title: A single-chain antibody fragment against human thyroglobulin: construction and evaluation of immunoreactivity.
abstract: Smaller recombinant antibody fragments are at the forefront of in vivo diagnosis and therapy. These units possess better distribution and faster clearance than larger molecules. Among these, single chain antibody fragments (scFv) are emerging as credible alternatives. These proteins are shown to have same specificities and affinities for their antigens as the parental monoclonal antibody (MAb). We have attempted to produce scFv against human thyroglobulin (H-Tg) using anti-Tg secreting hybridoma cells and PCR-based cloning approach. Hybridoma secreting anti-Tg MAb B10IV was established. cDNA was prepared from hybridoma cells. The V(H) and V(L) genes were amplified and cloned. The gene sequences were submitted to Genebank database (accession nos. AJ508533 and AM072962, respectively.) V(L) and V(H) genes were then linked together with a linker peptide and successfully cloned in pET28a and expressed as His-tag fusion protein in expression host BL21 (DE3). The scFv protein from IPTG-induced cells was purified under native conditions by immobilized metal affinity chromatography on a Ni-NTA agarose column. The yield expressed in Escherichia coli was approximately 8 mg/L. ScFv could be labeled with (125)I and its immunoreactivity evaluated in radioassays. Although scFv demonstrated specific binding to H-Tg, the immunoreactivity was low (10.3%) compared to the parental MAb B10IV, which showed immunoreactivity of 37.27%. Inhibition radioassays exhibited that scFv and MAb interact with the same epitope on the target antigen, indicating its specificity.
authors.affiliation: Radiation Medicine Centre, BARC, Tata Memorial Hospital Annex, Parel, Mumbai, India.
authors.forename: Rekha
authors.initials: R
authors.lastname: Rajawat
authors.forename: Archana
authors.initials: A
authors.lastname: Narkar
authors.forename: Archana
authors.initials: A
authors.lastname: Damle
authors.forename: G B Sunil
authors.initials: GB
authors.lastname: Kumar
authors.forename: K P
authors.initials: KP
authors.lastname: Mishra
mesh_terms: D000918:Antibody Specificity; D001483:Base Sequence; D002846:Chromatography, Affinity / Q000379:methods; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli; D006801:Humans; D006825:Hybridomas / Q000276:immunology / Q000378:metabolism; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011869:Radioligand Assay; D017422:Sequence Analysis, DNA; D057127:Single-Chain Antibodies / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology*; D013954:Thyroglobulin / Q000276:immunology*
chemical_list: D018076:DNA, Complementary; D057127:Single-Chain Antibodies; D013954:Thyroglobulin