title: Construction and bacterial expression of a recombinant single-chain antibody fragment against Wuchereria bancrofti SXP-1 antigen for the diagnosis of lymphatic filariasis.
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.
authors.affiliation: Centre for Biotechnology, Anna University,Chennai600 025,India.
authors.forename: R
authors.initials: R
authors.lastname: Kamatchi
authors.affiliation: Centre for Biotechnology, Anna University,Chennai600 025,India.
authors.forename: J
authors.initials: J
authors.lastname: Charumathi
authors.affiliation: Centre for Biotechnology, Anna University,Chennai600 025,India.
authors.forename: R
authors.initials: R
authors.lastname: Ravishankaran
authors.affiliation: Centre for Biotechnology, Anna University,Chennai600 025,India.
authors.forename: P
authors.initials: P
authors.lastname: Kaliraj
authors.affiliation: Centre for Biotechnology, Anna University,Chennai600 025,India.
authors.forename: S
authors.initials: S
authors.lastname: Meenakshisundaram
mesh_terms: D000818:Animals; D000947:Antigens, Helminth / Q000097:blood* / Q000235:genetics / Q000276:immunology; D004605:Elephantiasis, Filarial / Q000097:blood / Q000175:diagnosis* / Q000469:parasitology; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D051379:Mice; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology; D057127:Single-Chain Antibodies / Q000032:analysis* / Q000235:genetics / Q000276:immunology; D014958:Wuchereria bancrofti / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification
chemical_list: D000947:Antigens, Helminth; D011994:Recombinant Proteins; D057127:Single-Chain Antibodies