ELISA protocol for rapid screening of potential antitubercular 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 5μg/well of ES-31 antigen and at 1:25 dilution of anti-DSS-goat-serum. Isoniazid showed 42% inhibition of ES-31 serine protease at 0.4μg/well, while orlistat showed inhibition of 60% at 0.5μg/well. Inhibition of Mtb H37Ra bacilli is further confirmed in axenic culture. 35% and 29% inhibition by isoniazid at 0.4μg/well and orlistat at 0.5μg/well were observed respectively on bacterial growth. Conclusion: Simple ELISA protocol based on assay of antigenic reactivity of mycobacterial ES-31 serine protease, a drug target, has been standardized for rapid screening of potential anti-tubercular drugs.

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.

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.

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 (1×107 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 1×104 bacilli/ml while auramine-O method showed upto 1×102 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 (1×103 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-31antigen with sensitivity of 74% and 70% respectively. The assay for ICcocktail 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 μg/ ml and levels of free and IC cocktail antigens were 1.70 ± 1.04 and 1.13 ± 0.047 μg/ ml in AFB positive patients’ sera. Conclusions: Peroxidase enzyme immunoassay for circulating antigen was found to be a useful serodiagnostic assay and in particular in AFB –ve cases responding to ATT.

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.

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.

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.

Serodiagnosis of childhood tuberculosis by ELISA.

OBJECTIVE: Diagnosis of childhood tuberculosis remains an enigma despite many recent technological developments. The present study has been taken up with the aim to assess the diagnostic potential of mycobacterium tuberculosis excretory-secretory ES-31 antigen and affinity purified anti ES-31 antibodies in the serodiagnosis of different spectrum of childhood tuberculosis. METHODS: Mycobacterium tuberculosis H37Ra excretory-secretory antigen (ES-31) and affinity purified goat anti ES-31 antibodies were used in stick penicillinase ELISA for IgG antibody detection and stick Sandwich penicillinase ELISA for detection of circulating free and immune complexed antigen in the sera of 230 children. RESULTS: Analysis of tubercular antibody, circulating free and immune complexed antigen (CIC-Ag) was done in both pulmonary and extrapulmonary form of childhood tuberculosis and overall sensitivity of 81.4% with a specificity of 93% was achieved for detection of antitubercular IgG antibodies. Of the five cases of pulmonary tuberculosis showing absence of IgG antibody, 3 showed the presence of CIC-Ag and one was found positive for both free and CIC-Ag. Similarly out of 8 cases of extrapulmonary childhood tuberculosis missed by IgG detection 5 were found to be positive for CIC-Ag and 1 showed the positive reaction for both free and immune complexed antigens. CONCLUSION: IgG antibody to excretory-secretory antigen ES-31 is found to be having good specificity with acceptable sensitivity in detecting different forms of childhood tuberculosis. Further detection of circulating free and/or immunecomplexed antigen can be used as an adjunct tool in the diagnosis of childhood tuberculosis.

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

The ES-31 (31 kDa protein) antigen was isolated from culture filtrate of Mycobacterium tuberculosis H37Ra and was shown to have potential in immunodiagnosis of pulmonary tuberculosis. Serum samples from 38 confirmed sputum positive pulmonary tuberculosis patients were grouped into AFB+, AFB++, AFB+++ based on 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 tubercular antibody was considerably decreased compared to elevated levels of CIC-Ag (Antibody: 1360 to 816 and CIC-Ag: 534 to 1744) from low bacillary sample to high bacillary samples, whereas there is no significant change in the titre of circulating free antigen. Low levels of detectable antibody is observed possibly due to removal of antibody from circulation by immune complex formation as confirmed by its elevated levels in sputum AFB+++ positive patients.

Seroreactivity of 31 kDa and 41 kDa mycobacterial secretory proteins isolated from culture filtrate in extra pulmonary tuberculosis.

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 infecting agent in particular in extrapulmonary tuberculosis. Immune response to excretory-secretory (ES) proteins of Mycobacterium tuberculosis (M.tb) 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 H37Ra 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-31 and ES-41 secreted by M.tb H37 Ra bacilli were employed in stick indirect penicillinase 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 81.5% (22/27) and 84.6% (22/26) respectively in IgG antibody detection compared to 70% (19/27) and 69.2% (18/26) shown by ES-31. This study is of interest that different antigen protein fractions of M.tb 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.

Analysis of IgG subclasses and IgE antibodies across the clinical spectrum of bancroftian filariasis in an endemic area.

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 filaria infected endemic population. The levels of four IgG subclasses 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 IgG4 antibodies (P < 0.02), Occult filarial cases had higher levels of IgG4 and IgG3 (P < 0.02) and also of IgG4 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.

Sero-reactivity of two different antigenic protein fractions of M.tb H37Ra excretory-secretory antigen in pulmonary and extra-pulmonary tuberculosis.

Excretory-secretory proteins of Mycobacterium tuberculosis H37Ra, have been of diagnostic interest in pulmonary (PTB) and extrapulmonary tuberculosis (EPTB). Two different excretory-secretory antigenic proteins of M.tbH37Ra 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 infection (TBLN & TBM) whereas EST-DE1 was found to be better in detecting specific antibodies in bone & joint and abdominal tuberculosis.

Purification and characterization of a 31 kDa mycobacterial excretory-secretory antigenic protein with a diagnostic potential in pulmonary tuberculosis.

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 active 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.

Seroreactivity of purified Brugia malayi microfilarial soluble and excretory-secretory antigens in different clinical presentations of bancroftian filariasis.

Brugia malayi microfilarial excretory-secretory (mf ES) and phosphate buffer saline soluble (mf S) antigens were fractionated by fast protein liquid chromatography (FPLC) on superdex 200 HR 10/30 gel filtration column. The active antigen fractions were identified and explored in comparison with whole mf ES and mf S antigens to detect filarial IgG antibodies in different groups viz microfilaraemics, acute, chronic and occult filarial cases of Wuchereria bancrofti infection and endemic and non-endemic normals. One of the fractions of mf ES antigen (ESF-6) and two fractions of mf S antigen (SF-2 & 3) were identified to be useful to detect filarial antibodies. A pooled preparation of these antigen fractions gave a sensitivity of 86.6% (for microfilaraemic cases) and a specificity of 95% to detect filarial IgG antibodies by indirect ELISA. The pooled FPLC purified mf antigens also showed 55-88% of cases of different grades of clinical filariasis and 65% of tropical pulmonary eosinophilia cases as positive for filarial antibodies. The pooled FPLC purified B. malayi mf antigens with higher specificity are preferable to whole mf ES and mf S antigens to detect active filarial infection in microfilaraemia and as well in different clinical entities of bancroftian filariasis.

Surgical filariasis: immunoscreening for filarial IgG antibodies using Wuchereria bancrofti microfilarial excretory-secretory antigen.

A clinical study and immunoscreening was conducted on 363 suspected filarial patients attending the surgery out patient division of the MGIMS, Sevegram. The disease was significantly higher in males (86%) than in females (14%). Majority (52.9%) of the cases were in the age group of 11-30 years. The distribution of cases into three different grades of infection showed, 52.6%, 33.3% and 14.1% of the cases having acute (grade I), sub-acute (grade II) and chronic (grade III) stages of infection respectively. While 73% of the cases had genital manifestations, 23% were with lymphatic obstruction in limbs and the rest of the 4% suffered from manifestations like cellulitis, abscesses, haematuria and chyluria. Filarial IgG antibodies against microfilarial excretory-secretory (mf ES) antigen were detected in 89% of cases with genital manifestations, 87% of lymphoedema cases, 67% of lymphadenitis cases and 60% of cases with other clinical manifestations and 3% of endemic normals.