Rajawat Rekha, Narkar Archana, Damle Archana, Kumar G B Sunil, Mishra K P
Radiation Medicine Centre, BARC, Tata Memorial Hospital Annex, Parel, Mumbai, India.
Hybridoma (Larchmt). 2011 Jun;30(3):253-9. doi: 10.1089/hyb.2010.0114.
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.
较小的重组抗体片段处于体内诊断和治疗的前沿。这些单位比较大的分子具有更好的分布和更快的清除率。其中，单链抗体片段（scFv）正成为可靠的替代品。这些蛋白质对其抗原的特异性和亲和力与亲本单克隆抗体（MAb）相同。我们试图利用分泌抗甲状腺球蛋白（H-Tg）的杂交瘤细胞和基于PCR的克隆方法来生产抗H-Tg的scFv。建立了分泌抗Tg MAb B10IV的杂交瘤。从杂交瘤细胞中制备cDNA。扩增并克隆V(H)和V(L)基因。基因序列提交到基因库数据库（登录号分别为AJ508533和AM072962）。然后将V(L)和V(H)基因与连接肽连接在一起，并成功克隆到pET28a中，在表达宿主BL21(DE3)中表达为His标签融合蛋白。通过镍-硝酸镧琼脂糖柱上的固定金属亲和层析在天然条件下纯化IPTG诱导细胞中的scFv蛋白。在大肠杆菌中的产量约为8mg/L。scFv可用(125)I标记，并在放射分析中评估其免疫反应性。尽管scFv显示出与H-Tg的特异性结合，但与亲本MAb B10IV相比，其免疫反应性较低（10.3%），亲本MAb B10IV的免疫反应性为37.27%。抑制放射分析表明，scFv和MAb与靶抗原上的相同表位相互作用，表明其特异性。
