Skip to content
Licensed Unlicensed Requires Authentication Published by De Gruyter December 28, 2016

Characterization of immunoreactive proteins of Setaria cervi isolated by preparative polyacrylamide gel electrophoresis

Priyanka Priyadarshi, Piyush Dravid, Inayat Hussain Sheikh, Sunita Saxena, Ashish Tandon, Deep C. Kaushal, Shakir Ali and Nuzhat A. Kaushal
From the journal Acta Parasitologica


Filarial parasites are complex mixtures of antigenic proteins and characterization of these antigenic molecules is essential to identify the diagnostically important filaria-specific antigens. In the present study, we have fractionated the somatic extracts from adults of Setaria cervi (bovine filarial parasite) on preparative SDS-polyacrylamide gel electrophoresis and tested the immunoreactivity of the separated gel fractions with polyclonal antibodies against filarial excretory-secretory antigens as well as filarial patients sera. The SDS-PAGE analysis of gel eluted fractions revealed 1 protein band in F-1 fraction, 2 protein bands in F-2 fraction and 2–3 protein bands in all other fractions (F3- F11). Seven gel eluted fractions (F1, F2, F3, F4, F5, F6 and F11) showed high ELISA reactivity with the polyclonal antibody (against excretory-secretory antigen) and four of these fractions (F-1, F-2, F3 and F6) exhibited high ELISA reactivity with antibodies present in filarial patient sera. The reactivities of the gel fractions (F1 and F2), recognized by filarial patients sera, were also tested with the monoclonal antibody (detecting the filarial circulating antigen). The F1 and F2 gel eluted fractions were found to have the target antigen of monoclonal antibody as evident by high reactivity with the monoclonal antibody in ELISA and immunoblotting. The S. cervi gel eluted F1 fraction (containing single antigen) could detect antibodies in filarial patients sera and not in non-filarial sera thereby suggesting its usefulness for specific serodiagnosis of human filariasis.

  1. Conflict of interest:Authors declare no competing interests.


The authors would like to thank the Director, CSIR-CDRI and Scientist-in-charge, Parasitology, CSIR-CDRI for their help and encouragement. PP and SS are thankful to ICMR, New Delhi for financial support and two of the authors (PD, IHS) are thankful to CSIR, New Delhi, India. CSIR-CDRI Communication No.: 8552.


Alli R., Bhandari Y.P., Reddy M.V., Harinath B.C. 2000. Seroreactivity of purified Brugia malayi microfilarial soluble and excretory-secretory antigens in different clinical presentations of bancroftian filariasis. Indian Journal of Experimental Biology, 38, 791–796Search in Google Scholar

Almeida A.J., Bhopale M.K., Renapurkar D.M. 1990. Evaluation of Setaria cervi antigen in the diagnosis of human filariasis. Folia Parasitologica, 37, 323–329Search in Google Scholar

Bradford M.M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72, 248–254. 10.1016/0003-2697(76)90527-3Search in Google Scholar

Cabrera Z., Parkhouse R. 1986. Identification of antigens of Onchocerca volvulus and Onchocerca gibsoni for diagnostic use. Molecular and Biochemical Parasitology, 20, 225–231. 10.1016/0166-6851(86)90102-7Search in Google Scholar

Dissanayake S., Zheng H., Dreyer G., Xu M., Watawana L., Cheng G., Wang S., Morin P., Deng B., Kurniawan L. 1994. Evaluation of a recombinant parasite antigen for the diagnosis of lymphatic filariasis. The American Journal of Tropical Medicine and Hygiene, 50, 727–73410.4269/ajtmh.1994.50.727Search in Google Scholar

Ichimori K., King J.D., Engels D., Yajima A., Mikhailov A., Lammie P., Ottesen E.A. 2014. Global programme to eliminate lymphatic filariasis: the processes underlying programme success. PLoS Neglected Tropical Diseases, 8, e3328. 10.1371/journal.pntd.0003328Search in Google Scholar

Janardhan S., Pandiaraja P., Pandey V., Karande A., Kaliraj P. 2011. Development and characterization of monoclonal antibodies against WbSXP-1 for the detection of circulating filarial antigens. Journal of Helminthology, 85, 1–6. 10.1017/ S0022149X10000118Search in Google Scholar

Kaushal N.A., Hussain R., Ottesen E.A. 1984. Excretory-secretory and somatic antigens in the diagnosis of human filariasis. Clinical and Experimental Immunology, 56, 567–576. in Google Scholar

Kaushal N.A., Kaushal D.C., Ghatak S. 1987. Identification of antigenic proteins of Setaria cervi by immunoblotting technique. Immunological Investigations, 16, 139–149. 10.3109/08820138709030571Search in Google Scholar

Kaushal N.A., Kaushal D.C., Ghosh S., Talwar G.P. 1994. Monoclonal antibodies against antigenic epitopes common between Setaria cervi and Brugia malayi. Indian Journal of Experimental Biology, 32, 371–375. pubmed/7525468Search in Google Scholar

Kaushal N.A., Srivastava N., Mustafa H., Tandon A., Singh S.K., Kaushal D.C. 2009. Isolation of an antigen fraction from Setaria cervi adults having potential for immunodiagnosis of human filariasis. Immunological Investigations, 38, 749–761. 10.3109/08820130903204093Search in Google Scholar

Laemmli U.K. 1970. Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4. Nature, 227, 680–685. 10.1038/227680a0Search in Google Scholar

Lowry O.H., Rosebrough N.J., Farr A.L., Randall R.J. 1951. Protein measurement with the Folin phenol reagent. The Journal of Biological Chemistry, 193, 265–27510.1016/S0021-9258(19)52451-6Search in Google Scholar

Malhotra A., Kaushal N.A., Ghatak S. 1986. Protein and antigenic composition of adult and microfilarial stages of Setaria cervi. Immunological Investigations, 15, 505–519. 10.3109/0882 0138609026691Search in Google Scholar

Malhotra A., Kaushal N.A., Kaushal D.C., Ghatak S. 1987. Antigenic characterization of excretory-secretory products of Setaria cervi. Tropical Medicine and Parasitology. 38,106–110. in Google Scholar

Mohammadian T., Doosti M., Paknejad M., Siavoshi F., Massarrat S. 2010. Preparative SDS-PAGE Electroelution for Rapid Purification of Alkyl Hydroperoxide Reductase from Helicobacter pylori. Iranian Journal of Public Health, 39, 85–91Search in Google Scholar

More S.J., Copeman D.B. 1990. A highly specific and sensitive monoclonal antibody-based ELISA for the detection of circulating antigen in bancroftian filariasis. Tropical Medicine and Parasitology, 41, 403–406. pubmed/2075384Search in Google Scholar

Mustafa H., Srivastava N., Kaushal D.C., Kaushal N.A. 1996. Analysis and potential of excretory-secretory antigens of Setaria cervi for immunodiagnosis of human filariasis. Indian Journal of Experimental Biology, 34, 508–512Search in Google Scholar

Ravishankaran R., Shridharan R.N., Vishal L.A., Meenakshisundaram S., Karande A.A., Kaliraj P. 2016. Evaluation of immuno diagnostic assay for the exposure of stage specific filarial infection. Acta Parasitologica, 61, 232–240. 10.1515/ap-2016-0033Search in Google Scholar

Riyong D., Waikagul J., Panasoponkul C., Choochote W., Ito A., Dekumyoy P. 2010. Size and charge antigens of Dirofilaria immitis adult worm for IgG-ELISA diagnosis of bancroftian filariasis. Southeast Asian Journal of Tropical Medicine and Public Health, 41, 285–297Search in Google Scholar

Rubio de Krömer M.T., Krömer M., Lüersen K., Brattig N.W. 1998. Detection of a chemotactic factor for neutrophils in extracts of female Onchocerca volvulus. Acta Tropica, 71, 45–5610.1016/S0001-706X(98)00044-8Search in Google Scholar

Seelert H., Krause F. 2008. Preparative isolation of protein complexes and other bioparticles by elution from polyacrylamide gels. Electrophoresis, 29, 2617–2636. 10.1002/elps.20080 0061Search in Google Scholar

Singh A., Kamal S., Rathaur S. 2010. Filarial selenium glutathione peroxidase: a probable immunodiagnostic marker for lymphatic filariasis. Transactions of the Royal Society of Tropical Medicine and Hygiene, 104, 524–528. 10.1016/j.trstmh. 2010.02.007Search in Google Scholar

Singh V., Kaushal D.C., Rathaur S., Kumar N., Kaushal N.A. 2012. Cloning, overexpression, purification and characterization of Plasmodium knowlesi lactate dehydrogenase. Protein Expression and Purification, 84, 195–203.10.1016/j.pep .2012.05.008Search in Google Scholar

Srivastava S., Srikanth E., Liebau E., Rathaur S. 2010. Identification of Setaria cervi heat shock protein 70 by mass spectrometry and its evaluation as diagnostic marker for lymphatic filariasis. Vaccine, 28, 1429–1436. 10.1016/j.vaccine.2009.06.044Search in Google Scholar

Theodore J.G., Kaliraj P. 1990. Isolation, purification and characterization of surface antigens of the bovine filarial parasite Setaria digitata for the immunodiagnosis of bancroftian filariasis. Journal of Helminthology, 64, 105–11410.1017/S0022149X00012001Search in Google Scholar

Towbin H., Staehelin T., Gordon J. 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proceedings of the National Academy of Sciences of the United States of America, 76, 4350–435410.1073/pnas.76.9.4350Search in Google Scholar

Voller A., Bidwell D., Huldt G., Engvall E. 1974. A microplate method of enzyme-linked immunosorbent assay and its application to malaria. Bulletin of the World Health Organization, 51, 209–211Search in Google Scholar

WHO 2015. Lymphatic filariasis WHO. URL: entity/ mediacentre/ factsheets/fs102/en/index.htmlSearch in Google Scholar

Wickremanayake M.N., Ekanayake S., Karunanayake E.H. 2001. Wuchereria bancrofti: detection of microfilariae in asymptomatic microfilaremic individuals with Setaria digitata antigens. The Southeast Asian journal of tropical medicine and public health, 32, 230–234Search in Google Scholar

Weil G.J., Lammie P.J., Weiss N. 1997. The ICT Filariasis Test: A rapid-format antigen test for diagnosis of bancroftian filariasis. Parasitology Today, 13, 401–40410.1016/S0169-4758(97)01130-7Search in Google Scholar

Received: 2016-2-14
Revised: 2016-8-22
Accepted: 2016-8-31
Published Online: 2016-12-28
Published in Print: 2017-3-1

© 2017 W. Stefański Institute of Parasitology, PAS

Scroll Up Arrow