Publications in Tuberculosis
Lingaraja Jena, Pranita Waghmare, Supriya Kashikar, Satish Kumar, Bhaskar C. Harinath. Computational approach to understanding the mechanism of action of isoniazid, an anti-TB drug. International Journal of Mycobacteriology;3(2014) :276-282 Abstract: Tuberculosis (TB) is an ancient disease caused by Mycobacterium tuberculosis (MTB), which remains a major cause for morbidity and mortality in several developing countries. Most drug-resistant MTB clinical strains are resistant to isoniazid (INH), a first-line anti-TB drug. Mutation in KatG, a catalase-peroxidase, of MTB is reported to be a major cause of INH resistance. Normally upon activation by KatG, INH is converted to an active intermediate which has antimycobacterial action in MTB. This INH intermediate in the presence of NADH forms INH-NAD adduct which inhibits inhA (2-trans-enoyl-acyl carrier protein reductase) of MTB, thus blocking the synthesis of mycolic acid, a major lipid of the mycobacterial cell wall. In this docking study, the high binding affinity of INH-NAD adduct towards InhA was observed in comparison with INH alone. In this study, two resistant mutants of KatG (S315T and S315N) were modeled using Modeller9v10 and docking analysis with INH was performed using AutoDock4.2 and the docking results of these mutants were compared with the wild type KatG. Docking results revealed the formation of a single hydrogen (H) bond between the secondary amine nitrogen (–NH) of INH with Thr or Asn residues in place of Serine at 315 position of KatG mutant strains respectively, whereas in the case of the wild type, there was no H-bond formation observed between INH and Ser315. The H-bond formation may prevent free radical formation by KatG in mutant strains thus the development of resistance to the drug. This in silico evidence may implicate the basis of INH resistance in KatG mutant strains. Lingaraja Jena, Supriya Kashikar, Satish Kumar, Bhaskar C Harinath. Effect of Single Amino Acid Mutations on function of Mycobacterium tuberculosis H37Rv and H37Ra by Computational Approaches. Indian J Tuberc 2014;61 (3):200-206. Abstract: BACKGROUND: Studies have been reported on genomic analysis to explain virulence of MTB H37Rv vs. MTB H37Ra strain. Proteomic comparison analysis at our centre has shown variability of 244 proteins in MTB H37Rv. A single amino acid mutation in protein sequence may cause alteration in protein structure and function that may account for virulence and drug resistance properties of pathogenic organisms. AIM: To find the effect of single amino acid mutations on Mycobacterium tuberculosis H37Rv and H37Ra using computational approaches METHODS: Proteins with single amino acid mutation were analysed by different mutation analysis systems such as SIFT, PolyPhen, PROVEAN and HOPE. Subsequently, structure comparison of proposed modelled structure of mutant proteins of MTB H37Ra with native proteins of MTB H37Rv was performed. RESULTS: We observed that amongst 40 single amino acid mutated proteins of MTB H37Ra, five were found to be damaged by all the mutation analysis systems. Upon structure comparison, the RMSD values between the native and mutant type proteins were found to be significantly higher. All the five proteins showed important biological function in MTB H37Rv. Further, when these five proteins of MTB H37Ra were compared with corresponding proteins of other virulent strains of MTB (i.e. F11 and CDC 1551), similar observation was made. CONCLUSION: The data suggests the important role of single amino acid mutation in five proteins in causing changes in the virulence and pathogenicity of clinical strains of MTB. Waghmare P, Hutke V, Wandile K, Badole CM, Harinath BC. In house developed SEVA TB ELISA for ruling out tuberculosis in suspected cases of Extra pulmonary tuberculosis, a six month retrospective study in a tertiary care hospital. Biomedical Research 2014;25(3):361-364. Abstract: Evaluat ion of SEVA TB ELISA using cocktail of mycobacterial antigens ES-31 and EST-6 (containing ES-38 and ES-41 ) and their specific antibodies in diagnosis of suspected cases of extrapulmonary tuberculosis. Detection of circu lating free and immune complexed antigen to specific antibodies by sandwich ELISA and antibody to cocktail of antigens ES-31 and EST-6 by indirect ELISA and was carried out in clinically suspected cases of extra pulmonary tuberculosis during a period of six months. Absence of anti gen and antibody is considered as negative ELISA. 146 suspecte d cases of extrapulmonary tuberculosis were screened by SEVA TB ELISA on request by clin icians. 112 cases out of 146 showed negative ELISA test out of which 108 cases were not recommended ATT. The test showed 96% co- relation with ELISA negativity and no ATT treatment. Further four cases showing ELISA negativity were advised ATT. These cases need follow up for the development of TB disease or effectiveness ATT of treatment in context to clinical relief. This study showed usefulness of SEVA TB ELISA as a adjunct test for ruling out TB in clinically suspected cases of extrapulmonary tuberculosis . Lingaraja Jena, Supriya Kashikar, Satish Kumar, Bhaskar C. Harinath. Comparative proteomic analysis of Mycobacterium tuberculosis strain H37Rv versus H37Ra. International Journal of Mycobacteriology - 11 November 2023 (10.1016/j.ijmyco.2013.10.004) Abstract: Background:Mycobacterium tuberculosis (MTB) H37Ra is an attenuated tubercle bacillus closely related to the virulent type strain MTB H37Rv. In spite of extensive study, variation in virulence between the MTB H37Rv and MTB H37Ra strains is still to be understood. The difference in protein expression or structure due to mutation may probably be an important factor for the virulence property of MTB H37Rv strain.MethodsIn this study, a whole proteome comparison between these two strains was carried out using bioinformatics approaches to elucidate differences in their protein sequences. ResultsOn comparison of whole proteome using NCBI standalone BLAST program between these two strains, 3759 identical proteins in both the strains out of 4003 proteins were revealed in MTB H37Rv and 4034 proteins were revealed in MTB H37Ra; 244 proteins of MTB H37Rv and 260 proteins of MTB H37Ra were found to be non-identical. A total of 172 proteins were identified with mutations (Insertions/deletions/substitutions) in MTB H37Ra while 53 proteins of MTB H37Rv and 85 proteins of MTB H37Ra were found to be distinct. Among 244 non-identical proteins, 19 proteins were reported to have an important biological function; In this study, mutation was shown in these proteins of MTB H37Ra. ConclusionThis study reports the protein differences with mutations between MTB H37Rv and H37Ra, which may help in better understanding the pathogenesis and virulence properties of MTB H37Rv. Vinita Hutke, Gauri Wankhade, Pranita J Waghmare, Harinath BC.   ELISA protocol for rapid screening of potential anti tubercular drugs based on antigenic reactivity of mycobacterial ES-31 serine protease – a drug target supported by axenic culture of mycobacterium tuberculosis H37Ra strain in the presence of inhibitor”. Indian J Tuberc 2013 July;60(3):138-141. Abstract: Background : Mycobacterial ES-31 serine protease has been reported to be a drug target using protease and lipase inhibitors in axenic and macrophage cultures. Simple screening techniques are needed for rapid testing of anti-tubercular drugs. Aim : To demonstrate the usefulness of ELISA protocol based on antigenic reactivity of mycobacterial serine protease by indirect ELISA for detecting anti-tubercular activity. Material and Methods : Indirect ELISA for assessment of antigenic reactivity of mycobacterial ES-31 serine protease was standardized using ES-31Ag and anti-DSS-goat-serum and assessed the inhibition of the antigenic reactivity by isoniazid, an anti-tubercular drug and serine protease inhibitor and orlistat, a lipase inhibitor. Results : Optimal antigenic reactivity of mycobacterial ES-31 serine protease was observed at 5μg/well of ES-31 antigen and at 1:25 dilution of anti-DSS-goat-serum. Isoniazid showed 42% inhibition of ES-31 serine protease at 0.4μg/well, while orlistat showed inhibition of 60% at 0.5μg/well. Inhibition of Mtb H 37 Ra bacilli is further confirmed in axenic culture. 35% and 29% inhibition by isoniazid at 0.4μg/well and orlistat at 0.5μg/well were observed respectively on bacterial growth. Conclusion : Simple ELISA protocol based on assay of antigenic reactivity of mycobacterial ES-31 serine protease, a drug target, has been standardized for rapid screening of potential anti-tubercular drugs. Pranita J Waghmare, Gauri Wankhade, Anindita M, Kiran Wandile, CM Badole, BC Harinath. SEVA TB ELISA – Multi antigen and antibody assays for serodiagnosis of suspected cases of pulmonary and extra pulmonary tuberculosis in tertiary care hospital –A retrospective study. Asian Pacific Journal of Tropical Disease (2012):S827-S832. Abstract: Objective To assess the usefulness of in-house developed multiantigen and antibody assays, in diagnosis of both pulmonary and extra-pulmonary tuberculosis. Method Clinically suspected cases of 31 pulmonary and 171 extra-pulmonary tuberculosis (TB) were screened by ELISA using cocktail (ES-31 + EST-6) antigen and their specific antibodies (anti ES-31 + anti EST-6 IgG) for detection of antibody and and antigen (circulating antigen and immune complexed antigen) respectively and correlated with antituberculosis therapy in retrospective study. Results Out of 31 cases of pulmonary TB screened, 15 patients showed ELISA positivity out of which five cases were given antituberculosis therapy. Out of 171 cases of EPTB screened, 76 cases showed ELISA positivity out of which 18 were given antituberculosis therapy. Further 4 EPTB cases which showed AFB negativity were given ATT. The data was further analyzed based on PTB & EPTB, adults and children, OPD and IPD patients to understand false positivity in clinically suspected PTB and EPTB cases. There was significant correlation (108/202 cases) with ELISA negativity and no ATT advised in clinically suspected PTB and EPTB patients. Conclusions In house developed multi antigen and antibody assays have been observed to be quite useful as adjunct test in serodiagnosis of suspected cases of tuberculosis in particular extrapulmonary tuberculosis. Keywords Multi antigen and antibody assay; Serodiagnosis; PTB; EPTB; ES antigen Jena L, Kumar S, Harinath BC.  MycoProtease-DB: Useful resource for Mycobacterium tuberculosis complex and nontuberculous mycobacterial proteases. Bioinformation 2012; 8(24):1240-1242. Abstract: MycoProtease-DB is an online MS SQL and CGI-PERL driven relational database that domiciles protease information of Mycobacterium tuberculosis (MTB) complex and Nontuberculous Mycobacteria (NTM), whose complete genome sequence is available. Our effort is to provide comprehensive information on proteases of 5 strains of Mycobacterium tuberculosis (H37Rv, H37Ra, CDC1551, F11 and KZN 1435), 3 strains of Mycobacterium bovis (AF2122/97, BCG Pasteur 1173P2 and BCG Tokyo 172) and 4 strains of NTM (Mycobacterium avium 104, Mycobacterium smegmatis MC2 155, Mycobacterium avium paratuberculosis K-10 and Nocardia farcinica IFM 10152) at gene, protein and structural level. MycoProtease-DB currently hosts 1324 proteases, which include 906 proteases from MTB complex with 237distinct proteases & 418 from NTM with 404 distinct proteases. Flexible database design and easy expandability & retrieval of information are the main features of MycoProtease-DB. All the data were validated with various online resources and published literatures for reliable serving as comprehensive resources of various Mycobacterial proteases. Availability: The database is available for free at http://www.bicjbtdrc-mgims.in/MycoProtease-DB/ Gauri Wankhade, Vinita Hutke, Pranita J Waghmare, Arup Kr. Misra, Sushil Kumar Varma, Harinath BC. Inhibitory effect of isoniazid and orlistat combination on mycobacterial ES-31 serine protease in vitro and on the growth of M.tb bacilli in axenic culture. Indian J Tuberc 2012;59:156-161. (Abstract) Abstract: Background: Isoniazid and orlistat were reported to have inhibitory effect on mycobacterial ES-31 serine protease in vitro and bacterial cell growth in axenic culture. Aim: To study the cumulative effect and understand drug - drug interaction, if any, when isoniazid and orlistat used in combination. Material and Methods: Inhibition of mycobacterial ES-31 serine protease by different combinations of orlistat and isoniazid together and individually were studied using azocasein assay. Inhibition of secretion of excretory secretory ES-31 antigen in Sautan culture medium was studied under axenic condition and growth of M.tuberculosis H37Ra bacilli by CFU count on LJ-medium. Results: Orlistat and isoniazid both showed inhibitory activity of ES-31 serine protease in in vitro as well as in vivo. Individually, isoniazid showed 90% inhibition at 200 ng/ml while orlistat at 250 ng/ml showed 65% inhibition of mycobacterial ES-31 serine protease in vitro. A combination of orlistat (250 ng/ml) and isoniazid (200 ng/ml) showed 86% inhibition in vitro while 73% inhibition was observed by orlistat (25 ng/ml) and isoniazid (200 ng/ml) on bacterial growth in axenic culture. Conclusion: Significant inhibition by orlistat suggests that it could be tried in patients with intolerance to isoniazid or in those already developed isoniazid resistance. It may also be explored in the suspected TB patients as initial medication in place of antibiotics for clinical relief. Key words: Mycobacterial ES-31, Serine protease, Orlistat, Isoniazid, Inhibition G Wankhade, A Majumdar, P Kamble, Sajal De, BC Harinath.  Multi-antigen and antibody assays (SEVA TB ELISA) for the diagnosis of Tuberculous pleural effusion. Indian J Tuberc 2012;59:78-82. (Abstract) Abstract: OBJECTIVE: Prospective evaluation of inhouse developed SEVA TB ELISA using cocktail of Mycobacterial antigens ES-31 and EST-6 (containing ES-38 and ES-41) and their specific antibodies in the diagnosis of Tuberculous pleural effusion was done in a tertiary care hospital. METHODS: Detection of circulating free and immune-complexed (IC) antigens and antibody by sandwich and indirect peroxidase ELISA respectively was done in pleural fluid and sera specimens. Total 33 patients with pleural effusion, including 24 patients diagnosed as tuberculous pleural effusion based on clinico-radiological, microbiological and biochemical profile (protein, LDH and ADA) of pleural effusion and nine patients with non-tuberculous pleural effusion, were studied. RESULTS: Pleural fluid showing either antigen or immune-complexed antigen or antibody positive was considered as ELISA positive for tuberculous pleural effusion. Multi antigen and antibody assay (SEVATB ELISA) showed 100% specificity and 83% sensitivity in pleural fluid while 78% specificity and 92% sensitivity in serum of tuberculous pleuritis patients. CONCLUSION: This study showed usefulness of SEVATB ELISA, using cocktail of ES-31 and EST-6 antigens and their antibodies for antibody and antigen detection respectively in analysis of either sera or pleural fluid samples of suspected tuberculous pleuritis patients as an adjunct test to clinical diagnosis. Singhal S, Majumdar A, Harinath BC, Kamble P. Use of inhouse developed M.TB ES antigen ELISA in diagnosis of tuberculosis. Indian Medical Gazette, July 2011; CXLV(7):279-282. (Abstract) Abstract: Background: Precise and faster diagnosis of tuberculosis is the need of the day for speedy and successful management of tuberculosis patient, attending tertiary health care hospital in a rural area. Objectives: To evaluate the usefulness of SEVATB ELISA for detecting antibody, antigen and immunecomplexed antigen in pulmonary and extra pulmonary tuberculosis (EPTB) patients. Methods: 21 patients suspected of tuberculosis were screened by sputum AFB, and culture, chest radiographs and cytological examination, if patient is suspected case of tuberculosis. Based on clinical assessment, antitubercular activity (ATT) was started and cases were followed to judge the response of ATT, especially in EPTB. Sera from all these patients were subjected to SEVA TB ELISA at Mahatma Gandhi Institute of Medical Sciences and data was analyzed retrospectively. Results: Assay showed 100% correlation with acid fast bacilli positivity and antitubercular treatment. Only one case showed negative ELISA test and this case was not recommended antitubercular therapy. Out of 2 AFB and culture negative suspected PTB cases, ELISA was positive in 1 case and 1 case of EPTB could not be followed. Only one old pulmonary TB case with bronchiectasis showed negative ELISA test and this case was also not recommended ATT. S Haldar, M Bose, P Chakrabarti, H F Daginawala, BC Harinath, R S Kashyap, S Kulkarni, A Majumdar, H K Prasad, C Rodrigues, S Singh, R Srivastava, GM Taori, M Varma-Basil, JS Tyagi. Improved Laboratory Diagnosis of Tuberculosis – The Indian Experience. Tuberculosis. Sept 2011;91(5):414-426. (Abstract) Abstract: Tuberculosis (TB) is the leading cause of death worldwide attributable to a single infectious disease agent. India has more new TB cases annually than any other country. In 2008, India accounted for a fifth of the estimated 9.4 million TB cases globally. There is an overwhelming need for improving TB diagnostics in India through the use of cost effective, patient-friendly methods appropriate to different tiers of the country health system. Substantial progress has been made in India in the field of TB diagnosis and serious efforts have been made to herald the development of diagnostic tests for pulmonary TB, extra pulmonary TB and MDR-TB. Diverse approaches have been attempted towards improving smear microscopy, rapid culture and for differentiation between the Mycobacterium tuberculosis complex and non-tuberculous mycobacteria. Several laboratories have developed in-house PCR assays for diagnosing TB with high accuracy. Approaches for distinguishing M. tuberculosis and/or Mycobacterium bovis infection and disseminated Mycobacterium avium complex infection in HIV-AIDS patients have also been described. Serological tests to detect antigens or antibodies to M. tuberculosis specific components by using cocktails of Excretory/Secretory protein antigens, Ag85 complex antigens, Hsp 65 antigen, RD1 antigens and Rapid Reverse Line Blot Hybridization assays to detect MDR-TB (mutations to rifampicin, isoniazid and streptomycin) have also been developed. Other methods like measurement of adenosine deaminase activity and use of luciferase reporter phages have also been explored for TB diagnosis. These advances in the Indian context are detailed in the present chapter. The validation and application of these methods in laboratory and public health settings is likely to result in improved TB diagnosis and contribute to effective disease management in India.    Jena L, Kumar S, Harinath BC.   MTB-PCDB: Mycobacterium tuberculosis proteome comparison database. Bioinformation. 2011;6(3):131-133. (Abstract) Abstract The Mycobacterium tuberculosis Proteome Comparison Database (MTB-PCDB) is an online database providing integrated access to proteome sequence comparison data for five strains of Mycobacterium tuberculosis (H37Rv, H37Ra, CDC 1551, F11 and KZN 1435) sequenced completely so far. MTB-PCDB currently hosts 40252 protein sequence comparison data obtained through inter-strain proteome comparison of five different strains of MTB. 2373 proteins were found to be identical in all 5 strains using MTB H37Rv as reference strain. To enable wide use of this data, MTB-PCDB provides a set of tools for searching, browsing, analyzing and downloading the data. By bringing together, M. tuberculosis proteome comparison among virulent & avirulent strains and also drug susceptible & drug resistance strains MTB-PCDB provides a unique discovery platform for comparative proteomics among these strains which may give insights into the discovery & development of TB drugs, vaccines and biomarkers. Availability: The database is available for free at http://www.bicjbtdrc-mgims.in/MTB-PCDB/ A Majumdar, Gauri Wankhade, Pranita Kamble, Deepti Joshi, Harinath BC.A sensitive and specific ES-31 Antigen detection based fluorometric assay for confirmation of M.tb in cell culture’.Indian J of Experimental Biology, April 2011;49:304-306. (Abstract) Abstract: Confirmation of presence of M. tuberculosis bacilli on microscopic examination is very important in diagnosis of tuberculosis. The present study was undertaken to find the usefulness of mycobacterial ES-31 serine protease as a marker to detect tuberculosis bacilli using fluorescein isothiocyanate conjugated anti-ES-31 serine protease antibody. This immunofluorescence method was compared with Ziehl-Neelsen and auramine-O staining methods for detection of tuberculosis bacilli. Slides were prepared for each serially diluted tuberculosis H37Ra bacilli (1 x 10(7) bacilli/ml to 5 bacilli/ml). Slides for each dilution group were stained by ZN method, auramine-O and immunostaining methods using fluorescein isothiocyanate conjugated anti-ES-31 serine protease antibody. ZN staining method showed efficacy for detection of M. tuberculosis H37Ra upto 1 x 10(4) bacilli/ml while auramine-O method showed upto 1 x 10(2) bacilli/ml. The presence of bacilli was indicated by green fluorescence on immunostaining using anti-ES-31 antibody conjugate and this method was effective upto 10 bacilli/ml. The slides which were negative for ZN (1 x 10(3) cells/ml) and auramine-O (100 cells/ml) method showed positivity on restaining with immunofluorescent staining method. The results of this preliminary study showed that immunofluorescent staining method using specific anti-ES-31 antibody conjugate was more sensitive for detection of tuberculosis bacilli than ZN and auramine-O methods in samples of laboratory strain. The utility of this method will be studied further in clinical specimens. Anindita Majumdar, Gauri Wankhade, Pranit Kamble, Harinath BC. Effect of HIV protease inhibitors and orlistat on mycobacterial ES-31 serine protease, a potential drug target in Mycobacterium tuberculosis. Indian J of Tuberc 2011;58:4-10. (Abstract) Abstract: Background: Mycobacterial excretory secretory-31 (SEVA TB ES-31) antigen is shown to possess protease and lipase activities. Aim: To study the effect of commonly used HIV-protease inhibitors and lipase inhibitor Orlistat if any on mycobacterial ES-31 serine protease in vitro enzyme activity and on the growth of M.tb H37Ra bacilli in axenic culture. Methods: Effect of HIV-protease inhibitors namely Ritonavir, Lopinavir and Indinavir and Orlistat on protease activity of ES-31 was assessed using azocasein assay and on bacillary growth in axenic culture of Mycobacterium tuberculosis H37Ra. The concentration of ES-31 antigen in culture filtrate was determined by sandwich peroxidase ELISA using anti ES-31 antibody and the growth of bacilli by CFU count. Results: HIV-protease inhibitors such as Ritonavir, Lopinavir and Indinavir and lipase inhibitor Orlistat inhibited serine protease activity by 41.3 - 69.7% in vitro. These inhibitors also showed decreased bacterial growth in axenic culture and further confirmed by decreased concentration of ES-31 serine protease secretion in the culture fluid. Ritonavir showed maximum inhibition of 77% on the growth of the bacilli in axenic culture while anti obesity drug Orlistat showed 61% inhibition. Conclusion: SEVA TB ES-31 with serine protease and lipase activities may be a potential drug target in tuberculosis management. Key Words: Mycobacterium tuberculosis, HIV, Lopinavir, Ritonavir, Orlistat, Mycobacterial ES-31 serine protease. G Wankhade, A Majumdar, P Kamble, BC Harinath. Lipase activity of 31kDa Mycobacterial ES-31 serine protease. Biomedical Research 2011; 22 (1):45-48. (Abstract) Abstract: Mycobacterium tuberculosis is known to secrete number of proteins which play an important role in pathogenicity and diagnosis. A diagnostically important secreted antigen, Excretory Secretory-31 (SEVA TB ES-31) protein with serine protease activity was isolated from Mycobacterium tuberculosis H37Ra culture filtrate. Serine proteases like Chymotrypsin have been reported to possess lipase activity as the catalytic triad is similar in serine protease and some lipase enzymes. In this study, ES-31 showed presence of 44.5U/mg pr of lipase activity by titrimetric assay. The lipase activity was inhibited by lipase inhibitor, Orlistat by 100%, as well as by serine metalloprotease inhibitors, Phenyl methyl sulphonyl fluoride and Ethylene Diamine Tetraacetic acid by 100%. The serine protease activity of ES-31 was inhibited by Phenyl methyl sulphonyl fluoride, Ethylene Diamine Tetraacetic acid, and orlistat by 78.3%, 64.3%, 89.4% respectively. Inhibition of serine protease activity of ES-31 by lipase inhibitor and inhibition of lipase activity of ES-31 by serine protease inhibitor suggests that the catalytic site of ES-31 is sensitive to both types of the inhibitors and ES-31 may be a chymotrypsin-like protein with drug target potential. Harinath BC. Immunodiagnostics for Tuberculosis – Problems and progress. Indian J Tuberc July 2010;57:123-127. (Editorial) Editorial Tuberculosis (TB) is the seventh leading cause of death among infectious diseases. Sputum smear microscopy has remained the corner stone of TB diagnosis in the global strategy to control the disease. The global targets for TB control, adopted by World Health Assembly, are to cure 85% of the newly detected smear positive TB cases and to detect 70% of the estimated incidence of sputum smear positive TB cases. 70% of case detection still leaves behind a gap of 30% of cases yet to be detected. Further the sensitivity of sputum smear microscopy is limited requiring >10000 bacilli per ml of sputum and not useful in extra pulmonary TB and paediatric tuberculosis. Culture is the most sensitive method for detecting TB, but it can take several weeks to yield results and demands advanced technical infrastructure that is not widely accessible in resource - limited health systems. A novel sample processing methodology (Universal sample processing methodology, USP) was developed by Chakravorty and Tyagi. Specimens containing ~300 to 400 bacilli per ml can be reproducibly detected as positive by USP smear microscopy. Automated Liquid Culture Systems such as BACTEC and MGIT reduce the delays in obtaining results to days rather than weeks. However they are expensive and require expertise and infrastructure. Improvements have been made in culture techniques. Bhattacharya et al4 developed a relatively rapid, low cost and safe bilayered medium with tetrazolium indicator achieving higher isolation rates. Performance of a filtration step was shown to improve the culture yield from paucibacillary body fluids including cerebrospinal fluid (CSF)5. Singh et al6 have described various methods for improving the performance of commercial liquid culture techniques in the Indian scenario. (Cont..) Majumdar A, Kamble PD, Badole CM, Harinath BC. Prospective study of inhouse developed SEVA TB Peroxidase enzyme immunoassay for cocktail antigen and antibody in the diagnosis of Tuberculosis in suspected patients attending a tertiary care hospital located in rural area. Asian Pacific Jr of Tropical Medicine, May 2010; 3(5):.356-359. (Abstract) Abstract: Objective: To evaluate inhouse developed SEVA TB peroxidase enzyme immunoassay using cocktail of mycobacterial excretory-secretory antigens (ES-31, ES-43 & EST-6) for antibody detection and their affinity purified antibodies for antigen detection in tuberculosis suspected patients. Methods: Inhouse developed SEVA TB peroxidase enzyme immunoassay was evaluated prospectively in 73 suspected pulmonary and 46 extra-pulmonary tuberculosis patients during November 2008-March 2009 in a tertiary hospital located in rural area. Results: Assay on prospective analysis showed 100% correlation of pulmonary tuberculosis (PTB) and extra-pulmonary tuberculosis (EPTB) acid fast bacilli positivity and antitubercular treatment in 11 cases. Thirty nine PTB and 12 EPTB cases showed negative for ELISA test and were also not given antitubercular therapy. However 30 PTB and 27 EPTB cases showing ELISA positivity were neither acid fast bacilli positive nor antitubercular therapy treated. These cases may possibly have dormant infection and need further diagnosis. In EPTB cases ELISA was observed to be more useful than AFB smear test. Conclusions: This inhouse developed user-friendly peroxidase ELISA can be used as an adjunct test of smear microscopy or culture techniques for routine screening of patients suspected of PTB or EPTB. Majumdar A, Kamble PD, Harinath BC. Detection of circulating free and immune-complexed antigen in pulmonary tuberculosis using cocktail of antibodies to Mycobacterium tuberculosis excretory secretory antigens by peroxidise enzyme immunoassay. Indian J Tuberc 2010;57: 67-74. (Abstract) Abstract: Background: Decreased sensitivity has been a limiting factor of antigen assay for detection of tuberculosis. Assay of more than one antigen may improve sensitivity of an assay. Aim: To develop a simple, rapid and less-expensive serodiagnostic method compared to culture method for Pulmonary Tuberculosis. Method: A cocktail of affinity purified antibodies against Mycobacterium tuberculosis H37Ra antigens (SEVA TB ES-31, ES-43 and EST-6) was explored for detection of circulating free and Immune-Complexed (IC) cocktail antigen by microtitre plate Peroxidase sandwich ELISA. The assay was evaluated in 27 clinical sera of sputum acid fast bacilli (AFB) positive and 10 AFB negative but anti-tuberculosis therapy responded pulmonary tuberculosis patients and 20 normal sera as controls. Results: Assay of cocktail antigen showed marginal improvement in sensitivity compared to assay of ES-31 antigen alone. The assay for circulating free cocktail antigen showed a sensitivity of 77.7% for AFB positive cases and 70% for AFB negative cases compared to assay of ES-31antigen with sensitivity of 74% and 70% respectively. The assay for IC-cocktail antigen showed sensitivity of 77.7% for AFB positive and 80% for AFB negative cases compared to assay of IC ES-31 antigen with sensitivity of 77% and 70% respectively. Specificity of antigen assay was found to be 90%. Detection of IC-antigen as adjunct assay improved the sensitivity of detection in AFB-ve but ATT responded cases. Peroxidase enzyme immunoassay of cocktail antigen showed a sensitivity of detection of 0.25 μg/ ml and levels of free and IC cocktail antigens were 1.70 ± 1.04 and 1.13 ± 0.047 μg/ ml in AFB positive patients’ sera. Conclusions: Peroxidase enzyme immunoassay for circulating antigen was found to be a useful serodiagnostic assay and in particular in AFB –ve cases responding to ATT. M Anindita, D Thamke, DK Mendiratta, BC Harinath. Potential of Mycobacterial Excretory Secretory Protein Antigens (SEVA TB ES-31, ES-43, EST-6 and ES-20) as Biomarkers to detect Mycobacterium Tuberculosis Bacilli. Indian Journal of Clinical Biochemistry, January 2010; 25(1):15-19. (Abstract) Abstract: There is a need for a simple and reliable method to identify Mycobacterium tuberculos is from nontuberculous mycobacteria (NTM). The utility of mycobacterial ES-31, ES-43, EST-6 or ES-20 antigen as a biomarker for differentiation of Mycobacterium tuberculosis bacilli from nontuberculous mycobacteria was explored using Fluorescein isothiocyanate conjugated antibodies against these antigens. Detection of these antigens was done from M.tb H37Ra and H37Rv DSS antigen. The presence of antigen in bacilli using FITC labelled antibody was indicated by green fluorescence on the cell surface while, its absence by no fluorescence under microscope. In M.tb H37Ra and H37Rv bacilli, fluorescence was observed on addition of FITC labelled anti ES-31 and anti ES-43 antibody; whereas no fluorescence was observed in case of EST-6 and ES-20 antibody conjugates. However all the antigens were detected in detergent soluble sonicate antigen of tubercle bacilli on addition of FITC conjugates. Fluorescence was not observed for ES-31, ES-43, EST-6 and ES-20 antigen in any of the tested NTM as well as in Escherichia coli. SEVA TB ES-31 and ES-43 may be used as biomarkers to distinguish M.tuberculosis bacilli from NTM. M. Anindita, V. Upadhye, D. Thamke, D.K. Mendiratta, B.C. Harinath. Mycobacterial ES-31 serine protease – a biomarker for mycobacterium tuberculosis- a preliminary report. Indian J Tuberc 2009;56: 141-143. (Abstract) Abstract: There is a need for simple and reliable method to identify Mycobacterium tuberculosis from AFB smear positive cases. Utility of mycobacterial ES-31 serine protease as a marker to detect Mycobacterium tuberculosis bacilli was explored using Fluorescein isothiocyanate conjugated anti-ES-31 serine protease antibody. The presence of ES-31 serine protease in bacilli was indicated by green fluorescence on the cell surface. Green fluorescence was observed with M.tb. H37Ra bacilli and M.tb. H37Rv bacilli while no Fluorescence was observed with M. chelonae, Nocardia farcinicum as well as in E. coli showing the usefulness of ES-31 serine protease as a marker for identification of mycobacterium tubercle bacilli in cultures. Upadhye Vijay, Majumdar Anindita, Gomashe Ashok, Joshi Deepti, Gangane Nitin, Thamke Dipak, Mendiratta Deepak and Harinath, B. C. (2009). 'Inhibition of Mycobacterium tuberculosis secretory serine protease blocks bacterial multiplication both in axenic culture and in human macrophages', The Scandinavian Journal of Infectious Diseases. 2009, 41(8):569-576. (Abstract) Abstract: To study the possible importance of mycobacterial ES-31 serine protease for bacterial cell growth, the effect of serine and metalloprotease inhibitors, anti-tubercular drugs such as isoniazid and anti-ES-31 antibody, was evaluated on mycobacterial ES-31 serine protease in vitro and on bacilli in axenic and macrophage cultures. Serine protease inhibitors such as pefabloc, 3,4 dichloroisocoumarin, phenyl methyl sulfonyl fluoride (PMSF) and metalloprotease inhibitors such as ethylene diamine tetracetic acid (EDTA) and 1,10 phenanthroline inhibited 65-92% serine protease activity in vitro. Isoniazid showed 95% inhibition on mycobacterial ES-31 serine protease. These inhibitors also showed decreased bacterial growth in axenic culture and inhibition was further confirmed by a decreased amount of ES-31 serine protease in culture filtrate. In human macrophage culture, highly inhibitory pefabloc, 1,10 phenanthroline and isoniazid inhibited infectivity of virulent as well as avirulent M. tuberculosis bacilli to macrophages. It was observed that addition of mycobacterial ES-31 serine protease to macrophage culture enhanced the entry of bacilli and their multiplication in human macrophages. However, the addition of anti-ES-31 serine protease antibody strongly inhibited the mycobacterial growth as observed by decreased CFU count, showing the importance of mycobacterial ES-31 serine protease for entry of bacilli and their multiplication. Upadhye VJ, Gomase AV, Kumar S, Harinath BC. Isolation, characterization and kinetic studies on SEVA TB ES-31 antigen, a metallo-serine protease of interest in serodiagnosis. Indian J Tuberc 2009;56:22-29. (Abstract) Summary: Background: SEVA TB Excretory secretory -31 (ES-31) antigen, a glycoprotein isolated from M. tb H37Ra culture filtrate, was found to be useful in the serodiagnosis of pulmonary tuberculosis (TB), extrapulmonary TB and in HIV-TB coinfection. Further, it has been shown to be a zinc containing serine protease. Aim: To isolate and purify SEVA TB ES-31 antigen from M. tb H37Ra culture filtrate and study of its enzyme properties and peptide sequence. Methods: ES-31 antigen was purified from culture filtrate of M. tuberculosis H37Ra strain by ammonium sulphate precipitation, SDS-PAGE fractionation and FPLC. Protease activity of ES-31 antigen was studied using azocasein as substrate. ES-31 antigen was further fractionated by two dimensional polyacrylamide gel electrophoresis (2D PAGE) followed by LCMS-T analysis. Result: Mycobacterial metallo-serine protease was purified 3096 fold from M. tb H37Ra culture filtrate protein. Purified enzyme showed optimum activity at pH 7.0 at 370C. Of the four substrates explored, the enzyme has shown maximum activity with azocasein and had a Km value of 0.01 mM with specific activity of 6250 x 10 -6 U/mg protein. Further, analysis of ES-31 antigen by 2D PAGE showed two protein spots (A and B). Conclusion: Kinetic studies on SEVA TB ES-31 protein, an immunogen with metallo serine protease activity are reported for the first time. Purified enzyme had a Km value of 0.01 mM with azocasein as substrate. Further, study on structure and biological role of serine protease will be of interest. Majumdar A, Upadhye V, Harinath BC. A prospective study of inhouse developed SEVA TB ELISA using cocktail of antigen and their immunoglobulins in the diagnosis of the tuberculosis suspected patients in a tertiary care hospital located in rural area. Biomedical Research 2009; 20(1): 59-63. (Abstract) Abstract: An inhouse developed SEVA TB ELISA using cocktail of Mycobacterium Excretory-Secretory antigens (ES- 31, ES-43 & EST-6) for antibody detection and their affinity purified antibodies for antigen detection was prospectively evaluated during January-August 2008 in TB suspected patients in tertiary hospital located in rural area. Antibody or antigen positivity is considered as ELISA positive. 238 suspected cases of pulmonary and extra-pulmonary tuberculosis were screened. ELISA test showed 100% correlation (12 patients) with AFB positivity. ELISA test also showed 100% correlation (42 patients) with anti-tubercular therapy (ATT) treated cases. 110 out of 238 cases showed negative ELISA test and these cases were also not recommended ATT. 86 out of 238 cases were neither AFB positive nor ATT treated, but these cases were ELISA positive. These cases may possibly have dormant infection and need further follow up for showing disease, if any, in due course of time and its usefulness for confirmation of clinical suspicion. This study has shown usefulness of SEVA TB ELISA in diagnosis of suspected patients of Pulmonary and Extrapulmonary tuberculosis. Majumdar A, Upadhye V, Harinath BC. Peroxidase enzyme immunoassay for circulating SEVA TB ES-31 Antigen in pulmonary tuberculosis sera. Biomedical Research 2008; 19 (3): 201-206. (Abstract) Abstract: Antigen assay is a better indicator of active infection than antibody detection hence affinity purified anti ES-31 antibody against Mycobacterium tuberculosis H37Ra Excretory-Secretory 31kDa serine protease antigen (SEVA TB ES-31) was explored for detection of circulating free and Immune-Complexed (IC) ES-31 antigen by microtitre plate Peroxidase sandwich ELISA. The assay was evaluated in 27 clinical sera of sputum acid fast bacilli (AFB) positive and 10 AFB negative but anti-tuberculosis therapy responded pulmonary tuberculosis patients in a tertiary hospital and 20 normal sera as control. The assay for circulating free ES-31 antigen in this preliminary study showed a sensitivity of 74% for AFB positive cases and 70% for AFB negative cases with 90% specificity. The assay for IC ES-31 antigen showed sensitivity of 77% for AFB positive and 70% for AFB negative cases with 90% specificity. Detection of IC-antigen as adjunct assay improves the sensitivity of detection. Peroxidase enzyme immuno assay showed a sensitivity of detection of 0.25 µg/ ml and levels of free and IC ES-31 antigens were 0.71 ± 0.64 and 0.82 ± 0.40 µg/ ml in AFB positive patients' sera. Niraj Shende, Sonika Gupta, Vijay Upadhye, Satish Kumar & Bhaskar C Harinath  Detection of in vitro and in vivo released antigens of diagnostic interest in Mycobacterium tuberculosis by immunoblotting. Indian Journal of Experimental Biology, 46:22-26. (Abstract) . Abstract: Identification of in vitro and in vivo released mycobacterial antigens are of considerable interest in diagnosis of Mycobacterium tuberculosis. Isolation of in vitro released antigen from M. tb excretory-secretory culture filtrate protein and in vivo released circulating tuberculous antigen from smear positive pulmonary tuberculosis sera by ammonium sulphate precipitation is reported. The antigens were resolved by SDS–PAGE and immunoblotting was performed using pooled serum of smear positive, smear negative pulmonary tuberculosis sera and normal sera to identify reactive tuberculous antigens. In vitro and in vivo released mycobacterial antigens showed reactivity at 100, 31, 43 and 20 kDa with smear positive and smear negative pulmonary tuberculosis patients. Further, the in vitro released antigen showed strong reactivity exclusively at 55 kDa antigen with smear positive and 24 kDa antigen with smear negative pulmonary tuberculosis sera. In vivo released antigen reacted exclusively at 170 and 16 kDa with smear positive and 19 kDa antigen with smear negative pulmonary tuberculosis patients. Antigens of 24 and 19 kDa which are reactive with sputum negative sera will be of diagnostic interest and need further study in patients with low bacillary load. The in vitro and in vivo released mycobacterial 100, 31, 43 and 20 kDa antigens, reactive with patients sera are of diagnostic interest in tuberculosis. Shende N, Upadhye V, Kumar S, Harinath BC. A low molecular weight ES-20 protein released in vivo and in vitro with diagnostic potential in lymph node tuberculosis. Indian J Med Microbiol 2008;26:29-33 . (Abstract) Abstract: Purpose: To determine role of antigens released in vivo and in vitro in immunodiagnosis of tuberculosis (TB). Methods: In vivo released circulating tuberculosis antigen (CTA) was obtained from TB sera by ammonium sulphate precipitation and in vitro released excretory-secretory (ES) antigens from Mycobacterium tuberculosis culture filtrate. CTA and ES antigens were fractionated by SDS-PAGE and electro-eluted gel fractions were analyzed for antigen by ELISA. Results: Low molecular weight proteins CTA-9 and ES-9 showed high titre of antigen activity. To explore the diagnostic potential of low molecular weight ES antigen, M. tuberculosis ES antigen was further fractionated by gel filtration chromatography followed by purification on anion exchange column using fast protein liquid chromatography and a highly seroreactive ESG-5D (ES-20) antigen was obtained. Competitive inhibition showed that CTA-9 and ES-9 antigens inhibit the binding of ES-20 antigen to its antibody. Seroanalysis showed sensitivity of 83 and 80% for ES-20 antigen and antibody detection, respectively, in pulmonary TB and 90% in lymph node TB. Conclusions: Seroreactivity studies using M. tuberculosis ES-20 antigen showed usefulness in detection of TB; in particular, lymph node TB. Vijay Upadhye, Santa-Saha (Roy), Niraj Shende, Satish Kumar, Bhaskar C. Harinath. Isolation of Mycobacterium tuberculosis protein antigens ES-31, ES-43 and EST- 6 of diagnostic interest from Tubercle Bacilli by affinity chromatography. Indian Journal of Experimental Biology 2007: 45: 599-602. (Abstract) Abstract Immunodiagnostically useful M. tuberculosis H37Ra protein antigens ES-31, ES-43 and EST-6 were isolated from detergent soluble sonicate (DSS) antigen using monospecific antibodies by affinity chromatography and compared with similar antigens isolated from M. tuberculosis culture filtrate for seroreactivity in tuberculosis sera by Indirect Enzyme Linked Immunosorbent Assay. Recovery of affinity purified ES-31, ES-43 and EST-6 antigen from DSS antigen was approximately 3, 3.5 and 4% respectively, compared to 10, 9 and 6.3% from culture filtrate. Affinity purified ES-31, ES-43 and EST-6 antigens from both culture filtrate as well as DSS antigen showed similar seroreactivity with overall sensitivity 85, 80 and 75% respectively and specificity of 85% at optimum concentration of 50 pg protein of each antigen. The results suggest that DSS antigen may be a promising antigen source for isolating antigens of diagnostic interest obviating the need for cumbersome, time-consuming culture techniques of mycobacteria. Shende N, Gupta S, Upadhye V, Kumar  S and Harinath BC. Isolation and analysis of circulating tuberculous antigens in mycobacterium tuberculosis. Indian Journal of Tuberculosis. Indian J Tuberc 2007; 54:125-129.(Abstract) Abstract: BACKGROUND: Serological techniques like enzyme linked immunosorbent assay (ELISA) and immunoblotting are useful for detection of mycobacterial antigens of diagnostic importance in tuberculosis. AIM: To isolate and identify circulating tuberculous antigens reactive with sputum positive and sputum negative pulmonary tuberculosis (PTB) sera. METHODS: Circulating tuberculous antigen was isolated by ammonium sulphate fractionation from the sera of sputum positive and sputum negative (clinically and radiologically diagnosed) PTB cases. The circulating antigen fractions and individual patients' serum samples were resolved by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting was performed using anti M.tb sonicate IgG as a probe to detect antigens. RESULTS: Anti M.tb sonicate IgG was found to be reactive with mycobacterial proteins 170 kDa, 140 kDa, 85 kDa, 55 kDa, 43 kDa, 20 kDa and 16 kDa in the antigen fraction isolated from sputum positive tuberculosis sera by immunoblotting. However only 85 kDa, 55kDa, 43 kDa and 20 kDa antigenic proteins were found to be recognized by anti sonicate IgG in the antigen isolated from sputum negative sera. These observations were further confirmed by analysis of individual S+ and S- PTB serum by immunoblotting. CONCLUSION: Seroreactive studies of circulating tuberculous antigens showed the presence of 170 kDa, 140 kDa, 85 kDa, 55 kDa, 43 kDa, 20 kDa and 16 kDa protein antigens in sputum positive sera, while 85 kDa, 55 kDa, 43 kDa and 20 kDa antigens were found to be present in sputum negative PTB which need further evaluation for their use in serological diagnosis of tuberculosis. Vijay Upadhye, Niraj Shende, Satish Kumar, B.C. Harinath. Detection of antibody and antigen in extrapulmonary tuberculosis patients sera using a cocktail of mycobacterial excretory secretory antigens and their antibodies. Biomedical Research 2007; 18(3):214-219. (Abstract) Abstract: Increased incidence of extrapurmonary tubercurosis (EpTB) as co-infection is observed due to rise in human immunodeficiency virus (HIV) infection. However, the precise diagnosis of EPTB is faced with difficulty due to the Iack of tissue biopsy or fine-needle aspiration cytology (FNAC) facilities in rural hospitals. Enzymes linked immunosorbent assay (ELISA) will be simple, convenient and doesn’t require sophisticated laboratory. Mycobacterial antigens ES-31, ES-43 and EST-6 antigens were isolated from Mycobacterium tuberculosis (M. tuberculosis) H37Ra bacilli by affinity chromatography. Cocktail of these antigens and their affinity purified antibodies were explored for detection of antibody and antigen by Indirect and Sandwich ELISA respectively. In a preliminary study with bacteriology confirmed EPTB cases (n=32), assay of antibody/free antigen/immunecomplexed (IC) antigen showed a sensitivity of 100% and specificity of 90%. Based on this study, sera from suspected EPTB patients (n=164) diagnosed by clinical and other laboratory investigations, non-tubercular disease patients (n=75) and healthy controls (n=75) were screened. A sensitivity and specificity of 72% & 91 % for antibody detection, 70% & 94% for circulating free antigen and 63% & 98% for circulating IC antigen detection were observed . On combining the positivity of antibody, circulating free and IC antigen, overall sensitivity of 96% and specificity of 91% were observed in EPTB. Tuberculous antibody detection to cocktail antigen was found to be useful in detection of EPTB. However, circulating free and IC-antigen detection may be a better marker for detection of different groups of EPTB. Vijay Upadhye, Santa-Saha (Roy), Niraj Shende, Satish Kumar, Bhaskar C. Harinath. Isolation of Mycobacterium tuberculosis protein antigens ES-31, ES-43 and EST- 6 of diagnostic interest from Tubercle Bacilli by affinity chromatography. Indian Journal of Experimental Biology 2007: 45: 599-602. (Abstract) Abstract: Immunodiagnostically useful M. tuberculosis H37Ra protein antigens ES-31, ES-43 and EST-6 were isolated from detergent soluble sonicate (DSS) antigen using monospecific antibodies by affinity chromatography and compared with similar antigens isolated from M. tuberculosis culture filtrate for seroreactivity in tuberculosis sera by Indirect Enzyme Linked Immunosorbent Assay. Recovery of affinity purified ES-31, ES-43 and EST-6 antigen from DSS antigen was approximately 3, 3.5 and 4% respectively, compared to 10, 9 and 6.3% from culture filtrate. Affinity purified ES-31, ES-43 and EST-6 antigens from both culture filtrate as well as DSS antigen showed similar seroreactivity with overall sensitivity 85, 80 and 75% respectively and specificity of 85% at optimum concentration of 50 pg protein of each antigen. The results suggest that DSS antigen may be a promising antigen source for isolating antigens of diagnostic interest obviating the need for cumbersome, time-consuming culture techniques of mycobacteria. Shende N, Upadhye V, Kumar S, Gangane N and Harinath BC. Study of M.tuberculosis ES-31 and ES-20 antigen levels in different pathogenic grades of lymph node tuberculosis. Int J Tuberc Lung Dis 2007; 11(2); 222-226.. (Abstract) Abstract: OBJECTIVE: To study tuberculous excretory-secretory (ES) 31 and ES-20 antigens in different pathogenic grades of lymph node tuberculosis (TB). DESIGN: The study group included lymph node TB patients showing granuloma with mature epithelioid cells based on cytology findings (strong immune response group, SI) and patients showing no granuloma formation and acellular necrosis (weak immune response group, WI). Sandwich ELISA was performed using affinity purified antibodies against Mycobacterium tuberculosis ES-31 and ES-20 antigens to assay free and immune complexed antigen levels in the serum of these patients. RESULTS: Higher levels of immune complexed ES-31 (geometric mean titre [GMT] 848) and ES-20 (GMT 1818) antigens than free ES-31 (GMT 462) and ES-20 (GMT 647) were observed in WI patients. There were higher levels of immune complexed ES-20 antigen levels (GMT 1818) in WI patients than in SI lymph node TB patients; the difference was significant (P < 0.05).  CONCLUSION: Elevated levels of immune complexed ES-20 antigen in patient's serum may be a useful immunological marker for weak immune response patients in lymph node TB.  Das AK, Banerjee S, Kumar S, Reddy MVR, Harinath BC. Immunoscreening of clinically suspected pulmonary and extrapulmonary tuberculosis cases using ES-31 and ES-41 antigens – Hospital case study. The Indian Practitioner, Dec. 2006; 59(12):801804. (Abstract) Abstract: Immunoscreening was done by ELISA for 22 sera samples from the patients admitted in Kasturba Hospital, Sevagram. These patients were clinically suspected for pulmonary and extrapulmonary tuberculosis, as no confirmatory finding could be arrived at pathological, radiological, bacteriological and biochemical investigations done on their clinical samples. Given different line of symptomatic and antibiotic trial, no relief was observed. Blood samples were examined for presence of tuberculosis specific antibody and antigen. Two different antigen preparations namely M.tb. H37Ra ES-31 and ES-41 were used for the detection of tubercular IgG antibody whereas affinity purified anti ES-31 antibody was used for tubercular circulating free and immune complexed (CIC) antigen . The positivity observed for IgG antibody, tubercular free and CIC- antigen were 50%, 82% and 86% respectively. On considering the presence of any of the IgG antibody, tubercular free or CIC-antigen as confirmation for tubercular infection, all 22 patients were detected. Based on the ELISA results and clinical suspicion, the patients were put on ATT and followed for 5 months for compliance. All showed relief of symptoms and ATT was continued till full course. It can be envisaged from this study that when routine measures fail for confirmation of tubercular aetiology, serology has its place and may be applied for detection of tuberculosis in clinically suspected cases. Swati Bera, Niraj Shende, Satish Kumar and B.C. Harinath. Detection of Antigen and Antibody in Childhood Tuberculous Meningitis. Indian Journal of Pediatrics, Aug 2006; 73:675-679. (Abstract) Abstract: Objective: Mycobacterium tuberculosis excretory secretory 31 kDa, a serine protease antigen (M. tb ES-31), prepared from Mycobacterium tuberculosis H[37]Ra culture medium has been shown to have potential in detecting tuberculosis. Precise diagnosis and management of tuberculous meningitis, in children in particular, is essential to curtail mortality and morbidity. Methods. In this study, M. tb ES-31 antigen, was used in Indirect ELISA to detect tuberculous IgG antibody, in sera and CSF samples while affinity purified anti ES-31 goat antibody was used in sandwich ELISA for detection of tuberculous antigen. In sixty-five samples each of CSF and sera from cases with neurotuberculosis and control with non-tuberculous diseases were collected from Kasturba Hospital, Sevagram. Results. Among the 20 patients suffering from neurotuberculosis the IgG antibody was detected in 17(85%) of CSF and 16(80%) of sera samples, while antigen was detected in 18 (90%) in CSF and 16 (80%) in sera. Overall specificity of the assay for both IgG antibody and antigen detection in CSF was 96% while in sera it was 94% for IgG antibody and 96% for antigen detection. Conclusion. This study showed the usefulness of mycobacterial serine protease antigen and its antibody in detecting neurotuberculosis. Bhaskar Chinnaiah Harinath, Satish Kumar, Santa Saha Roy, Suvarna Hirudkar, Vijay Upadhye, Niraj Shende. A cocktail of affinity – purified antibodies reactive with diagnostically useful mycobacterial antigens ES-31, ES-43, and EST-6 for detecting the presence of Mycobacterium tuberculosis. Diag Microbiol Infect Dis 2006; 55(May):65-68.. (Abstract) Abstract: A cocktail of affinity-purified antibodies against diagnostically useful Mycobacterium tuberculosis H37Ra excretory-secretory protein antigens ES-31, ES-43, and EST-6 was explored for detection of circulating free and immune-complexed (IC) antigen in sera of patients with confirmed tuberculosis (TB) by sandwich enzyme-linked immunosorbent assay and compared with monospecific anti-ES-31 antibody. Out of 68 smear-positive TB cases studied, using cocktail antibody, a sensitivity of 97% (66/68) for immune-complexed cocktail antigen and 91% (62/68) for free cocktail-antigen detection was observed, compared to 91% (62/68) for immune-complexed ES-31 and 79% (54/68) for free ES-31 antigen when anti-ES-31 antibody was used alone. Thus, combinatorial use of antibodies showed improved sensitivity and was thus observed to be better than single antibody. The specificity was observed to be 99% for immune-complexed antigen using cocktail antibody. Furthermore, analysis of different groups of TB sera showed that circulating immune-complexed antigen is a sensitive marker than free antigen. Santa Saha-Roy, Niraj Shende, Satish Kumar and BC Harinath. Effectivity of crude versus purified mycobacterial secretory proteins as immunogen for optimum antibody production. Indian J of Experimental Biology, December 2005;43:1196-1198. (Abstract) Abstract: Monospecific antibodies have been successfully utilized in antigen detection, which is better indicator of active infection. Mycobacterium tuberculosis excretory secretory (M tb ES) antigens such as ES 31, ES 41 and ES 43 (3l kDa, 41 kDa and 43 kDa protein, respectively) have been shown to be present in Mycobacterium tuberculosis H37Ra culture filtrate and are of diagnostic interest. To study the immunogenic potential of crude versus purified antigen, goat was immunized with M tb detergent soluble sonicate (DSS) antigen as well as purified antigen fraction (ESAS 7) containing ES 31 antigen. Both anti-DSS IgG antibody and anti ESAS 7 IgG antibody were found to be reactive with ES 31 antigen upto I ng concentration of antibody by ELISA. Crude DSS antigen was found to be quite effective in producing high titre antibodies and showed further high reactivity with other ES antigens (ES 41 and ES 43) of diagnostic interest. Sonika Gupta, Niraj Shende, Amerjeet Singh Bhatia, Satish Kumar and Bhaskar C Harinath. IgG subclass antibody response to mycobacterial serine protease at different stages of pulmonary tuberculosis. Med Sci Monit, 2005;11(2): CR585-588. (Abstract) Abstract: Summary: Tuberculosis (TB) is a chronic bacterial infection caused by M. tuberculosis. Studies of antibody response in TB have focussed mainly on their usefulness as a diagnostic serological tool, with little attention given to analysis of antibodies at the isotype and subclass level in relation to disease pathogenesis. Hence the present study was done to analyse IgG subclass response at different stages of tuberculosis, in order to understand the immunological events associated with disease development. Sera samples were collected from 104 subjects: 79 tuberculosis patients (fresh, relapse and chronic cases) and 25 healthy normals. IgG subclass antibody response was analysed by indirect plate peroxidase ELISA against previously reported mycobacterial serine protease (ES-31) antigen. Fresh cases of tuberculosis showed increased IgG1 and IgG3 antibodies, while a few cases showed moderately increased IgG2. IgG1 and IgG3 were found to be elevated with increased bacillary load. Relapse and chronic cases showed increased IgG1 and IgG3, while positivity to IgG2 was decreased. Chronic cases showed a moderate increase in IgG4 antibody. Thus IgG1 and IgG3 were predominant in all forms of tuberculosis. The elevated levels of IgG1 and IgG3 antibodies to mycobacterial serine protease in active tuberculosis observed in this study provide an additional marker for diagnosis of tuberculosis. Furthermore, the higher level of these antibodies with high bacillary load patients and in chronic cases of tuberculosis may provide valuable insight into their possible role in disease progression. N. Shende, S. Gupta, AS Bhatia, S. Kumar, BC Harinath. Detection of free and immune complexed serine protease and its antibody in patients of tuberculosis with and without HIV co-infection.  Int. J of Tuberc. Lung Disease 2005; 9(8): 915-919. (Abstract) Abstract: OBJECTIVE: To understand the usefulness of detecting tu­berculous IgG antibodies against mycobacterial excretory­-secretory 31 kDa serine protease antigen (SEVA TB ES-31) and circulating free and circulating immune-complexed (CIC) serine protease in TB patients with and without HIV infection. DESIGN: Serum was collected from 144 individuals: pa­tients with TB, with TB-HIV co-infection and HIV infec­tion only, and ill and healthy controls. SEVA TB ES-31 antigen, a serine protease isolated from Mycobacterium tuberculosis H37Ra culture fluid, was used in indirect penicillinase ELISA to detect tuberculous antibodies. Similarly, affinity purified anti-ES-31 antibody was used in sandwich ELISA to detect circulating free and CIC serine protease. RESULTS: There was less sensitivity for tuberculous antibody in HIV-infected TB patients (46%) than in those with TB alone (87%) using mycobacterial serine protease. However, the sensitivity of detection of TB in the presence of HIV increased to 87% by concomitant detec­tion of circulating free and CIC serine protease antigen. Gupta S, Shende N, Kumar S, Harinath BC Detection of antibodies to a cocktail of mycobacterial excretory-secretory antigens in tuberculosis by ELISA and Immunoblotting. Current Science June 2005;88(11):1825-1827. (Abstract) Abstract The seroreactivity of a cocktail of purified mycobacterial excretory–secretory (ES) antigens ES-31, ES-41 and ES-43 was assessed by ELISA and immunoblotting in patients with pulmonary tuberculosis. The ES-31 antigen was isolated by affinity chromatography and ES-41 and ES-43 were isolated by fast protein liquid chromatography from Mycobacterium tuberculosis H37Ra culture filtrate. Seven of 27 pulmonary tuberculosis sera were not reactive to ES-31 antigen by ELISA. However, 6 out of 7 turned positive, when a cocktail of ES-31, ES-41 and ES-43 antigens was used in ELISA. Seroreactivity pattern of cocktail antigen was studied in immunoblotting using tuberculous sera. Addition of ES-41 and ES-43 antigens helped in increasing the sensitivity compared to ES- 31 alone. Further, ELISA was observed to be more sensitive than immunoblotting using a c ocktail of antigens. S. Bhatia, Sonika Gupta, Neeraj Shende, Satish Kumar and B.C. Harinath. Serodiagnosis of Childhood Tuberculosis by ELISA. Indian J of Pediatr2005;16(1);72(5):383-7. (Abstract) Abstract: OBJECTIVE: Diagnosis of childhood tuberculosis remains an enigma despite many recent technological developments. The present study has been taken up with the aim to assess the diagnostic potential of mycobacterium tuberculosis excretory-secretory ES-31 antigen and affinity purified anti ES-31 antibodies in the serodiagnosis of different spectrum of childhood tuberculosis. METHODS: Mycobacterium tuberculosis H37Ra excretory-secretory antigen (ES-31) and affinity purified goat anti ES-31 antibodies were used in stick penicillinase ELISA for IgG antibody detection and stick Sandwich penicillinase ELISA for detection of circulating free and immune complexed antigen in the sera of 230 children. RESULTS: Analysis of tubercular antibody, circulating free and immune complexed antigen (CIC-Ag) was done in both pulmonary and extrapulmonary form of childhood tuberculosis and overall sensitivity of 81.4% with a specificity of 93% was achieved for detection of antitubercular IgG antibodies. Of the five cases of pulmonary tuberculosis showing absence of IgG antibody, 3 showed the presence of CIC-Ag and one was found positive for both free and CIC-Ag. Similarly out of 8 cases of extrapulmonary childhood tuberculosis missed by IgG detection 5 were found to be positive for CIC-Ag and 1 showed the positive reaction for both free and immune complexed antigens. CONCLUSION: IgG antibody to excretory-secretory antigen ES-31 is found to be having good specificity with acceptable sensitivity in detecting different forms of childhood tuberculosis. Further detection of circulating free and/or immunecomplexed antigen can be used as an adjunct tool in the diagnosis of childhood tuberculosis. Sonika Gupta, Niraj Shende, Satish Kumar and B.C. Harinath. Isolation of excretory secretary protein 6 kDa antigen (ES-6) and its seroreactivity in patients with different stages of pulmonary tuberculosis and healthy house hold contacts. Biomedical Research 2005; 16(1);23-27. (Abstract) Abstract: An Excretory Secretory protein antigen of 6 kDa (ES-6) was isolated from Mycobacterium tuberculosis H37Ra culture filtrate by gel filtration using fast protein liquid chromatography. Seroreactivity of ES-6 antigen was compared with earlier reported diagnostically useful ES-31 and ES-43 antigens at different stage of pulmonary tuberculosis and in household contacts of the patients. The ES-31 and ES-43 antigens showed good immune response in chronic and relapse cases respectively while ES-6 antigen has shown comparatively low immune response in these cases. However ES-6 showed increased seroreactivity in household contacts of pulmonary tuberculosis patients. These results suggest the heterogeneous responses of antigens in different disease conditions and immune response to ES-6 antigen may be associated with latent infection for predicting active disease in course of time, as observed in the follow up of these individuals. Shende N, Gupta S, Kumar S, Harinath BC. Levels of antibody, free antigen and immune complexed antigen by ELISA in different grades of sputum positive patients of pulmonary tuberculosis. Indian J of Pathology and Microbiology July 2004; 47(3): 438-440. (Abstract) Abstract: The ES-31 (31 kDa protein) antigen was obtained in purified form from culture filtrate of Mycobacterium tuberculosis H37Ra and was found to have potential in immunodiagnosis of pulmonary tuberculosis. Serum samples from 38 confirmed sputum positive pulmonary tuberculosis patients were collected. These samples grouped into AFB+, AFB++, AFB+++ depending upon sputum bacillary load. Analysis of tubercular antibody, circulating free and immune complexed antigen (CIC-Ag) was done in serum samples by Indirect and sandwich ELISA using ES-31 antigen and affinity purified anti ES-31 antibody respectively. The analysis of Geometric mean titre (GMT) of all the three groups showed that GMT of tuberculosis antibody was considerably decreased compared to elevated levels of CIC-Ag (Antibody: 1360 to 816 and CIC-Ag: 534 to 1744 ) moving across from low bacillary loaded sample to high bacillary loaded samples. Whereas there is no significant change in the titre of circulating free antigen. Low levels of detectable antibody in high bacillary loaded samples i.e. AFB+++ group is possibly due to removal of antibody from circulation by immune complex formation as confirmed by its elevated levels in high bacteremic patients. Gupta S, Shende N, Kumar S, Harinath BC. Antibody response to M.tb H37Ra excretory secretory ES-43 and ES-31 antigens at different stages of pulmonary tuberculosis.  Biomedical Research, 2004; 15(1):76-79. (Abstract) Abstract: The secreted protein antigens provide first stimulus in vivo for humoral and cellular immune response to mycobacteria and may play useful role in serodiagnosis. A secretory protein ES-31 isolated from Mycobacterium tuberculosis H37Ra culture filtrate has been earlier shown diagnostically useful in pulmonary tuberculosis. Similarly ESAS-6 antigen found to be reactive in our earlier studies, was further fractionated by Fast Protein Liquid Chromatography (FPLC) and obtained a 43 kDa protein labelled as ES-43 antigen. The immunoreactivities of purified ES-31 kDa and ES-43 kDa antigens were assessed in sera at different stages of pulmonary tuberculosis viz. Fresh, chronic and relapse cases by indirect stick penicillinase ELISA. The ES-31 antigen showed higher reactivity in chronic cases, while ES-43 antigen was primarily recognized by serum antibodies in relapse cases, whereas both antigens showed comparatively decreased antibody response in fresh cases. The study does show that the immune response varies with different antigens at different stages of tuberculosis. It will be of interest to monitor tuberculosis patients by using antibody response to ES-31 and ES-43 antigens, as a predictive tool for relapse cases. Gupta S, Kumar S, Harinath BC. Mycobacterium tuberculosis excretory secretory (ES) protein antigens of interest in diagnosis, prognosis and prediction of disease development. J. MGIMS, 2004 Sept.; 9(ii): 16-21. Review.  (Abstract) Abstract: The Excretory Secretory (ES) proteins of Mycobacterium tuberculosis released into culture medium have been of considerable diagnostic interest. The ES proteins ES-31, ES-41, ES-43 and ES-6 have been isolated from M. Tuberculosis H37Ra culture filtrate by various sophisticated biochemical methods at our centre. The seroreactivity of these purified antigens have been assessed in different stages of pulmonary tuberculosis (fresh, relapse and chronic), extra pulmonary tuberculosis and in household contacts. These studies demonstrated the heterogeneous antibody response to these purified antigens in different disease conditions. Analysis of immune response to these purified antigens showed ES-31 antigen having good diagnostic potential in pulmonary tuberculosis and in certain groups of extra pulmonary tuberculosis in particular tuberculous lymphadenopathy, tuberculous meningitis, whereas ES-41 was found to be useful in abdominal and bone & joint tuberculosis. ES-43 was primarily recognized by serum antibodies in relapse cases and ES-6 antigen showed elevated   levels of antibody in household contacts. Further immunomonitoring of TB patients under ATT, showed that ES-31 is useful in determining the effectiveness of therapy and patient’s compliance. E Raji Nair, S Banerjee, S Gupta, S Kumar, MVR Reddy  & BC Harinath. Seroreactivity and characterization of 31 kDa M.tb.  secretory protease of diagnostic interest in tuberculosis. Trends Clin Biochem Lab Medicine 2003;330-338. (Abstract) Banerjee S, Gupta S, Shende N, Kumar S & Harinath BC. Serodiagnosis of tuberculosis using two ELISA system. Ind. J. Clin. Biochem 2003; 18(2): 48-53. (Abstract) Abstract: Serodiagnosis by ELISA has been widely explored over the years, in the diagnosis of tuberculosis. Two ELISA systems were evaluated for detection of mycobacterial antibodies in pulmonary and extra pulmonary tuberculosis. The two test assays explored were ERBA LISA (TB IgG) test (Anda Biologicals) which uses A60 antigen complex found in the cytosol of typical and atypical mycobacteria, and SEVA TB (IgG) ELISA, which uses a 31 kDa, glycoprotein antigen purified from M.tb H37Ra culture filtrate. Sera from 98 proven tuberculosis [pulmonary TB (48), tuberculous lymphadenopathy (30), tuberculous meningitis (15) & genito-urinary TB (5)] were studied along with 32 healthy controls. The overall positivity obtained ,using ERBA LISA (TB IgG) test and SEVA TB (IgG) ELISA test was 72.9% and 91.6% in pulmonary tuberculosis, 43.3% and 76.6% in tuberculous lymphadenopathy respectively. The sensitivity of ERBA LISA test in tuberculous meningitis and genito-urinary TB was significantly low (26.6% & 40% respectively) compared to sensitivity obtained using SEVA TB ELISA (86.6% & 60% respectively) with overall specificity of 60% and 87.5%. Thus SEVA TB IgG ELISA test was found to be more sensitive than ERBA LISA in detecting IgG antibodies in tuberculous sera, in particular in extra pulmonary tuberculosis cases. Bhatia AS, Kumar S & Harinath BC. Immunodiagnosis of tuberculosis: An update. Ind. J. Clin. Biochem 2003; 18(2):1-5. (Abstract) Abstract: Tuberculosis is still a major health problem in most developing countries and its incidence is rising in many developed countries. This resurgence has been attributed to the HIV epidemic and TB has been declared as a global health emergency by WHO in 1993. The diagnosis of tuberculosis mainly depends upon initial clinical suspicion and radiographic findings with subsequent bacteriological confirmation by sputum smear examination and culture. Lack of sensitivity in smear examination, non specificity of radiological findings, extended turn around time of Mycobacterium tuberculosis culture and difficulties in diagnosing paucibacillary, childhood and extrapulmonary tuberculosis has necessitated to explore the utility of immunodiagnosis of tuberculosis as a convenient and cost effective test to supplement clinical information for definite diagnosis. Many commercial tests are available in the market for diagnosis of TB. Most of these tests are based on the detection of IgG, IgA and IgM antibodies to ,specific mycobacterial antigen or mixture of antigens. Indigenous immunoassay systems have explored excretory-secretory ES-31 mycobacterial antigen for immunodiagnosis of TB. Many a time there is lack of consistent elevation in all the three Ig classes in active infection thus making it more important to determine the ideal antibody isotype assay for reliable diagnosis of tuberculosis and to save the costs of the patient for unnecessary investigations. S. Banerjee, S. Kumar & B.C. Harinath, Isolation and characterization of in vivo released 41 kDa mycobacterial antigen in pulmonary and bone and joint tuberculosis and its identification with H37Ra in vitro released antigen. Int. J. Tuberc Lung Dis 2003; 7(3): 278-283. (Abstract) Abstract: OBJECTIVE: To isolate and characterize in vivo released 41 kDa mycobacterial antigen in pulmonary and bone and joint tuberculosis (TB) and its identification with in vitro released ES-41 kDa antigen. DESIGN: Circulating antigen was isolated from confirmed pulmonary tuberculosis serum (PTS) and bone and joint tuberculosis serum (BJS) by trichloroacetic acid precipitation and further fractionation by fast protein liquid chromatography (FPLC). RESULTS: Fractionation of PTS and BJS by gel filtration column gave six protein fractions each. PTS-G3 and BJS-G3 showed maximum antigenic activity with ELISA. Further fractionation of PTS-G3 and BJS-G3 on cation exchange FPLC gave four different fractions each, of which BJS-G3B was seroreactive similarly to in vitro released 41 kDa antigen (ES-41) isolated from culture medium whereas PTS-G3C was slightly less seroreactive. BJS-G3B could inhibit binding of in vitro released ES-41 to affinity purified antibodies in inhibition ELISA at lower concentrations than PTS-G3C (2 vs. 20 ng/ml), showing the identical nature of the antigens. Biochemical characterisation showed that circulating antigen PTS-G3C, BJS-G3B and in vitro released ES-41 antigen were lipoproteins in nature. CONCLUSION: This study helped to demonstrate the presence of 41 kDa antigen in the serum of pulmonary and bone and joint TB patients and its identification with H37Ra in vitro released 41 kDa antigen. S. Banerjee, S. Gupta , N. Shende, S. Kumar & B.C. Harinath, Cocktail of ES-31 and ES-41 antigens for screening of pulmonary and extrapulmonary tuberculosis  Biomedical Research 2002; 13(2/3): 135-137. (Abstract) Abstract: A serological test with good diagnostic potential in pulmonary and in particular extra pulmonary tuberculosis, a paucibacillary condition would contribute to early detection and help in better management of patients. Two different secretory proteins of Mycobacterium tuberculosis (M. tb) H37Ra viz; ES-31 with diagnostic potential in pulmonary TB, tuberculous lymphadenopathy (TBLN) and tubercular meningitis (TBM) and ES-41 having potential in abdominal and bone and joint tuberculosis have been reported earlier from our laboratory. In present communication we report the use of cocktail preparation of ES-31 and ES-41 antigens for detecting IgG antibodies in pulmonary and different forms of extra pulmonary tuberculosis. Cocktail preparation has shown better sensitivity of 92%, 88% and 90% in pulmonary TB, TBLN &TBM, respectively compared to ES-31 alone and 81.5%,& 84.1% in abdominal and bone & joint TB compared to ES-41 when used alone. The results envisaged that cocktail preparation of ES-31 and ES-41 antigens is equally promising for the detection of IgG antibodies compared to using ES-31 or ES-41 alone and thus a single assay is useful for screening of broad spectrum of tubercular sera. S Banerjee, S Gupta, Satish Kumar, A V Shrikhande, MVR Reddy  & BC Harinath. Seroreactivity of 31 kDa and 41 kDa mycobacterial secretory proteins isolated from culture filtrate in extra pulmonary tuberculosis. Indian J Patho Microbiol 2003; 43(2): 261-264. (Abstract) Sonika Gupta, Niraj shende, Swati Banerjee, Satish Kumar, MVR Reddy and BC Harinath. Analysis of SEVA TB ES-31 antigen specific immunoglobulins IgM, IgA and IgG in sera of sputum and culture positive pulmonary tuberculosis. Indian J. of Clin. Biochemistry. 2002 Jan; 17(1): 5-8. (Abstract) Abstract: Tuberculosis remains major health problem in India and developing countries. Immunodiagnosis has important role in screening, diagnosis and management of tuberculosis. SEVA TB ES-31 antigen has shown potential in detecting tuberculous IgG antibody in earlier studies from our laboratory. In the present study we have analysed SEVA TB ES-31 antigen specific immunoglobulins IgM, IgA and IgG in clinically and bacteriologically confirmed pulmonary tuberculosis cases to determine the usefulness of specific immunoglobulin class in the diagnosis of patients attending the hospital. Of the 30 cases of pulmonary tuberculosis 25 (83.3%) were positive for IgG, 19 (63.3%) for IgM and 16 (53.3%) for IgA. On combining IgG and IgM positivity, sensitivity was increased to 93.3%. While combining IgG and IgA positivity, sensitivity increased to 90%. However specificity was decreased to 66.6% and 70% for both of these combinations respectively. It could be envisaged from this study that IgG antibody detection against ES-31 antigen showed acceptable sensitivity (83.3%) and specificity (86.6%) compared to IgM or IgA alone or in combination. When immune responses were analysed according to degree of sputum positivity, IgG response was observed to be predominant in all grades, compared to IgM or IgA antibody. The addition of IgM or IgA as an adjunct test increases the sensitivity but at the cost of specificity. Hence the detection of IgG alone is more useful compared to IgM or IgA assay, in detecting tuberculosis disease cases coming to the hospital. Swati Banerjee, E Raji Nair, Satish Kumar, N Gangane, MVR Reddy & BC Harinath. Seroreactivity of two different antigenic protein fractions of M.tb H37Ra excretory secretory antigen in pulmonary and extra pulmonary tuberculosis. J Comm Dis. 2001;33(2): 63-71. (Abstract) Abstract: Excretory-secretory proteins of Mycobacterium tuberculosis H37 Ra, have been of diagnostic interest in pulmonary (PTB) and extra pulmonary tuberculosis (EPTB). Two different excretory-secretory antigenic proteins of M.tbH37 Ra viz., EST-DE1, (a 6% TCA soluble and DEAE anion exchange purified antigen) and ESAS-7 (50% ammonium sulphate solubilized and SDS-PAGE fractionated antigen) were studied in stick indirect penicillinase ELISA for detecting tuberculous IgG antibodies in serum samples of pulmonary as well as extrapulmonary tuberculosis (tuberculous lymphadenopathy (TBLN), tuberculous meningitis (TBM), bone & joint tuberculosis (B&J TB), abdominal tuberculosis (Abd. TB) patients. The ESAS-7 antigen has shown comparatively better seroreactivity (90%) than that of EST-DE1 antigen in pulmonary tuberculosis cases. The overall specificity of 93.2% using ESAS-7 antigen was also found better compared to 86.4% obtained using EST-DE1 antigen. Further, in extra pulmonary tuberculosis group, using ESAS-7 antigen 84% (21/25) of histopathologically confirmed TBLN cases and 90% (9/ 10) clinically diagnosed and ATT responded TBM cases showed positive reaction for tuberculous IgG antibody. The per cent positivity using EST-DE1 antigen was however comparatively low in TBLN and TBM cases, (76% and 80% respectively). In histopathologically proven bone and joint tuberculosis and abdominal tuberculosis cases EST-DE1 antigen showed better sensitivity of 75% and 83.3% respectively in IgG antibody detection compared to that of ESAS-7 antigen (50% and 66% respectively). From the present study, it can be envisaged that ESAS-7 antigenic fraction has a good potential in the diagnosis of pulmonary and certain extra-pulmonary tuberculosis infections (TBLN & TBM) whereas EST-DE1 was found to be better in detecting specific antibodies in bone & joint and abdominal tuberculosis. Swati Banerjee, E Raji Nair, Satish Kumar, MVR Reddy & BC Harinath. Assay of tubercular antibody, circulating free and immune complexed antigen in the diagnosis of pulmonary tuberculosis. Indian J Clin Biochem 2001;16(2):203-206. (Abstract) Abstract: Analysis of tubercular antibody, circulating free and immune complexed antigen (CIC-Ag) was done in confirmed pulmonary tuberculosis sera by ELISA, using ES-31 antigen and affinity purified anti ES-31 antibody. Twenty three of 25 (92°k) tuberculosis sera were positive for IgG antibody to ES-31 antigen. Using anti ES-31 antibody, free tubercular antigen could be detected in 20 of 25 (80%) cases whereas circulating immune complexed antigen (CIC-Ag) in 18 of 25 (72%) cases by sandwich ELISA. Of the two sera showing absence of antibody, one showed presence of free and CIC-Ag whereas the other showed the presence of free antigen. Thus antigen assay may be used as an adjunct tool for confirmation of pulmonary tuberculosis. E Raji Nair, S Banerjee, S Kumar, MVR Reddy & BC Harinath. Isolation of Mycobacterium tuberculosis 31 kDa antigen protein of diagnostic interest from culture filtrate using anti ES-31 antibody by affinity chromatography. Ind. J. Clin. Biochem. 2001; 16: 132-135. (Abstract) Abstract: Proteins secreted into the culture medium by Mycobacterium tuberculosis(Mtb) are shown to be source of antigens of immunodiagnostic interest. An in vitro released 31 kDa antigen ESAS-7F isolated from M.tb H37Ra culture filtrate by salt precipitation SDS-PAGE and cation exchange fast protein liquid chromatography (FPLC) was shown earlier to be a diagnostically important antigen fraction. In this report, we describe the isolation of ESAS-7F antigen using nonspecific antibody coupled to sepharose CL-4B column. The percentage recovery of ESAS-7F antigen using affinity chromatography was approximately 8% of the total ES antigen proteins compared to 0.05% obtained by conventional purification steps using salt precipitation, SDS-PAGE and FPLC. Similar seroreactivity was observed by the antigen isolated by both the methods in indirect ELISA. Affinity chromatography helped in an increased recovery of ESAS-7F antigen and obviates the need for time consuming conventional purification steps. E Raji Nair, Swati Banerjee,  Satish Kumar, MVR Reddy & BC Harinath. Purification and characterization of a 31 kDa mycobacterial excretory-secretory antigenic protein with a diagnostic potential in pulmonary tuberculosis. Indian J Chest Dis Allied Sci 2001; 43:81-90. (Abstract) Nair E. R, Banerjee S, Kumar S, Reddy M. V. R. and Harinath B. C. “Biochemical characterization and Seroreactivity of in vitro and in vivo release 31 kDa antigen protein of diagnostic interest in tuberculosis.” Proceedings of the first international symposium in Recent Advances in Molecular biology, Allergy and Immunology, Vadodara, 3-5th September, 2000, 208-218. (Abstract) Abstract: The excretory-secretory proteins of Mycobacterium tuberculosis released in vitro and in vivo are of considerable diagnostic interest. A 31 kDa mycobacterial protein E5AS-7F was isolated from M.tb H37 Ra culture medium by 50% ammonium sulphate solubilization and purified by SDS-PAGE followed by fast-protein liquid chromatography using cation-exchange resource ‘S’, 1 ml column. ESAS-7F protein showed good immunodiagnostic potential with about 95% sensitivity and specificity in pulmonary tuberculosis by penicillinase dip-stick indirect ELISA using as little as 25 pg protein per test. Circulating tubercular antigen protein CTA2-7D was isolated from bacteriologically confirmed pulmonary tuberculosis sera by ammonium sulphate precipitation, and purified by fractionation on Ultrogel AcA 34, SDS-PAGE and cation-exchange fast-protein liquid chromatography. CTA2-7D protein showed seroreactivity similar to ESAS-7F antigen and inhibited binding of ESAS-7F to affinity purified antibodies in inhibition ELISA. Biochemical characterization showed in vitro released ESAS-7F antigen as a glycoprotein while in vivo released CTA2-7D antigen as lipoglycoprotein. Further studies on ESAS-7F antigen showed that it is a 31 kDa protein with metallo-serine protease activity and has N-terminal sequence of first 5 amino acids as Asp-Thr-Gly-glu-Ser (NTGQS)/Glu. Nair E. R, Banerjee S, Kumar S, Reddy M. V. R. and Harinath B. C. “Isolation of M.tb 31 kDa antigen protein of diagnostic interest from culture filtrate using affinity purified ES-31 antibody by affinity chromatography.” Proceedings of the first international symposium on Recent Advances in Molecular biology, Allergy and immunology, Vadodara, 3-5th September, 2000, 204-207. (Abstract) Abstract: Proteins secreted into the culture medium by Mycobacterium tuberculosis are shown to be source of antigens of immunodiagnostic interest. An in vitro released 31 kDa antigen ESAS-7F isolated from M.tb H37Ra culture filtrate by SDS-PAGE and cation exchange fast protein liquid chromatography (FPLC) was shown earlier to be a diagnostically important antigen fraction. In this report, we describe the isolation of ESAS-7F antigen using monospecific antibody coupled to sepharose CL-4B column. Crude culture filtrate proteins (ES) and partially purified ammonium sulphate solubilised proteins (ESAS) were applied to the column separately. The percentage recovery of ESAS-7F antigen was approximately 8% and 13% of the total ES and ESAS antigen proteins applied to the column respectively. Also recovery of antigenic activity in eluted ESAS-7F antigen fraction was assayed in indirect ELISA and purity of the antigen was checked by SDS-PAGE. Affinity chromatography helped in an increased recovery of ESAS-7F antigen and obviates the need for time consuming conventional purification steps where the percentage yield of ESAS-7F antigen from ES and ESAS was 0.05% and 0.25% respectively. Nair ER, Banerjee S, Kumar S & Harinath BC. Isolation and characterization of a 31 kDa Mycobacterial antigen from tuberculous sera and its identification with in vitro released culture filtrate antigen of M.tb H37Ra Bacilli. Scandinavian Journal of Infectious Diseases 2000; 32: 551-556. (Abstract) Abstract: Antigens released in vivo are of considerable interest in the immunodiagnosis of infectious diseases. Circulating antigen was isolated from bacteriologically confirmed tuberculous sera by ammonium sulphate precipitation. The protein fraction between 36% and 75% ammonium sulphate was reactive with tuberculosis (TB) sera showing the presence of circulating tubercular antigen (CTA). Fractionation of CTA on ultrogel AcA 34 gel filtration column gave 3 protein fractions CTA1, CTA2 and CTA3. CTA2 showed maximum antigenic activity by sandwich enzyme-linked immunosorbent assay (ELISA). SDS-PAGE fractionation and seroreactivity studies showed the presence of highly reactive tubercular antigen in CTA2-7 protein fraction by sandwich ELISA. Further fractionation of CTA2-7 on cation exchange fast-protein liquid chromatography (FPLC) gave 4 antigenic fractions, of which CTA2-7D was seroreactive similar to 31 kDa antigen (ESAS-7F) isolated from in vitro culture medium. Furthermore, CTA2-7D could inhibit binding of in vitro released ESAS-7F to affinity-purified antibodies in inhibition ELISA. CTA2-7D antigen may be used as a target antigen in confirming active tubercular infection. Biochemical characterization showed circulating antigen CTA2-7D to be a lipoglycoprotein is released in vivo. ESAS-7F as a glycoprotein is released in vitro culture. Pramanik J, Lodam AN, Badole CM, Reddy MVR, Patond KR and Harinath BC. Detection of tubercular antibody and antigen in sera of bone and joint tuberculosis.   Ind.  J. Clin. Biochem. 2000; 15  (1):  22–28. (Abstract) Abstract: Trichloroacetic acid (TCA) solubilized and DEAE fractionated Mycobacterium tuberculosis H37Ra excretory-secretory (ES) antigen viz., Mtb EST DE1 and affinity purified goat antibodies to the TCA solublized ES antigen (Mtb EST) were explored in detecting tubercular antibody and antigen respectively in sera of bone and joint tuberculosis by indirect and sandwich ELISA. Out of total 36 bone 8 joint tuberculosis cases, tubercular antibody was detected by indirect ELISA in 30 patients (sensitivity 83%), while circulating tubercular antigen was detected by sandwich ELISA in 27 patients (sensitivity 75%). Out of 34 non tubercular disease control cases, 10 patients showed positive reaction for antibody while only 4 patients showed positive reaction for antigen. In another group of 34 healthy subjects who were screened, 4 individuals showed positive reaction for tubercular antibody and 2 cases for antigen. This study shows that antigen detection assay using affinity purified anti Mtb EST antigen antibody is superior with overall specificity of 91% as compared to antibody detection assay with 75% specificity in bone & joint tuberculosis. Lodam AN, Reddy MVR and Harinath BC. Immunodiagnosis of pulmonary tuberculosis by concomitant detection of antigen and antibodies of excretory-secretory protein of Mycobacterium tuberculosis H37Ra. J. Biosci. 1998; 23 (1): 91-93. (Abstract) Abstract: With a view to diagnosing tuberculosis in populations in endemic areas, excretory-secretory antigen fraction (Mtb EST-6) of purified Mycobacterium tuberculosis H37Ra and affinity purified polyclonal antibodies against Mtb EST were used to detect both antibodies and circulating antigen in the sera of patients and disease-free individuals. Indirect stick penicillinase ELISA system using Mtb EST-6 detected antigen-specific IgG antibody in 84% of sputum positive, 77% of sputum negative pulmonary tuberculosis patients and 7% of healthy and 11% of subjects with nontuberculosis diseases. Similarly, a sandwich penicillinase ELISA system using affinity purified anti Mtb EST antibodies detected circulating antigen in 83% and 61% of sputum positive and negative pulmonary tuberculosis subjects. In contrast only 24% of healthy and 18% of disease controls showed seropositivity. Antibody assay showed higher sensitivity and specificity (83% and 91% respectively) compared to antigen detection (sensitivity of 70% and specificity of 79%). However, by concomitant use of both assays it was possible to enhance the specificity of detection to 98%, though sensitivity was reduced marginally to 70%. The present study confirms the presence of both antigen and specific antibodies in the circulation during clinical disease and draws attention to the utility of Mtb EST-6 as a diagnostic marker of pulmonary tuberculosis. Nair ER, Kumar S, Reddy MVR and Harinath BC. Mycobacterium tuberculosis H37Ra ESAS-7 an excretory-secretary antigen fraction of immunodiagnosic potential in pulmonary tuberculosis; Ind. J. Clin. Biochem. 1998; 13 (2): 98-105. (Abstract) Abstract: A mycobacterial excretory-secretory protein fraction ESAS-7 purified by 50% ammonium sulphate precipitation followed by SDS-PAGE fractionation was evaluated by penicillinase enzyme linked immunosorbent assay (ELISA) for its sensitivity and specificity in the diagnosis of pulmonary tuberculosis. At a "cut off" serum dilution of 600, 38 (90%) of 42 sera from bacteriologically confirmed tuberculosis cases, 15 (100%) of 15 sera from bacteriogically negative but anti tubercular therapy (ATT) responded cases, 3 (7%) of 43 sera from normal healthy subjects and 4 (8%) of 48 sera from non tuberculous disease control cases gave positive reaction for tubercular antibody to ESAS-7 antigen fraction containing predominantly 33-kDa protein with a sensitivity of 90% in bacteriologically confirmed cases and specificity of 92%. Further, this diagnostic assay using the ESAS-7 antigen is more sensitive requiring as little as one nanogram antigen per test compared to use of 100 nanogram EST-6 antigen reported earlier. Thus use of ESAS-7 antigen for antibody detection has good diagnostic potential with improved specificity in pulmonary tuberculosis. Harinath BC, Satish kumar and Reddy MVR. In vitro released antigens in diagnosis and immunomonitoring of filaria and tuberculosis. Ind. J. Clin. Biochem. 1997; 12 (suppl): 8-12. (Abstract) Abstract: In vitro released antigens by living parasites or bacteria under in vitro maintenance or short term culture showing specific humoral immune response have been explored in development of immunodiagnostics for infectious diseases such as filariasis and tuberculosis in our laboratory. ELISA using B. malayi mf ES antigen has been explored for detecting IgG antibody by Indirect ELISA and antigen by Inhibition ELISA and in immunomonitoring of carriers as well as clinical filarial cases. A ten year follow up of mf carriers with DEC therapy showed disappearance of antigen and antibody followed by reappearance in few cases in an endemic area. None of the cases followed developed clinical symptoms suggesting the need for long term monitoring and treatment of microfilaraemic carriers. Further immunomonitoring was found to be useful in confirming filaria aetiology in the absence of microfilaremia and determining appropriate period of treatment of acute, early clinical and occult filarial infections for clinical relief and cure. Indirect Stick Penicillinase ELISA system using Mtb EST-6 antigen for detecting tuberculous IgG antibody and a Sandwich Penicillinase ELISA system using affinity purified antibody for detecting circulating antigen were explored in tuberculosis. A combination of both the assay systems with a sensitivity of 70% and specificity of 98% was found to be promising in the precise diagnosis of pulmonary tuberculosis. Further antigen detection was found to be useful in bone and joint tuberculosis. Lodam AN, Pramanik J, Reddy MVR and Harinath BC. Diagnostic potential of fractionated Mycobacterium tuberculosis H37Ra excretory-secretory (EST-DE1) antigen in pulmonary tuberculosis. Ind. J. Clin. Biochem. 1997; 12 (1): 71-77. (Abstract) Abstract: Tuberculosis is emerging as a major public health problem in developing and developed world. Early and precise diagnosis is of prime importance in successful control of infection .Indirect ELISA with penicillinase as marker was developed using purified at tuberculosis excretory-secretory (EST- DE1) antigen for detecting IgG antibodies in pulmonary tuberculosis. The assay System gave a overall sensitivity of 82% for both smear positive and pulmonary tuberculosis cases with a specificity of 84%. The positive and negative predictive values were 75% and 88% respectively. Further studies with EST- DE1 antigen revealed that ,it contains two of the active antigen fractions of Mtb EST antigen i.e,Mtb EST-4 (56-68 KDa) and Mtb EST-6 (37-45 KDa), as demonstrated by inhibition ELISA. Reactivity with monoclonal antibodies HGT 3a showed the presence of 38 KDa molecule in EST- DE1 antigen. Paramanik J, Lodam AN, Reddy MVR, Narang P and Harinath BC. Increased yield of excretory-secretory antigen with thyroxine supplementation in in vitro culture of tubercle bacilli. Ind. J. tub. 1997; 44: 185-190. (Abstract) Abstract Excretory-secretory antigen(ES antigen) obtained  from short term in vitro culture of Mycobacterium tuberculosis H37Ra strain is of immunodiagnostic interest .Attempts were made to increase the yield of ES antigen in culture medium. In this study, the effect of thyroid hormone supplement in culture medium on the bacillary growth and on the yield of ES antigen has been evaluated .The supplementation helped in getting better yield of ES antigen. Lodam AN, Reddy MVR, Narang P, Gupta OP and Harinath BC. Fractionation, analysis and diagnostic utility of Mycobacterium tuberculosis H37Ra excretory-secretory antigen in pulmonary tuberculosis. Ind. J. Biochem. Biophys. 1996; 33: 66-71. (Abstract) Abstract: Excreatory-secretory antigen of mycobacterium tuberculosis H37Ra(Mtb ES antigen) isolated from culture filtrate was partially purified by 6% trichloroacetic acid precipitation. The TCA supernatant fraction (Mtb ES antigen)was examined for diagnostic use in the detection of tuberculous IgG antibody in human sera by stick Indirect ELISA. Using Mtb ES antigen, tuberculous IgG antibody was detected in 90% of smear positive and 71% of smear negative pulmonary tuberculosis cases and 10% of healthy and disease controls .Further fractionation of Mtb ES antigen by SDS-PAGE yielded four active antigenic fractions viz., Mtb EST-3,4,6and 10.Diagnostic evaluation of these fractions showed Mtb EST-6 antigen fraction to be useful in detection of both smear positive and smear negative pulmonary tuberculosis cases with sensitivities of 94% and 78% respectively and specificity of 88%. Basak A, Sinha Choudhary S, Lodam A.N, Gupta OP, Narang P, and Harinath B C. Detection of IgG using Mycobacterium tuberculosis H37 Ra ES antigen in tuberculosis by Penicillinase stick ELISA. Proceedings of CSIR Golden Jubilee symposium on tropical diseases “Molecular Biology and control strategies”. 1994; 558. (Abstract) Abstract: Satish Kumar, Chenthamarakshan V, Reddy MVR, Narang P, Gupta OP and Harinath B C. Detection of tuberculous= IgG antibodies using Mycobacterium tuberculosis H37Ra excretory-secretory antigen and tuberculin purified protein derivative. Ind. J. Exptl Biol. 1994; 32: 163-167. (Abstract) Abstract: Different antigen preparations, viz. excretory-secretory antigen (ES Ag), phosphate buffer saline soluble antigen (PBS-S Ag) and sodium dodecyl sulphate soluble antigen (SDS-S Ag) of M. tuberculosis (M.tb) H37 Ra strain along with tuberculin purified protein derivative (PPD) were employed in stick indirect ELISA to detect IgG antibodies in sera of sputum positive pulmonary tuberculosis cases. Sera from healthy individuals and individuals with diseases other than tuberculosis (cross-reacting diseases) were used as controls. ES antigen and PPD showed higher antibody titres in tuberculosis cases (GMT-1378 each) compared to PBS-S Ag (GMT-454) and SDS-S Ag (GMT-974). Thereafter, an extensive study was done analysing higher number of sera in each group for the detection of tuberculous IgG antibodies using ES Ag and PPD. The ES Ag showed better sensitivity (87%) and specificity (85%). compared to the sensitivity (73%) and specificity (78%) achieved with PPD. The ES Ag also showed higher IgG antibody titre (GMT- 1068) than PPD (GMT-721). From the present study it can be envisaged that ES Ag has high diagnostic potential in tuberculosis. Bhaskar A, Pradan P, Chaturvedi P, Basak A, Lodam AN, Narang P, and Harinath BC. Immunodiagnosis of childhood pulmonary and extrapulmonary tuberculosis using mycobacterium tuberculosis ES antigen by penicillinase ELISA. Annals of Trop Pead. 1994; 14: 25-30. (Abstract) Abstract: The diagnostic potential for detection of IgG to Mycobacterium tuberculosis excretory-secretory (ES) antigen in childhood pulmonary and extrapulmonary tuberculosis was explored. IgG antibody to M. tuberculosis ES antigen was detected by indirect penicillinase ELISA. Twenty (80%) out of 25 pulmonary tuberculosis cases (clinically diagnosed and /or AFB-positive), five of nine tuberculous pleural effusion cases and only six of 69 cases in the control group were positive for IgG antibody to M. tuberculosis ES antigen. All CSF and sera were positive for IgG antibody in 12 cases of clinically diagnosed tuberculous meningitis (TBM). Out of 35 cases in the control group for TBM, all five cases of pyogenic meningitis but none of the 13 cases of viral encephalitis, five cases of enteric encephalopathy and 12 cases with no CNS infection were positive for anti-tubercular IgG antibody in CSF samples. Only two of them, i.e. one case of pyogenic meningitis and the other with no CNS infection, were positive for antibody in sera. The study demonstrated the potential of this assay in the diagnosis of tuberculosis in children where bacteriological confirmation is very difficult. Sood J, Gupta OP, Narang P, Cheirmaraj K, Reddy MVR and Harinath BC. Penicillinase ELISA for detection of tubercular antigen in tuberculosis. J. Com. Dis. 1991; 23 (3): 173-177. (Abstract) Abstract: Stick sandwich enzyme linked immunosorbant assay (ELISA) using rabbit PPDRT 23 immunoglobulins and enzyme pencillinase has been explored for the detection of tubercular antigen in sera and CSF samples of pulmonary tuberculosis and tubercular meningitis (TBM) respectively .The analysis of sera showed 73.3% of pulmonary tuberculosis cases, 16.2% of healthy controls and 44.4% of Hansen’s disease positive for tubercular antigen. The accuracies of positive and negative predictive values were 69% and 82% respectively .The analysis of CSF samples showed the presence of tubular antigens in 76.4% of TBM, 16.6% of polygenic meningitis cases, 19.4% of neurological diseases other than meningitis and 16.1% non-neurological disease controls. The accuracies of positive and negative predictive values were 48% and 94% respectively. Hence this simple test using economical and indigenous reagents can be applied for the diagnosis of pulmonary and extra-pulmonary tuberculosis.