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Construction and bacterial expression of a recombinant single-chain antibody fragment against Wuchereria bancrofti SXP-1 antigen for the diagnosis of lymphatic filariasis

Published online by Cambridge University Press:  22 December 2014

R. Kamatchi
Affiliation:
Centre for Biotechnology, Anna University, Chennai600 025, India
J. Charumathi
Affiliation:
Centre for Biotechnology, Anna University, Chennai600 025, India
R. Ravishankaran
Affiliation:
Centre for Biotechnology, Anna University, Chennai600 025, India
P. Kaliraj*
Affiliation:
Centre for Biotechnology, Anna University, Chennai600 025, India
S. Meenakshisundaram*
Affiliation:
Centre for Biotechnology, Anna University, Chennai600 025, India
*
*Fax: +91 44 22350299 E-mail: [email protected]
*Fax: +91 44 22350299 E-mail: [email protected]

Abstract

Global programmes to eliminate lymphatic filariasis (GPELF) require mapping, monitoring and evaluation using filarial antigen diagnostic kits. To meet this objective, a functional single-chain fragment variable (ScFv) specific for filarial Wuchereria bancrofti SXP-1 (Wb-SXP-1) antigen was constructed for the diagnosis of active filarial infection, an alternative to the production of complete antibodies using hybridomas. The variable heavy chain (VH) and the variable light chain (kappa) (Vκ) genes were amplified from the mouse hybridoma cell line and were linked together with a flexible linker by overlap extension polymerase chain reaction (PCR). The ScFv construct (Vκ–Linker–VH) was expressed as a fusion protein with N-terminal His tag in Escherichia coli and purified using immobilized metal affinity chromatography (IMAC) without the addition of reducing agents. Immunoblotting and sandwich enzyme-linked immunosorbent assay (ELISA) were used to analyse the antigen binding affinity of purified ScFv. The purified ScFv was found to recognize recombinant and native Wb-SXP-1 antigen in microfilariae (Mf)-positive patient sera. The affinity of ScFv was comparable with that of the monoclonal antibody. The development of recombinant ScFv to replace monoclonal antibody for detection of filarial antigen was achieved. The recombinant ScFv was purified, on-column refolded and its detection ability validated using field samples.

Type
Research Papers
Copyright
Copyright © Cambridge University Press 2014 

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References

Capra, J.D. & Kehoe, J.M. (1974) Variable region sequences of five human immunoglobulin heavy chains of the VHIII subgroup: definitive identification of four heavy chain hypervariable regions. Proceedings of the National Academy of Sciences of the USA 71, 845848.CrossRefGoogle ScholarPubMed
Dattamajumdar, A.K., Jacobson, D.P., Hood, L.E. & Osman, G.E. (1996) Rapid cloning of any rearranged mouse immunoglobulin variable genes. Immunogenetics 43, 141145.CrossRefGoogle ScholarPubMed
Glockshuber, R., Malia, M., Pfitzinger, I. & Plueckthun, A. (1990) Comparison of strategies to stabilize immunoglobulin Fv-fragments. Biochemistry 29, 13621367.CrossRefGoogle ScholarPubMed
Holliger, P. & Hudson, P.J. (2005) Engineered antibody fragments and the rise of single domains. Nature Biotechnology 23, 11261136.CrossRefGoogle ScholarPubMed
Hudson, P.J. (1998) Recombinant antibody fragments. Current Opinion in Biotechnology 9, 395402.CrossRefGoogle ScholarPubMed
Huston, J.S., Levinson, D., Mudgett-Hunter, M., Tai, M.S., Novotny, J., Margolies, M.N., Ridge, R.J., Bruccoleri, R.E., Haber, E. & Crea, R. (1988) Protein engineering of antibody binding sites: recovery of specific activity in an anti-digoxin single chain Fv analog produced in Escherichia coli. Proceedings of the National Academy of Sciences of the USA 85, 58795883.CrossRefGoogle Scholar
Janardhan, S., Pandiaraja, P., Pandey, V., Karande, A. & Kaliraj, P. (2011) Development and characterization of monoclonal antibodies against Wb-SXP-1for detection of circulating filarial antigens. Journal of Helminthology 85, 16.CrossRefGoogle ScholarPubMed
Kabat, E.A., Wu, T.T., Reid-Miller, M., Perry, H.M. & Gottesmann, K.S. (1987) Sequences of proteins of immunological interest. Washington, DC, US Department of Health and Human Services, US Government Printing Office.Google Scholar
Lalitha, P., Ravichandran, M., Suba, S., Kaliraj, P., Narayanan, R.B. & Jayaraman, K. (1998) Quantitative assessment of circulating filarial antigen in human lymphatic filariasis: a field evaluation of monoclonal antibody based ELISA using blood collected on filter strips. Tropical Medicine & International Health 3, 4145.CrossRefGoogle ScholarPubMed
Lalitha, P., Eswaran, M., Gnanasekhar, B., Rao, K.V.N., Narayanan, R.B., Scott, A.L., Nutman, T.B. & Kaliraj, P. (2002) Development of antigen detection ELISA for the diagnosis of brugian and bancroftian filariasis using antibodies to recombinant filarial antigens BmSXP-1 and WbSXP-1. Microbiology and Immunology 46, 327332.CrossRefGoogle Scholar
Lee, M.H., Park, T.I., Park, Y.B. & Kwak, J.W. (2002) Bacterial expression and in vitro refolding of single chain Fv antibody specific for human plasma apolipoprotein B-100. Protein Expression and Purification 25, 166173.CrossRefGoogle ScholarPubMed
Nuchprayoon, S., Porksakorn, C., Junpee, A., Sanprasert, V. & Poovorawan, Y. (2003) Comparative assessment of an Og4C3 ELISA and an ICT filariasis test: A study of Myanmar migrants in Thailand. Asian Pacific Journal of Allergy and Immunology 21, 253257.Google Scholar
Orlandi, R., Gussow, D.H., Jones, P.T. & Winter, G. (1989) Cloning immunoglobulin variable domains for expression by polymerase chain reaction. Proceedings of the National Academy of Sciences of the USA 86, 38333837.CrossRefGoogle ScholarPubMed
Pandey, V., Madhumathi, J., Karande, A. & Kaliraj, P. (2011) Antigen detection assay with parasite specific monoclonal antibodies for diagnosis of lymphatic filariasis. Clinica Chimica Acta 412, 18671873.CrossRefGoogle ScholarPubMed
Rao, K.V.N., Eswaran, M., Ravi, V., Gnanasekhar, B., Narayana, R.B., Kaliraj, P., Jayaraman, K., Marson, A., Raghavan, N. & Scott, A.L. (2000) The Wuchereria bancrofti orthologue of Brugia malayi SXP1 and the diagnosis of bancroftian filariasis. Molecular and Biochemical Parasitology 107, 7180.CrossRefGoogle ScholarPubMed
Sambrook, J. & Russell, D.W. (2001) Molecular cloning: a laboratory manual. Cold Spring Harbor, New York, Cold Spring Harbor Laboratory Press.Google Scholar
Swietnicki, W. (2006) Folding aggregated proteins into functionally active form. Current Opinion in Biotechnology 17, 367372.CrossRefGoogle Scholar
Theodore, J.G. & Kaliraj, P. (1996) Wuchereria bancrofti recombinant antigen-derived poly- and monoclonal antibodies for the detection of circulating antigen(s) in the sera of lymphatic filarial patients. Journal of Helminthology 70, 6974.CrossRefGoogle ScholarPubMed
Wang, Z., Raifu, M., Howard, M., Smith, L., Hansen, D., Goldsby, R. & Ratner, D. (2000) Universal PCR amplification of mouse immunoglobulin gene variable regions: the design of degenerate primers and an assessment of the effect of DNA polymerase 3′ to 5′ exonuclease activity. Journal of Immunological Methods 233, 167177.CrossRefGoogle Scholar
Weil, G.J., Curtis, K.C., Fakoli, L., Fisher, K., Gankpala, L., Lammie, P.J., Majewski, A.C., Pelletreau, S., Won, K.Y., Bolay, F.K. & Fisher, P.U. (2013) Laboratory and field evaluation of a new rapid test for detecting Wuchereria bancrofti antigen in human blood. American Journal of Tropical Medicine and Hygiene 89, 1115.CrossRefGoogle ScholarPubMed
Yang, J., Chen, R., Wei, J., Zhang, F., Zhang, Y., Jia, L., Yan, Y., Luo, W., Cao, Y., Yao, L., Sun, J., Xu, Z. & Yang, A. (2010) Production and characterization of a recombinant single chain antibody against Hantaan virus envelope glycoprotein. Applied Microbiology and Biotechnology 86, 10671075.CrossRefGoogle Scholar