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 H37 Ra strain in the presence of inhibitor.

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 5 microg/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 0.4 microg/well, while orlistat showed inhibition of 60% at 0.5 microg/well. Inhibition of Mtb H,37Ra bacilli is further confirmed in axenic culture. 35% and 29% inhibition by isoniazid at 0.4 microg/well and orlistat at 0.5 microg/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.

Multi-antigen and antibody assays (SEVA TB ELISA) for the diagnosis of tuberculous pleural effusion.

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.

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.

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.

Improved laboratory diagnosis of tuberculosis--the Indian experience.

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.

A sensitive and specific ES-31 antigen detection based fluorometric assay for confirmation of Mycobacterium tuberculosis in cell culture.

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.

Effect of HIV protease inhibitors and Orlistat on mycobacterial ES-31 serine protease, a potential drug target in Mycobacterium tuberculosis.

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.

Detection of circulating free and immune-complexed antigen in pulmonary tuberculosis using cocktail of antibodies to mycobacterium tuberculosis excretory secretory antigens by peroxidase enzyme immunoassay.

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-31 antigen 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 ICES-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 microg/ml and levels of free and IC cocktail antigens were 1.70 +/- 1.04 and 1.13 +/- 0.047 microg/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.

Potential of mycobacterial excretory secretory protein antigens (sevatb ES-31, ES-43, EST-6 and ES-20) as biomarkers to detect Mycobacterium tuberculosis bacilli.

There is a need for a simple and reliable method to identify Mycobacterium tuberculosis 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 H(37)Ra and H(37)Rv 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 H(37)Ra and H(37)Rv 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.

Inhibition of Mycobacterium tuberculosis secretory serine protease blocks bacterial multiplication both in axenic culture and in human macrophages.

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.

Mycobacterial ES-31 serine protease--a biomarker for mycobacterium tuberculosis--a preliminary report.

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.

A low molecular weight ES-20 protein released in vivo and in vitro with diagnostic potential in lymph node tuberculosis.

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 analysed 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.

Isolation of Mycobacterium tuberculosis protein antigens ES-3 1, ES-43 and EST-6 of diagnostic interest from tubercle bacilli by affinity chromatography.

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.

Serum immune complexes as diagnostic and therapeutic markers in lymphatic filariasis.

In the present study we examined the utility of measuring antigen-specific soluble immune complexes (ICs) for the differential diagnosis and therapeutic monitoring of Bancroftian filariasis. Antigen-specific ICs were detected in significantly elevated levels in 90% of lymphatic filarial subjects suffering from disease manifestations, and very low levels in 8-10% of microfilaria (MF) carriers. The high incidence of antigen-specific ICs in the circulation of filarial subjects with clinical manifestations in contrast to the negligible presence in MF carriers facilitates serological differentiation of these two important stages of lymphatic filarial infection. A sudden spurt in circulating levels of antigen-specific ICs was recorded in all MF carriers on diethylcarbamazine (DEC) therapy, and the magnitude of the increase correlated positively with the pretreatment levels of circulating MF. Thus, it is evident from the present study that circulating ICs (CICs) are potential serological markers for both the differential diagnosis of filarial infection and therapeutic monitoring of MF carriers.

Study of M. tuberculosis ES-31 and ES-20 antigen levels in different pathogenic grades of lymph node tuberculosis.

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.

Antigen-specific immune complexes in urine of patients with lymphatic filariasis.

Lymphatic filarial subjects with disease manifestations exhibit significantly elevated levels of immune complexes (ICs) in their circulation. The objective of the study was to explore the possible excretion of filaria-specific soluble ICs in urine of subjects with lymphatic filariasis. Paired urine (overnight) and serum samples were analyzed for complement activating filarial antigen containing immune complexes by enzyme-linked immunosorbent assay (ELISA). Antigen-specific ICs were detected in urine samples of 34% of subjects with filarial disease manifestations while the frequency of occurrence was low in microfilaremic subjects. The titer of urine ICs is significantly high in subjects with chronic filariasis as compared to microfilaria (mf) carriers. The occurrence of filaria-specific ICs in urine and their passage through the filtering structures of the kidney is suggestive of the focal or diffuse damage in those subjects. Detection of ICs in urine may provide a noninvasive means of assessing the extent of renal damage in patients with lymphatic filariasis.

Detection of antigen and antibody in childhood tuberculous meningitis.

OBJECTIVE: Mycobacterium tuberculosis excretory secretory 31 kDa, a serine protease antigen (M. tb ES-31), prepared from Mycobacterium tuberculosis H37Ra 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.

Detection of enzymes dehydrogenases and proteases inBrugia malayi filarial parasites.

Lymphatic filariasis caused mainly by infection fromW. bancrofti andB. malayi remains a major cause of clinical morbidity in tropical and subtropical countries. Analysis ofB. malayi mf, infective larval and adult worm lysates for the activity of enzymes led to the demonstration of activities of three key enzymes of carbohydrate metabolism viz., Malate dehydrogenase (MDH), Malic enzyme (ME) and Glucose-6-phosphate dehydrogenase (G6PDH) in all the three stages of the parasite. The specific activity of all the three dehydrogenases was significantly high in mf lysate compared to their activity in lysates of the other two stages (P<0.001). Analysis by native polyacrylamide gel to their activity inlysates of the other two stages (P<0.001). Analysis by native polyacrylamide gel electrophoresis (PAGE) using 7.5% non-gradient gel showed the presence of two isoforms of each of the three enzymes (MDH, ME & G6PDH) in mf lysate, while only one form of each enzyme was present in L(3) larval and adult worm lysates. Further proteolytic enzyme activity was demonstrated both in microfilarial and infective larval lysates ofB. malayi. While both mf and L(3) larval lysates showed optimal protease activity at alkaline pH of 9.0, the mf lysate showed increased activity also at pH 3.0. The infective larval lysate was markedly inhibited by Tosylamide-L-Phenylalanine chloromethyl ketone (TPCK), a thiol protease inhibitor, while the protease activity in mf lysate was significantly inhibited by both TPCK and a serine protease inhibitor Phenyl Methyl Sulphonyl Flouride (PMSF). In sodium do-decyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), using gelatin copolymerized gel, the microfilarial lysate showed 3 protease molecules of 40 kDa, 180 kDa and 200 kDa and the L(3) larval lysate had 6 protease molecules of 18, 25, 37, 49, 70 and 200 kDa size.

Effectivity of crude versus purified mycobacterial secretory proteins as immunogen for optimum antibody production.

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 (31 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 1 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.

Detection of free and immune-complexed serine protease and its antibody in TB patients with and without HIV co-infection.

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. CONCLUSION: Detection of free and CIC tuberculous serine protease antigen along with antibody is more useful for detecting TB in the presence of HIV co-infection.