• B C Harinath

      Articles written in Journal of Biosciences

    • Detection of filarial infection usingWuchereria bancrofti microfilariae culture antigen and filter paper blood samples in enzyme linked immunosorbent assay

      Ashok Malhotra M V R Reddy J N Naidu S N Ghirnikar B C Harinath

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      Blood collected on filter paper by finger-prick gave results comparable to intravenous serum samples when analysed by enzyme-linked immunosorbent assay (ELISA). All the 100 microfilaraemia, 5 out of 100 endemic normals and none of the 10 nonendemic normal filter paper blood samples showed the presence of filarial antibody when tested by this method,using culture antigen and anti-immunoglubulins, class G, M and A — penicillinase conjugate. When the same samples were screened for the presence of IgM antibody, 91 out of 100 microfilaraemia, 13 out of 100 endemic normal and none of the 10 nonendemic normal samples showed a positive reaction. Enzyme linked immunosorbent assay, using culture antigen and filter paper blood samples, appears to work in large field studies for detection of filarial infection.

    • Evaluation of fractionatedWuchereria bancrofti microfilarial excretory-secretory antigens for diagnosis of bancroftian filariasis by enzyme linked immunosorbent assay

      M V R Reddy Ashok Malhotra G B K S Prasad B C Harinath

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      TheWuchereria bancrofti microfilarial excretory-secretory antigens were fractionated into ES1, ES2, ES3 and ES4 by ultra-membrane filtration and evaluated for their diagnostic utility by enzyme linked immunosorbent assay. Three of the four fractions showed antigenic activity (ES2, ES3 and ES4). The antigen fractions ES2 and ES4 were highly active in the detection of filarial IgM antibody in clinical filariasis and microfilaraemia respectively. The chemical characterization of the ES2 and ES4 antigen fractions showed that they were glycoproteins

    • Monoclonal antibodies againstWuchereria bancrofti microfilarial excretory-secretory antigens

      M V R Reddy W F Piessens B C Harinath

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      A battery of monoclonal antibodies were produced againstWuchereria bancrofti microfilarial excretory-secretory antigens and their specificity was studied using different filarial antigens. Among the 1116 wells plated out, 42 % of the wells developed hybrids and 5 % of the hybrids showed antiWuchereria bancrofti microfilarial excretory-secretory antigens. Specificity studies on the antibodies produced from 63 cloned and expanded hybrids showed 10 clones which were specifically positive only toWuchereria bancrofti microfilarial excretory-secretory antigens.

    • Diagnostic utility of monoclonal antibodies raised against microfilarial excretory-secretory antigens in bancroftian filariasis

      M V R Reddy P Rama Prasad W F Piessens B C Harinath

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      Two monoclonal antibodiesWuchereria bancrofti E 33 andWuchereria bancrofli E 34 raised againstWuchereria bancrofti microfilarial excretory-secretory antigens were studied for their diagnostic utility.Wuchereria bancrofti E 34 monoclonal antibody was found to be relatively specific and sensitive in detection of circulating filarial antigen. WhenWuchereria bancrofti E 34 monoclonal antibody was used alongwith immunoglobulin G fraction of human filarial serum immunoglobulins in double antibody sandwich enzyme linked immunosorbent assay. 68% of microfilaraemic sera (26 out of 38). 12% of clinical filarial sera (3 out of 25), 13% endemic normal sera (2 out of 15) and none of the 20 non-endemic normal sera showed the presence of filarial antigen. The filarial antigen detected byWuchereria bancrofti E 34 monoclonal antibody in double antibody sandwich enzyme linked immunosorbent assay is possibly associated with the active stage (microfilaraemia) of infection.

    • Stick enzyme-linked immunosorbent assay using the avidin-biotin system for detection of circulating antigen in bancroftian filariasis

      K A Parkhe P Ramaprasad B C Harinath

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      Detection of filarial antigen in different groups of sera was carried out by sandwich as well as inhibition enzyme-linked immunosorbent assays using antibody-coated sticks. Both systems were found to be equally sensitive in detecting antigen in 90% of microfilariae carriers. Incorporation of avidin-biotin in the sandwich assay system increased the sensitivity of antigen detection from 10−6 to 10−16 pg. A 67% decrease in the number of false negative results was observed when the sensitive avidin-biotin inhibition enzyme-linked immunosorbent assay system was used for analysis of filaria blood samples.

    • Analysis, characterization and diagnostic use of circulating filarial antigen in bancroftian filariasis

      K A Parkhe M V R Reddy K Cheirmaraj P Ramaprasad B C Harinath

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      Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of circulating filarial antigen fraction-2 isolated from plasma of microfilaraemic patients withWuchereria bancrofti infection has shown 21 bands with molecular weights ranging from 12 to 120 kDa. The gel (12 cm) was sliced at an interval of one cm and the eluates of all the gel slicesviz., CFA2-1 to CFA2-12 showed the presence of filarial antigen by sandwich enzyme-linked immunosorbent assay. The low molecular weight circulating filarial antigen fractions were found to share a common epitope withWuchereria bancrofti microfilariae excretory-secretory antigen and urinary filarial antigen. The 3 antigen fractions CFA2-1, CFA2-9 and CFA2-12 showed higher sensitivity in detecting filarial immunoglobulin M antibodies than immunoglobulin G antibodies. However CFA2-9 fraction was found useful in serological differentiation of microfilaraemics from those with disease manifestations when filarial immunoglobulin G antibodies were detected. The antigenic epitope of CFA2-1 appears to be a carbohydrate, whereas CFA2-9 appears to be protein in nature.

    • Differential reactivity of filarial antigens with human sera from bancroftian filariasis endemic zone

      K Cheirmaraj M V R Reddy B C Harinath

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      The reacting pattern of circulating filarial antigen fraction-2 fromWuchereria bancrofti and soluble antigen from adultBrugia malayi with bancroftian filarial sera were analysed by immunoblotting technique and enzyme linked immunosorbent assay. Microfilaraemic sera reacted specifically with proteins of molecular weight 200, 120, 97, 56, 54, 43, 26 and 17 kDa of circulating Filarial antigen fraction-2 and 44, 40, 38, 31, 22 and 18 kDa ofBrugia malayi adult soluble antigen. Clinical filarial sera identified protein molecules of 56, 54 and 42 kDa of circulating filarial antigen fraction-2 and 19, 16 and 14 kDa ofBrugia malayi adult soluble antigen. Some components of both the antigen preparation were also identified by endemic normal serai.e.proteins 120, 97, 62, 43 and 33 kDa of circulating filarial antigen fraction-2 and 170, 120, 43, 31 and 12 kDa ofBrugia malayi adult soluble antigen. One of the sodium dodecyl sulphate-polyacrylamide gel electropherosis fractions of circulating filarial antigen fraction-2 (CFA2-8) andBrugia malayi adult soluble antigen fraction-6 when used in enzyme linked immunosorbent assay could differentiate microfilaraemic sera from endemic normal and clinical filarial sera. The other antigen fractions (CFA2-2, 6 and 7 andBmA-2) showed a high geometric mean titre of filarial immunoglobulin G antibodies in endemic normal sera when compared to microfilaraemia and clinical filarial sera. These proteins need to be further studied to assess their involvement in protecting from filarial infection in an endemic area.

    • Immunoprophylaxis against filarial parasite,Brugia malayi: potential of excretory-secretory antigens in inducing immunity

      K Cheirmaraj V Chenthamarakshan M V R Reddy B C Harinath

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      The role of excretory-secretory antigens in inducing immunity in the host againstBrugia malayi microfilariae and infective larvae was studied byin vitro antibody dependent cell-mediated reaction as well asin vivo inoculation of filarial parasites within a microchamber in the host. The immune sera of jirds raised againstBrugia malayi microfilarial and infective larval excretory-secretory antigens(Bm Mf ESA andBm L3 ESA) promoted the adherence of peritoneal exudate cells toBrugia malayi microfilariae and infective larvaein vitro and induced cytotoxicity to the parasites within 48 h. The antiBm Mf ESA serum was more effective than antiBm L3 ESA serum in inducing cytotoxicity to microfilariae and both antisera had a similar cytotoxic effect on infective larvae. In the microchambers implanted in the immune jirds, host cells could migrate and adhere to the microfilariae and infective larvae and kill them within 48–72 h. Further,Mastomys natalensis immunized againstBm Mf ESA and L3 ESA generated a high degree of protective response against circulating microfilariae. These results suggest that excretory-secretory antigens are effective in inducing resistance against filarial parasites and thus have potential in immunoprophylaxis.

    • Diagnostic utility of fractionated urinary filarial antigen

      V Chenthamarakshan U M Padigel P Ramaprasad M V R Reddy B C Harinath

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      Urinary filarial antigen isolated from urine samples of microfilaraemic patients was analysed for its antigenic activity by immunoblotting and enzyme linked immunosorbent assay techniques. SDS-PAGE fractionation of urinary filarial antigen showed 11 protein bands, of which two showed reactivity with immunoglobulin-G fraction of filarial serum immunoglobulin in immunoblotting. Antigenic analysis of SDS-PAGE fractions of urinary filarial antigen by inhibition enzyme linked immunosorbent assay using filarial serum immunoglobulin-G andWuchereria bancrofti microfilarial excretory-secretory antigen revealed 3 fractions, numbers 5, 6 and 9 with significant activity. In indirect enzyme linked immunosorbent assay using fractions 5 and 6, filarial immunoglobulin-G antibody was detected in about 90% of microfilaraemics, 80% clinical filariasis and 20% of endemic normal individuals. Further, there was no phosphorylcholine epitope in these fractions. Fractions 5 and 6 can be a candidate antigens for the immunodiagnosis of filariasis.

    • Detection of filarial antigen by inhibition enzyme linked immunosorbent assay using fractionatedBrugia malayi microfilarial excretory secretory antigen

      V Chenthamarakshan M V R Reddy B C Harinath

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      Detection of filarial antigen in blood or urine samples would provide an accurate indication of active infection. The absence of a simple, well established animal model and limitations in getting the required amount of parasite material from human sources have been the main obstacles for the diagnosis ofWuchereria bancrofti infection. An inhibition ELISA has been developed for detection of filarial antigen using a partially purifiedBrugia malayi mf ES antigen (BmE DE1) and its affinity purified antibodies. Filarial antigen was detected in the sera of 88% of microfilaraemic, 60% of chronic filarial, 17% of endemic normal and none of the non- endemic normal subjects. The sensitivity and specificity of the assay were 88% and 89% respectively. Moreover, undiluted urine samples from 82% of microfilaraemic and 17% of endemic normal, but none of the non- endemic normal samples showed the presence of filarial antigen. With the limitations on the availability of sufficient homologous parasite materials, the inhibition ELISA using BmE DE1 and anti BmE DE1 antibodies shows promise for the detection of active infection in bancroftian filariasis in man. Moreover, its detection in urine makes it more possible to test patients in field areas.

    • Immunoprophylactic studies with a 43 kDa human circulating filarial antigen and a cross reactive 120 kDaBrugia malayi sodium dodecyl sulphate soluble antigen in filariasis

      V Chenthamarakshan K Cheirmaraj M V R Reddy B C Harinath

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      Bancroftian filariasis is a major public health problem affecting about 120 million people all over the world. Immunoprophylaxis may serve as an additional adjunct along with chemotherapy and anti larval measures for successful filaria control. Circulating filarial antigen fraction (CFA2-6) containing 43 kDa antigen and adultBrugia malayi sodium dodecyl sulphate (S DS) soluble antigen fraction BmA-2 with a 120 kDa molecule were earlier shown to be reactive with endemic normal sera by immunoblotting and indirect ELISA techniques. BmA-2 was found to be highly cross reactive with CFA2-6. Sera raised against both the antigen fractions showed about 90 % cytotoxicity to the parasites in the presence of jird peritoneal cells inin vitro as well as byin situ micropore chamber implantation technique. Further inin vivo studies using animal model, jirds CFA2-6 and BmA-2 could induce about 90% protection to infection in immunized animals. In passive transfer studies of immunity it has been observed that BmA-2 induced protection is mainly antibody mediated.

    • Immunodiagnosis of pulmonary tuberculosis by concomitant detection of antigen and antibodies of excretory-secretory protein ofMycobacterium tuberculosis H37Ra

      A N Lodam M V R Reddy B C Harinath

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      With a view to diagnosing tuberculosis in populations in endemic areas, excretory-secretory antigen fraction(Mtb EST-6) of purifiedMycobacterium tuberculosis H37Ra and affinity purified polyclonal antibodies againstMtb EST were used to detect both antibodies and circulating antigen in the sera of patients and disease-free individuals. Indirect stick penicillinase ELISA system usingMtb EST-6 detected antigen-specific IgG antibody in 84% of sputum positive, 77% of sputum negative pulmonary tuberculosis patients and 7% of healthy and 11% of subjects with nontub~rculosis diseases. Similarly, a sandwich penicillinase ELISA system using affinity purified antiMtb EST antibodies detected circulating antigen in 83% and 61% of sputum positive and negative pulmonary tuberculosis subjects. In contrast only 24% of healthy and 18% of disease controls showed seropositivity. Antibody assay showed higher sensitivity and specificity (83% and 91% respectively) compared to antigen detection (sensitivity of 79% and specificity of 79%). However, by concomitant use of both assays it was possible to enhance the specificity of detection to 98%, though sensitivity was reduced marginally to 70%. The present study confirms the presence of both antigen and specific antibodies in the circulation during clinical disease and draws attention to the utility ofMtb EST-6 as a diagnostic marker of pulmonary tuberculosis.

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