPD antigen demonstrated four mycobacterial antigens possessing molecular weights of 35, 45, 66 and 82 kDa respectively. The CSF from ‘confirmed’ TBM patients contained 45, 66 and 82 kDa antigens, of which 82 kDa antigen appeared conspicuous. In 16 out of 25 patients with ‘probable’ TBM, 82 kDa antigen was also demonstrated in the NCM strips. In the remaining 9 patients in the ‘probable’ TBM, 82 kDa antigen was absent. The CSFs from control group showed both 38 and 66 kDa antigens but 82 kDa was absent in all of them. Based on this observation in NCM, we regard that; the presence of 82 kDa antigen in the heat inactivated CSF in a patient will distinguish tuberculous from non-tuberculous etiology. All those TBM patients with 82kDa antigen in their heat-inactivated CSFs were regarded as positive for tuberculous etiology. Accordingly five ‘confirmed’ patients with TBM and 16/25 patients with ‘probable TBM were treated with ATT during their hospital stay and they showed optimal neurologic recovery at the time of discharge from the hospital. Nine out of the 25 ‘probable’ TBM did not show 82 kDa antigen in their CSfs. Hence they were not regarded as TBM and ATT was withdrawn. These nine patients were extensively investigated to establish the cause of meningitis. Despite of that the etiology of meningitis remained undetermined in them.
7. Discussion The data of this study highlight three relevant observations. Firstly, heat stable 82 kDa mycobacterial antigen was
A. Mathai et al. / Diagnostic Microbiology and Infectious Disease 39 (2001) 225–228
227
Fig. 1. Showing the immunoblot in PPD, TBM and non TBM groups. Note the presence of 82 kDa antigen in only CSFs of TBM patients
seen in the CSF of 5/5 ‘confirmed’ and 16/25 ‘probable’ TBM patients and was absent in the CSF of control group. Thus assay carried high degree of specificity for tuberculous etiology. Secondly, the assay can easily be performed in any routine clinical laboratory and does not require any expensive equipment. Thirdly, heat inactivation of CSF specimens considerably reduced the risk of handling of infectious material in the laboratory. This becomes relevant, because of the increasing prevalence of tuberculosis among HIV patients. The rabbit antibody to M tuberculosis can easily be stored at -20°C for at least 6 months without any decrease in their titers. The applications of the recent techniques like PCR, for the routine laboratory diagnosis of TBM, still remain speculative. In this be study PCR was performed simultaneously in all 30 CSFs from patients with TBM and showed an overall sensitivity of 33.3% and also required 72 h to perform in the laboratory. By contrast, the technique as described in this study required only 48 h to perform and
Table 1 Showing the results of Immunoblot and PCR Immunoblot Confirmed TBM (n ⫽ 5) Probable TBM (n ⫽ 25) Control Group Sensitivity (%) Confirmed Probable Specificity (%) Duration of the assay
PCR
5 16 0
3 7 3
100 64 100 48 h
60 28 66 72 h
more importantly the assay could be done in any routine clinical laboratory (Table 1) and is best suited to laboratories in developing countries. Hence we advocate this new method for rapid laboratory diagnosis of TBM. Acknowledgments The authors are grateful to the Director, Sree Chithra Tirunal institute for Medical Sciences and Technology, Thiruvananthapuram, India for providing all the facilities to undertake this study and also for the kind permission to publish this paper. The authors are also indebted to Miss Susha Kumari for her excellent secretarial assistance. References Berenguer, J., Moreno, S., Laguna, F. (1992). Tuberculous meningitis in patients infected with the human immuno deficiency virus. N Engl J Med 36, 668 – 672. Bonington, A., Strang, J. I. G., Klapper, P. E., Hodd, S. V., Rubombora, W., Penny, M., Willers, R., & Wilkins, E. G. L. (1998). Use of Roche Amplicor Mycobacterium tuberculosis PCR in early diagnosis of Tuberculous meningitis. J Clin Microbiol 36, 1251–1254. Krambovitis, E., Mc Illmurray, M. B., Lock, P. E., Hendrickse, W., & Hozel, H. (1984). Rapid diagnosis of tuberculous meningitis by latex particle agglutination. Lancet, ii1229 –1231. Sada, E., Riuz Palacio, G. M., Lopex- Videl, Y., & Ponce De Leon, S. (1983). Detection of mycobacterial antigens in cerebrospinal fluid of patients with tuberculous meningitis by enzyme –linked immunosorbent assay. Lancet ii:651– 652. Kox, F. F., Kuijper, S., & Kolk, H. J. (1995). Early diagnosis of tuberculous meningitis by polymerase chain reaction. Neurology, 45, 2228 – 2232. Mathai, A., Radhakrishnan, V. V., Sehgal, S., & Mohan, P. K. (1989). Enzyme linked immunosorbent assay for IgG antibody to tuberculin
228
A. Mathai et al. / Diagnostic Microbiology and Infectious Disease 39 (2001) 225–228
purified protein derivative in cerebrospinal fluid specimens of patient with tuberculous meningitis. Indian J Med Microbiol, 7, 98 –104. Radhakrishnan, V. V., Mathai, A., Rao, B. S., & Sehgal, S. (1990). Immunoelectrophoresis of mycobacterial antigens. Indian J Exp Biol, 28, 812– 815.
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head bacteriophage T4. Nature (London), 227, 680 – 685. Towbin, H., Stanelin, J., & Gorden, J. (1979). Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc Natl Acad Sci (USA), 76, 4350 – 4354.