1

Jamnalal Bajaj Tropical Disease Research Centre and Department of Biochemistry, Mahatma Gandhi Institute of Medical Sciences, Sevagram, Wardha-442102, Maharashtra, India.

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.

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2

Indian J Chest Dis Allied Sci 2001; 43:81-90.

Mycobacterial Excretory-Secretory Antigenic Protein

with a Diagnostic Potential in Pulmonary Tuberculosis

Jamnalal Bajaj Tropical Disease Research Centre and Department of Biochemistry, Mahatma Gandhi Institute of  Medical Sciences, Sevagram, Wardha, Maharashtra

 ABSTRACT

Proteins secreted into the culture medium by Mycobacterium tuberculosis are shown to be a source of antigens of immunodiagnostic importance. In our earlier study, we had reported a 31-37 kDa seroreactive gel-eluted antigenic fraction (ESAS-7), isolated from culture filtrate proteins of Mycobacterium tuberculosis H₃₇Ra. In this report, we describe further purification of excretory-secretory ESAS-7 antigen fraction by fast protein liquid chromatography (FPLC) on Resource 'S' cation-exchange column and isolation of a more reactive and purified protein antigen fraction ESAS-7F. ESAS-7F antigen was characterized as a 31 kDa molecular weight glycoprotein containing a metallo-serine protease activity. N -terminal sequence analysis showed the first five amino acids as NTGQS (Asp-Thr-Gly-Glu-Ser). The present study helped in the isolation of a well characterized 31 kDa mycobacterial glycoprotein antigen with protease activity and diagnostic potential in detection of tuberculosis infection.

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3

Jamnalal Bajaj Tropical Disease Research Centre and Department of Biochemistry, Mahatma Gandhi Institute of Medical Sciences, Sevagram, Wardha-442102, Maharashtra, India

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.

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4

R. Alli, S. Kulkarni, M. V R. Reddy and B. C. Harinath

Department of Biochemistry & JB Tropical Disease Research Centre, MGIMS, Sevagram (Wardha)-442102, Maharashtra, India

ABSTRACT

A comparative analysis was made on the utility of SEVAFILACHEK-stick based immunoassays and commercially available ICT-filariasis test to detect active infection in different groups of bancroftian filariasis. The SEVAFILACHEK immunoassays were found to be useful to detect filarial infection in microfilaraemia and in a significant number of clinical filarial cases with acute, chronic and occult clinical manifestations. In the clinical cases, microfilariae are not usually detected in peripheral circulation. Employing SEVAFILACHEK assays 6 and 5 of the7 samples of patients with chronic filarial disease, and 6 and 5 of 6 microfilaraemic cases gave positivity for filarial IgG antibodies and antigen respectively. Four of the 6 occult filarial samples were positive for antibodies and antigen. Filarial antigen was detected by ICT-filariasis test in blood samples of all the 6 microfilariaemic cases, 1 chronic filarial and 2 occult filarial samples. The main advantage of ICT assay is its rapid format and convenience for field use.

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5

Indian J Patol Microbiol 2003,Vol 46,No.2

Abstract: Despite rapid advances in molecular genetics for detection of mycobacteria, it is clear that interest in serodiagnosis remains high, especially for those situations in which a specimen may not contain the infection agent in particular in extra pulmonary tuberculosis. Immune response to excretory-secretory (ES) proteins of Mycobacterium tuberculosis (Mtb) has been of diagnostic interest in tuberculosis. In earlier study from our laboratory, a secretory protein M.tb ES-31 has been shown to have diagnostic potential in pulmonary tuberculosis. Further, another M.tb H ₃₇ Ra ES protein (ES-41) was isolated and purified by trichloroacetic acid solubilization followed by Fast Performance Liquid Chromatography (FPLC). These two protein fractions viz., ES-37 and ES-41 secreted by M.tb H₃₇Ra bacilli were employed in stick indirect pencillinase ELISA to study seroreactivity in extra pulmonary tuberculosis namely tuberculous lymphadenopathy, tuberculous meningitis, abdominal tuberculosis and bone & joint tuberculosis. While using ES-31 antigen 88% (22/25) of tuberculous lymphadenopathy and 90% (9/10) of tuberculous meningitis cases showed positive reaction for tuberculous IgG antibody, ES-41 showed 80% positivity in both groups. In abdominal and bone & joint tuberculosis cases, ES-41 antigen showed better sensitivity of 8l.5% (22/ 27) and 84.6% (22/26) respectively in IgG antibody detection compared to 70% (19/27) and 69.2% (13/ 26) shown by ES-31. This study is of interest that different antigen protein fractions of Mtb exhibit differential seroreactivity, as ES-31 protein showed good potential in detecting tuberculous IgG antibodies in tuberculous lymphadenopathy (TBLN) & tuberculous meningitis (TBM), while ES-41 in abdominal and bone & joint tuberculosis cases.

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6

J Comm Dis. 2001;33(2): 63-71.

(Received for publication: May 2000)

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.

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7

TRANSACTIONS OF THE ROYAL SOCIETY OF TROPICAL MEDICINE AND HYGIENE (2001) 95,168-169

B. Balaji Ganesh', A. M. Kader, G. S. Agarwal', M. V. R. Reddy' and B. C. Harinath' 'Department of Biochemistry and JBTDRC, Mahatma Gandhi Institute of Medical Sciences, Sevagram, Institute of Vector Control and Zoonoses, Hosur and ³ Defence Research Development Establishment, Gwalior, India

Early and accurate diagnosis of filarial infection is essential for successful implementation of a filariasis control programme. The diagnosis of lymphatic filariasis has conventionally relied on microscopy detection of microfilariae (mf) in peripheral blood, which is an unreliable method lacking sensitivity in the detection of low microfilaraemia (DREYER et al., 1996). A number of immunoassays using homologous and heterologous antigens for the detection of lymphatic filariasis have been reported (WEIL et al., 1987; RAMAPRASAD et al., 1988; Chenthamarakshan et al., 1996) but are either too cumbersome or costly for routine screening of endemic populations in developing countries. Hence, there is a need for a simple and reliable diagnostic assay for lymphatic filariasis in the field and in laboratories in an endemic area.

We have assessed a simple, dot-blot ELISA using a purified 66-kDa Brugia malayi mf soluble protein antigen (Bm mf S-2) for detection of circulating filarial antibodies in bancroftian filariasis and compared it with an assay reported earlier in this laboratory, i.e., stick-penicillinase ELISA using B. malayi microfilarial excretory-secretory (Bm mf ES) antigens (REDDY et al., 1996).

Sera were collected from 30 microfilaraemic cases and 25 healthy individuals (endemic normals) with no mf in night blood and no clinical history of filariasis, from Sevagram and surrounding villages in Maharashtra, India, which are endemic for the nocturnally periodic Wuchereria bancrofti. Bm mf S-2 was prepared by purification of Bm mf soluble (Bm mf S) antigens by gel filtration. About 1 million mf obtained by peritoneal lavage of infected jirds were homogenized, sonicated and extracted overnight in 0-05 M phosphate-buffered saline (PBS) as described by KALIRAJ et al. (1982). The extract was centrifuged, concentrated by ultra-membrane filtration and further fractionated by fast-performance liquid chromatography on Superdex 75 HR 10/30 gel filtration column (Pharmacia Biotech, Sweden) as per the manufacturer's instruction. A 66-kDa protein that was isolated from the second individual peak was coated on nitrocellulose membrane pads adhered to plastic combs at a concentration of 20 ng per pad and blocked with 5% skimmed milk powder in 0-01 M PBS overnight. The combs were then washed in 0-01 M PBS with 0^.5%

Tween-20 (PBS-T), dried and incubated with optimally diluted sera (1:200) in PBS-T at 37°C for 1 h. After 3 washes the combs were incubated with anti-human IgG horse radish peroxidase conjugate at 37^°C for 1 h, washed 4 times and incubated with diaminobenzidine substrate for a few seconds. The enzyme substrate reaction was terminated by washing under tap water. Formation of a clear dot indicated a positive reaction. The same panel of sera was also analysed for circulating filarial IgG antibodies against Bm mf ES antigen by stick ELISA using anti-human IgG-penicillinase conjugate and starch-iodine-penicillin substrate as described earlier (REDDY et al., 1996).

Twenty seven (90-0%) of the 30 microfilaraemic sera screened were positive by dot-blot assay while only 22 (73-3%) were detected by stick ELISA. Five of the 25 (20%) endemic-normal samples showed positive dots as compared to only 3 (12%) of these positive by stick ELISA. The Figure shows the results of the dot-blot assay, screening 6 microfilaraemic and 6 endemic-normal sera. The dot-blot assay thus showed a sensitivity of 90% and specificity of 80%. The fact that stick ELISA using ES antigens showed a sensitivity of only 73% indicates that Bm mf S-2 antigen may be more useful in detection of microfilaraemic cases, although Bm mf ES antigen showed greater specificity.

There is urgent need for simple and cost-effective diagnostic tests for screening large populations. Rapid diagnostic tests are becoming popular and convenient for mass screening in disease control programmes. However, the imported rapid immunochromatographic test (ICT Filariasis) for filariasis is not easily available and affordable for routine screening in National Control Programmes. Dot-blot assay described here is inexpensive, costing <5 rupees (7.5p; £1 = 67 rupees) for materials per test, and is simple to perform in field laboratories for detection of mf carriers in endemic populations.           

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8

Indian Journal of Clinical Biochemistry, 2002, 17 (1)5-8

Mahatma Gandhi Institute of Medical Sciences

Sevagram, Wardha-442 102, Maharashtra, INDIA

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.

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9

Proc. of International Symposium on

Recent Advances in Molecular Biology, Allergy and lmmunology 

3-5th Sept., 2000,Baroda,India

Isolation of M.tb. 31 kDa Antigen Protein of Diagnostic Interest from Culture Filtrate using Affinity Purified ES-31 Antibody by Affinity Chromatography

Nair ER, Banerjee S, Kumar S, Reddy MVR and Harinath BC*

Jamnalal Bajaj Tropical Disease Research Centre and Dept. of Biochemistry, Mahatma Gandhi Institute of Medical Sciences, Sevagram, Wardha-442 102, Maharashtra, INDIA

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.

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10

Proc. of International Symposium on 

Recent Advances in Molecular Biology, Allergy and lmmunology 

3-5th Sept., 2000,Baroda,India

Biochemical Characterization and Seroreactivity of in vitro and in vivo released 31 kDa antigen Protein of Diagnostic Interest in Tuberculosis

JB Tropical Disease Research Centre and Department of Biochemistry, Mahatma Gandhi Institute of Medical Sciences, Sevagram, Wardha -442 102, Maharashtra, INDIA

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.

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11

Biomedical Research 2002; 13 (1); 39-42

Diagnostic evaluation of circulating filarial antigen assay using

B. Bhunia, S. Kulkarni, M. V. R Reddy and B. C. Harinath

Abstract

A total of 125 sera samples of different groups of bancroftian filariasis and endemic normals were treated with glycine -HCI buffer (0.1 M, pH 2.8) and heat (65^°C for 15 min). Both untreated and acid -heat treated sera samples were analysed for filarial antigen by an inhibition ELISA developed using Brugia malayi microfilarial excretory-secretory (Bm Mf ES) antigen-penicillinase and IgG antibodies of chronic filarial patients. The geometrical mean titre (GMT) of circulating filarial antigen increased significantly (p<0.05) in acute (n=25) and occult (n = 25) clinical filarial sera followed by acid-heat treatment. In contrast the GMT of filarial antigen decreased significantly (p <0.05) in the acid-heat treated microfilaraemic sera (n =25). The change in the GMT of filarial antigen in chronic clinical filarial cases (n =25) and endemic normals (n=25 ) was not significant. The filarial antigen positivity increased from 84% to 88% in acute filarial cases, 72 % to 80 % in chronic filarial cases, 48% to 78 % in occult filarial group where acid-heat treated sera were used. The antigen positivity decreased from 80% to 68% in microfilaraemic group when acid-heat treated sera were used and the positivity remained same in endemic normal group. The findings of the present study suggest for analysis of sera samples with and without acid-heat treatment to detect clinical filarial cases with increased sensitivity and at the same time ensuring that maximum number of microfilaraemics are detected.

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12

and extrapulmonary tuberculosis .

S Banerjee, S Gupta, N Shende, S Kumar, B C Harinath

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.

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13

Indian Journal of Clinical Biochemistry, 2003,18 (1) 61-64

MICROSCOPIC HAEMATURIA AS AN OCCULT FILARIAL INFECTION IN BHUBANESHWAR AN ENDEMIC AREA FOR BANCROFTIAN FILARIASIS .

R. Alli*, B. Bhunia*, G. P Choudhry**, M.V R. Reddy* and B. C. Harinath*

* Dept.of Biochemistry & J. B. Tropical Disease Research Centre, MGIMS, Sevagram (Wardha) 442 102, India.

** Dept. of Pathology, Regional Medical Research Centre (Indian Council of Medical Research), Chandrasekhapur, Bhubaneshwar751016, India.

RUNNING TITLE : Microscopic haematuria- an occult filarial infection

Sera samples of 7 microscopic haematuria cases collected before and after treatment with Diethylcarbamazine citrate, (DEC) , 9 microfilaraemic cases and 19 endemic normal individuals were analysed for filarial antigen and IgG antibody levels. Filarial antigen was detected in 5 of the 7 microscopic haematuria cases, of which 3 turned negative for antigen after treatment with DEC. While none of the 7 haematuria cases were positive for filarial IgG antibodies, before the DEC treatment, all of them turned positive after DEC treatment. The sensitivity and specificity values(to detect mf +ve cases ) were 89% and 90% respectively for the detection of filarial antigen and 78% and 95% respectively for the detection of filarial IgG antibodies.

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14

INT J TUBERC LUNG DIS 7(3):278-283

Isolation and characterisation of in vivo released 41 kDa mycobacterial antigen in pulmonary and bone and joint tuberculosis and its identification with H₃₇Ra in vitro released antigen

S. Banerjee, S. Kumar, B. C. Harinath

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.

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Analysis of IgG subclasses and IgE antibodies across the clinical

Abstract: Analysis of immune response in individuals with different clinical manifestations living in filaria endemic area will be of interest to understand the immunological events associated with the disease development in filarial infected endemic population. The levels of four IgG sub classes and IgE antibodies against Brugia malayi microfilarial excretory-secretory (Bm mf ES) antigen as well as circulating filarial antigen level were evaluated in 84 individuals belonging to different groups in an endemic area for bancroftian filariasis. Microfilaraemics showed significantly elevated levels of IgG₄ and IgG₃ antibodies compared to endemic normals (P<0.02). As many as 70% of this group were positive for IgG₄ & IgG₃ antibodies. While Acute filarial cases had pronounced IgG₁ antibodies (P<0.001), the Grade I chronic cases showed higher levels of IgG₃ and IgG₁ antibodies (P<0.02), Occult filarial cases had higher levels of IgG₄ and IgG₃ (P<0.02) and also of IgG₁ antibodies (P<0.001). IgE antibodies were found to be elevated in microfilaraemics as well as other clinical filarial groups. Circulating filarial antigen was detected in 95% of microfilaraemics, 60% of acute cases, 75% to 90% of different grades of chronic filarial cases, 100% of occult cases and none of the endemic normals.

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16

Indian Journal of Clinical Biochemistry, 2003, 18 (2) 48-53

Swati Banerjee, Sonika Gupta, Niraj Shende, Satish Kumar and Bhaskar C. Harinath Jamnalal Bajaj Tropical Disease Research Centre and Department of Biochemistry, Mahatma Gandhi Institute of Medical Sciences, SEVAGRAM, WARDHA-442102, MAHARASHTRA, INDIA

Running title: Serodiagnosis of tuberculosis

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.

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17

Indian Journal of Clinical Biochemistry, 2003, 18 (2) 1-5

Running Title: Immunodiagnosis of tuberculosis

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.

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18

Soma Gupta, M.V R. Reddy and B. C. Harinath

Jamnalal Bajaj Tropical Disease Research Centre & Department of Biochemistry, Mahatma Gandhi Institute of Medical Sciences, Sevagram, Wardha.

ABSTRACT

Lipid peroxidation product, malonaldehyde (MDA) and antioxidants were estimated in plasma and erythrocytes of 34 cases of oral submucous fibrosis (OSMF) of different grades with equal number of healthy controls to evaluate the association of reactive oxygen species (ROS) and OSMF while plasma MDA was found to be significantly higher in patients (3.3.+0.4 nmole/ml, P < 0.001) as compared to controls (2.4+0.5 nmole/ml), plasma beta carotene and vitamin E levels were found to be decreased significantly in patients (81.7 ± 14.3 μg/100 ml, P<0.001; 9.3+ 0.9 mg/L, P<0.01 respectively) with respect to healthy controls (110 + 20.8 μg/100m1 and 10.1+ 1.2 mg/L). The decrease in beta-carotene and vitamin E was found to be more significant in OSMF grade II and III than in grade I. After 6 weeks of oral administration of beta-carotene and vitamin E, patients showed increase in plasma level of these two antioxidants along with decrease in MDA level associated with clinical improvement.

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19

Indian Journal of Clinical Biochemistry, 2004, 19 (1) 83-87

A.K.Pradhan*, A.K.Shukla**, M.V.R. Reddy* and N. Garg*

ABSTRACT

Oxidative stress was assessed by estimating lipid peroxidation product (LPO) in the form of thiobarbituric acid reactive substances (TBARS), enzymatic antioxidants in the form of superoxide dismutase (SOD), catalase and nonenzymatic antioxidant vitamins e.g. vitamin C,β carotene and vitamin E in either serum or plasma or erythrocytes in 190 cases of age related cataract in the age group of 50-80 years. 190 cases were grouped into three morphological types namely, 73 cases of cortical, 77 cases of posterior sub capsular and 40 cases of nuclear cataract and values of LPO and antioxidants were compared with 78 cases of age matched healthy control groups. Plasma TBARS levels were significantly high but serum SOD, plasma vit C and β carotene levels were significantly low in cataract cases when compared with control groups. There were no significant differences in the erythrocyte levels of catalase and plasma levels of Vit E between cataract cases and control groups. No significant changes of parameters were seen among three different morphological types of age related cataract. The present study shows that the oxidative stress may play an important role in the age related cataract.

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20

Biomedical Research 2004; 15 (1): 76-79

Jamnalal Bajaj Tropical Disease Research Centre and Department of Biochemistry, Mahatma Gandhi Institute of Medical Sciences, Sevagram, Wardha 442102, Maharashtra, India .

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.

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21

Biomedical Research, 2004;15(1):76-79.

Analysis of seroreactivity of different endemic populations of bancroftian filariasis with field mf isolates and Brugia malayi antigens.

Yashodhar.P. Bhandary, K.N.Krithika,SandeepKulkarni,M.V.R.Reddy and B.C.Harinath.

J.B.Tropical Disease Research Centre, Jamnalal Bajaj Tropical Disease Research centre and Department of Biochemistry, Mahatma Gandhi institute of Medical Sciences, Sevagram

 Wardha-442102, Maharashtra, India.

Abstract

Seroreactivity of bancroftian filarial cases of different endemic zones with different geographical isolates of W.bancrofti  mf was checked by indirect fluorescent antibody test (IFAT). While the filarial sera of Calicut showed binding to the mf isolates of all four endemic zones studied (Calicut, Bhubaneshwar, Rourkela and Wardha), the sera of Bhubaneshwar and Rourkela were not reactive with mf of any of the four regions. The sera of microfilaraemics of Wardha did not bind to the mf from Rourkela region. Further analysis of antibody levels  against  B.malayi ES antigen in human sera from different endemic zones showed high GMT of filarial IgG antibody in microfilaraemic  sera  of Wardha region compared to Bhubaneshwar (P<0.05). The microfilaraemic and as well clinical filarial sera of Wardha also showed significantly high GMT of filarial IgG antibodies against B.malayi mfS antigen compared to the antibody levels in the respective categories of sera from Calicut region (P<0.05). These differences in the seroreactivity of filarial groups in different endemic zones to the mf isolates of different geographical regions and difference in filarial antibody responses in different host populations may reflect parasite specific variations in immunogenecity.

 22

J. MGIMS,2004 Sept.;9(ii): 16-21

MYCOBACTERIUM TUBERCULOSIS EXCRETORY SECRETORY (ES) PROTEIN ANTIGENS OF INTEREST IN DIAGNOSIS, PROGNOSIS AND PREDICTION OF DISEASE DEVELOPMENT

SONIKA GUPTA, SATISH KUMAR, and B. C. HARINATH  

  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.

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23

Journal of Parasitic Diseases June 2004; 28(1):29-36.

 A monoclonal antibody Bm Ab120 to 120  kDa B malayi antigen with diagnostic potential in bancroftian filariasis.

Balaji Ganesh B, Parab PB*, Katdare M*, Reddy MVR, and Harinath BC

J.B. Tropical disease Research Centre, Jamnalal Bajaj Tropical Disease Research centre and Department of Biochemistry, Mahatma Gandhi institute of Medical Sciences,

Wardha-442102, Maharashtra, India.  

* National Centre Cell Sciences, Pune University campus, Pune- 411 007

 A monoclonal antibody (Bm Ab 120) of IgM isotype against B.malayi microfilarial soluble antigen [Bmmf (S)] that specifically recognized a 120 kDa antigen present in microfilarial soluble extracts, microfilarial excretory- secretory proteins and adult soluble proteins but not in soluble infective larval extracts was produced. Bm Ab 120 was employed in a sandwich ELISA to screen individuals in areas endemic for bancroftian filariasis as well as those suffering from other parasitic infections from non-endemic area using two different sources of polyclonal antibodies viz, Filarial serum immunoglobulins (FSIgG) isolated from clinical patients and anti-Bmmf (S) antibodies raised in rabbits as captured antibodies. FSIgG- Bm Ab 120 combination showed 84% mf carriers, 66% of patients with clinical symptoms of filariasis and 18% of endemic normals as positive for filarial antigens. When anti-Bmmf (S) antibodies were used as capture antibodies along with Bm Ab 120 antibodies as tracer probe, 88% of mf carriers were detected as also 54% of clinical cases and 12% endemic normals. A small percentage of patients with Tropical Pulmonary Eosinophilia were also picked up by both the assays. None of the sera of other parasitic infections such as ascaris, hookworm, leishmania and malaria showed levels above the cut-off suggesting high specificity of the monoclonal antibody. Thus, Bm Ab 120 was found to be useful in identifying significant numbers of asymptomatic microfilaraemics, along with patients suffering form clinical and occult filariasis in bancroftian endemic areas.

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24

Indian J of Pathology and Microbiology July 2004; 47(3): 438-440.

Levels of antibody, free antigen and immune complexed antigen in different grades of sputum positive pulmonary tuberculosis patients by ELISA

Niraj Shende, Sonika Gupta , Satish Kumar and B.C. Harinath

JB Tropical Disease Research Centre,Jamnalal Bajaj Tropical Disease Research Centre,Department of Biochemistry ,Mahatma Gandhi Institute of Medical Sciences,Sevagram – 442102, Wardha, Maharashtra, India

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.

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25

Biomedical Research, 2005; 16(1): 23-27 

Isolation of excretory secretory protein 6 kDa antigen (ES-6) and its seroreactivity in patients with different stages of pulmonary tuberculosis and healthy household contacts. 

Gupta S, Shende N, Kumar S, Harinath BC.

JB Tropical Disease Research Centre,Jamnalal Bajaj Tropical Disease Research Centre,

Department of Biochemistry ,Mahatma Gandhi Institute of Medical Sciences,Sevagram – 442102, Wardha, Maharashtra, India  

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.

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26

Biomedical Research, 2005; 16(1): 19-22.

Isolation and characterization of a major form of superoxide dismutase from human lymphatic filarial parasite, brugia malayi.

Dabir S, Dabir P, Reddy MVR. 

JB Tropical Disease Research Centre,Jamnalal Bajaj Tropical Disease Research Centre,

Department of Biochemistry ,Mahatma Gandhi Institute of Medical Sciences,

Sevagram – 442102, Wardha, Maharashtra, India

Superoxide dismutase (SOD) is an enzyme that converts superoxide radicals into hydrogen peroxide and molecular oxygen and provides first line of defense against oxygen toxicity. We have isolated and characterized the extracellular form of superoxide dismutase from lymphatic filarial nematode parasite Brugia malayi. The enzyme activity was higher in adult worms (30.29 U/mg) than in microfilariae (23.19 U/mg). The parasite extracts were also analyzed by native polyacrylamide gel electrophoresis on 12% gel fallowed by staining for enzyme SOD activity using nitroblue tetrazolium (NBT), N, N, N, N tetra methyl ethylene diamine (TEMED) and riboflavin. A very broad band of enzyme activity was observed in both mf and adult worms extracts, with the latter showing more intense band. Fractionation of adult worm extracts by Fast Performance Liquid Chromatography (FPLC) using superdex 75 HR 10/30 column also showed polydispersed nature of SOD activity with 10 of the 15 fractions having varying levels of enzyme activity. The 9th fraction with highest SOD activity was found to be a 29 kDa molecule having cross reactivity with SOD of B. pahangi. Further it was characterized as CuZn SOD based on significant inhibition of its activity by potassium cyanide.

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27

1.                      Indian J Pediat 2005 May, 72, 383-87. 

Serodiagnosis of childhood tuberculosis by ELISA. 

Bhatia AS, Gupta S, Shende N, Kumar S.Harinath BC.

JB Tropical Disease Research Centre & Dept. of Biochemistry, MGIMS, 

Sevagram - 442102, Wardha, MS. India.

Objective:  Diagnosis of childhood tuberculosis remains an enigma despite the 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. 

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28

Current Science, 2005 June 10;88(11):1825-1827.

      Detection of antibodies to a cocktail of mycobacterial excretory–secretory 

antigens in tuberculosis by ELISA and immunoblotting.

Gupta S, Shende N, Kumar S, Harinath BC.

JB Tropical Disease Research Centre & Dept. of Biochemistry, MGIMS, 

Sevagram - 442102, Wardha, MS. India.

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.

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29

Current Science, 2005 June 25;88(12):1962-1966

Cloning and characterization of a small heat shock protein cDNA clone of Wuchereria bancrofti.

Dabir P, Dabir S, Krithika KN, Reddy MVR.

Department of Biochemistry & J.B. Tropical Disease Research Centre, 

MGIMS, Sevagram-442102, Wardha, MS, India

A 426 bp cDNA encoding a predicted small heat shock protein (smHSP) was isolated from Wuchereria bancrofti lambda zap L₃ cDNA expression library by immunoscreening with microfilaremic sera. The open reading frame of the cDNA clone encodes a predicted protein of 142 amino acids (aa), which had high sequence identity with other nematode smHSPs. The homologous regions conserved in several different nematodes species reflect its importance in parasites that require mammalian host as a part of their development. SmHSPs and alpha-crystallin constitute a family of related molecular chaperons that exhibit striking variability in size ranging from 10 to 43 kDa. Here we describe the cloning and characterization of this cDNA clone encoding predicted 15.5 kDa smHSP of W. bancrofti.

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30

Biomedical Research 2005;16(2):101 – 106.

Isolation and purification of circulating filarial antigens from microfilarial carriers.

Kulkarni S, Krithika KN, Reddy MVR.

Department of Biochemistry & J.B. Tropical Disease Research Centre, 

MGIMS, Sevagram-442102, Wardha, MS, India

Lymphatic filariasis caused by infection from Wuchereria bancrofti and Brugia malayi is characterized by wide spectrum of clinical manifestations that include microfilaraemic carriers with sub clinical infection and clinical cases with acute, chronic and occult presentations. The circulating filarial antigen (CFA) considered to be an indicator of active infection, was detected in most of the microfilaraemics, acute and occult filarial cases.  Circulating filarial antigen fraction was isolated from plasma of microfilaraemic (Mf) cases with W bancrofti infection by 36-75% ammonium sulphate precipitation followed by Ultrogel ACA-34 gel filtration chromatography. Further fractionation of CFA₂ by Fast Protein Liquid Chromatography (FPLC) using resource ‘Q’ anion exchanger column yielded eight protein fractions of which two fractions (CFA₂-A & E) were positive for filarial antigen. By SDS PAGE analysis the unbound protein fraction CFA₂-A was found to be a 69 kDa protein while CFA2-E fractions showed eighteen protein bands. Analysis of filarial IgG antibody levels against both CFA₂-A and CFA₂-E showed significantly higher levels of mean antibody levels in microfilaraemics, chronic and occult filarial sera compared to the levels in sera of endemic normals (EN) or non endemic normals (NEN)(P < 0.001)  While 9 of 10 (90%) microfilaraemics, 1 of  10 (10%) acute cases, 16 of 30 (53%)  chronic filarial cases, 7 of 10 occult cases (70%) and none of the endemic (10) & non endemic normals (10) were positive for filarial antibodies against CFA₂-A, 90% of microfilaraemics, 20% of acute cases, 80% of chronic,  100% of occult cases and none of the endemic and non endemic normals had detectable filarial antibodies against CFA₂-E.

In immunoblot study CFA₂-A, was recognized by microfilaraemics, chronic and as well occult filarial sera. While three protein molecules of CFA₂-E with 29 kDa, 68 kDa and 70 kDa were identified by microfilaraemics and acute sera and low molecular weight protein (18 kDa) of CFA₂-E was identified by chronic filarial sera.  The present study helped to identify the different filarial antigens present in microfilaraemic stage of infection.

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31

Int J Tuberc Lung Dis. 2005 Aug;9(8):915-9.

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

Shende N, Gupta S, Bhatia AS, Kumar S, Harinath BC.

 JB Tropical Disease Research Centre & Department of Biochemistry 

MGIMS, Sevagram-442102, Wardha, MS, India

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 far detecting TB in the presence of HIV co-infection.

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32

Indian Journal of Experimental Biology 2005; 43: 759-768.

Identification of 38kDa Brugia malayi microfilarial protease as a vaccine candidate for lymphatic filariasis.

Krithika KN, Dabir P, Kulkarni S, Anandharaman V, Reddy MVR

Department of Biochemistry & J.B. Tropical Disease Research Centre, 

MGIMS, Sevagram-442102, Wardha, MS, India

A FPLC purified 38kDa protease (Bm mf S-7) isolated from B. malayi microfilarial soluble antigen was identified. It showed pronounced reactivity with sera collected from 'putatively immune’ asymptomatic and amicrofilaraemic individuals residing in an endemic area for bancroftian filariasis. Further the immune protective activity of Bm mf S-7 antigen was evaluated in susceptible hosts, jirds (Meriones unguiculatus) against B. malayi filarial infection. The antigen showed 89% cytotoxicity against mf and 87-89% against infective (L₃) larvae in in vitro antibody dependent cellular cytotoxicity Assay (ADCC) and in situ micropore chamber methods. Bm mf S-7 immunized jirds after challenge infection showed 81.5% reduction in the adult worm burden. The present study has shown that, the 38kDa microfilarial protease (Bm mf    S-7) could stimulate a strong protective immune response against microfilariae and infective larvae in jird model to block the transmission of filariasis.  Analysis of IgG subclasses against Bm mf S-7 revealed a significant increase in IgG2 and IgG3 antibodies in endemic normals. Lymphocyte proliferation to Bm mf S-7 was significantly high in endemic normal group as compared to that in clinical and microfilarial carriers. Significantly enhanced levels of IFN-γ in the culture supernatant of PBMC of endemic normals followed by stimulation with Bm mf S-7 suggest that the cellular response in this group is skewed towards Th 1 type.

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33  Immunology and Cell Biology, 2005;83:520-524.  

Cloning and expression of a 12 kDa serospecific epitope of Wuchereria bancrofti.

Dabir P, Dabir S, Reddy MVR. 

Summary: The immunoscreening of a microfilarial cDNA library of Wuchereria bancrofti with microfilaraemic sera revealed many positive clones expressing filarial antigens. One immunoreactive clone, designated PMR1, was shown to encode a protein of 114 amino acid residues. The cDNA fragment was subcloned into an expression vector, Pinpoint XaT. The resulting recombinant (r)PMR1-biotin fusion protein was expressed in Escherichia coli (BL21 [DE3] pLys) and was affinity purified on avidin resin. Analysis of sera of different groups for filarial antibodies against rPMRI showed it to be highly reactive with microfilaraemic and clinical filarial sera compared to its reactivity with endemic and nonendemic controls. This indicates that the gene sequence of cDNA is expressing an immunodominant epitope, which could be useful in serodiagnosis of lymphatic filariasis.

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34  Microbiol Immunol, 2005;49(10):909-914.

Analysis of polymorphism of 18S rRNA gene in Wuchereria bancrofti microfilariae.

Bhandari Y, Dabir P, Krithika KN, Kannayakanahalli MD, Shouche YS, Reddy MVR.

Abstract: The polymorphism of the 18S rRNA gene in Wuchereria bancrofti microfilariae (mf) collected from three different zones in India was analyzed by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP). The RFLPs of the amplified products obtained after digestion with restriction enzymes SspI, MspI and HhaI showed no difference in the banding patterns among the mf isolates from different endemic zones. Further the sequencing of PCR products did not show any differ­ence in the nucleotide sequence either. The phylogenetic analysis of the sequences of W. bancrofti mf iso­lates from different endemic zones has shown branching with the earlier reported sequences of W. bancrofti and its close relative Brugia malayi.

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35  Med Sci Monit, 2005;11(2): CR585-588.

IgG subclass antibody response to mycobacterial serine protease at different stages of pulmonary tuberculosis.

Gupta S, Shende N, Bhatia AS, Kumar S, Harinath BC.

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.

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36   Indian J of Experimental Biology, December 2005;43:1196-1198. 

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

Santa Saha-Roy, Niraj Shende, Satish Kumar and BC Harinath.

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.

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37  Indian Journal of clinical Biochemistry, 2006; 21 (1): 36-42.  

Detection of dehydrogenases and proteases in Brugia malayi parasites. 

Abstract: Lymphatic filariasis caused mainly by infection from W. bancrofti and B. malayi remains a major cause of clinical morbidity in tropical and subtropical countries. Analysis of B.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 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 L3 larval and adult worm lysates. Further proteolytic enzyme activity was demonstrated both in microfilarial and infective larval lysates of B.malayi. while both mf and L3 larval lysates showed optimal protease activity at alkaline pH 9.0 . the mf lysate showed increased activity also at pH 3.0. The infective larval lysate was markedly inhibited by Tosylamide –L-Phenylalanine chromethyl 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 L3 larval lysate had 6 protease molecules of 18,25, 37, 49, 70 and 200 kDa size.  

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38  Genetics and Evolution 2005. (PMID: 16199210).  

44. The National Medical Journal of India. 2007; 20(1):11-13.

Antioxidant vitamin levels in sickle cell disorders.

Debes Ray, Pradeep Deshmukh, Kalyan Goswammi, Neelam Garg.

ABSTRACT

Background. Sickle cell disorder is a haemoglobinopathy prevalent in the Vidharbha region of Maharashtra, central India. With recent evidence of oxidative stress in sickle haemoglobinopathy, a possible deficiency of antioxidant vitamins was suspected.

Methods. We measured plasma vitamin E, vitamin C and beta-carotene levels in persons with heterozygous ( n=80) and homozygous sickle cell state ( n=20), and suitable healthy controls for these groups ( n=100 and 66, respectively) in a community-based study in the villages near our institution.

Results. Subjects with heterozygous sickle cell trait had lower vitamin E levels than their respective controls (p<0.05). Subjects with homozygous sickle cell disease had lower levels of all three vitamins (p<0.05). Vitamins E and C levels showed a significant positive correlation in both forms of sickle cell disorder.

Conclusion. Our findings suggest that there is depletion of the antioxidant vitamins, particularly in severe forms of sickle cell disorder. A trial of administration of therapeutic doses of  vitamin E in this condition is warranted.

45. Biomedical Research 2007; 18(3):214-219.

Detection of antibody and antigen in extrapulmonary tuberculosis patients sera using a cocktail of mycobacterial excretory secretory antigens and their antibodies.

Vijay Upadhye, Niraj Shende, Satish Kumar, B.C. Harinath

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.

46.Indian Journal of Tuberculosis. Indian J Tuberc 2007; 54:125-129.

Isolation and analysis of circulating tuberculous antigens in mycobacterium tuberculosis.

Shende N, Gupta S, Upadhye V, Kumar S and Harinath BC

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.

47. Indian Journal of Experimental Biology. Vol. 45, July 2007, pp. 599-602.

Isolation of Mycobacterium tuberculosis protein antigens ES-31, ES-43 and EST-6 of diagnostic interest from Tubercle Bacilli by affinity chromatography.

Vijay Upadhye, Santa Saha-Roy, Niraj Shende, Satish Kumar & B. C. Harinath.

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.

48. Experimental Parasitology 116(4), August 2007, 483-491.

Comparison of protective immune responses induced by Bm-alt-2 DNA, recombinant Bm-ALT-2 protein and prime-boost vaccine regimens in a jird model.

Thirugnanam S, Pandiaraja P, Ramaswamy K, Murugan V, Gnanasekar M, Nandakumar K, Reddy MVR and Kaliraj P. Brugia malayi:

ABSTRACT

Immunization of jirds with Bm-alt-2 elicited partial protection against challenge infection with the filarial parasite Brugia malayi. In this study, we initially compared the protective immune responses elicited following immunization with recombinant Bm-ALT-2 protein regimen and Bm-alt-2 DNA regimen. These studies showed that protein vaccination conferred approximately 75% protection compared to DNA vaccination that conferred only 57% protection. Analysis of the protective immune responses showed that the protein immunization promoted a Th2-biased response with an increase in IL-4, IL-5 and IgG1 responses, whereas, the DNA vaccine promoted a Th1-biased response with profound IFN-gamma and IgG2a responses. Since protein vaccination gave better results than DNA vaccination, we then wanted to evaluate whether a prime-boost vaccination that combined DNA prime and protein boost will significantly increase the protective responses induced by the protein vaccine. Our results suggest that prime-boost vaccination had no added advantage and was comparatively less effective (64% protection) than the Bm-ALT-2 protein alone vaccination. Prime boost vaccination generated mixed Th1/Th2 responses with a slightly diminished Th2 responses compared to protein vaccination. Thus, our results suggest that Bm-ALT-2 protein vaccination regimen may be slightly better than prime-boost vaccine regimen and the mechanism of protection appears to be largely mediated by a Th2-biased response.