Rodenburg C M, Mernaugh R, Bilbao G, Khazaeli M B
Department of Medicine, University of Alabama at Birmingham 35294-3300, USA.
Hybridoma. 1998 Feb;17(1):1-8. doi: 10.1089/hyb.1998.17.1.
Recombinant single chain Fv (scFv) antibodies offer many advantages over mouse monoclonal antibodies (MAbs) such as faster clearance from blood, improved tumor localization, reduced human anti-mouse antibody (HAMA) response, and the availability to manipulate the scFv through genetic approaches. The scFv antibody (designated RK10.2) was generated using anti-CEA T84.66 hybridoma cells as a source of genetic starting material and the Pharmacia Recombinant Phage Antibody System (RPAS). Escherichia coli clones expressing antigen-positive soluble scFv were identified using a modified colony-life selection procedure and antigen-coated filters. The resultant anti-CEA scFv (designated RK10.2) had a molecular weight of approximately 33.6 kDa and an isoelectric point of 5.2 at 15 degrees C. The RK10.2 scFv interacted with LS174 T cells bearing the CEA antigen and inhibited the anti-CEA MAb/CEA antigen interaction in ELISA and the anti-CEA MAb/LS174 T cell interaction in a RIA. The modified colony-lift approach circumvented the more time-consuming phage-display approach that is normally taken to affinity select for antigen-positive scFv clones.
重组单链Fv（scFv）抗体相对于小鼠单克隆抗体（MAb）具有许多优势，如从血液中清除更快、肿瘤定位改善、人抗小鼠抗体（HAMA）反应降低，以及可通过基因方法对scFv进行操作。scFv抗体（命名为RK10.2）以抗CEA T84.66杂交瘤细胞作为遗传起始材料来源，并使用Pharmacia重组噬菌体抗体系统（RPAS）产生。使用改良的菌落印迹选择程序和抗原包被的滤膜鉴定表达抗原阳性可溶性scFv的大肠杆菌克隆。所得的抗CEA scFv（命名为RK10.2）在15℃时分子量约为33.6 kDa，等电点为5.2。RK10.2 scFv与携带CEA抗原的LS174 T细胞相互作用，并在ELISA中抑制抗CEA MAb/CEA抗原相互作用以及在RIA中抑制抗CEA MAb/LS174 T细胞相互作用。改良的菌落印迹方法规避了通常用于亲和选择抗原阳性scFv克隆的耗时更长的噬菌体展示方法。
