Kim S H, Chun J H, Park S Y
Antibody Engineering Lab., Central Research Center of the Korea Green Cross Corp., Kyunggi-Do, 449-903, Korea.
Hybridoma. 2001 Aug;20(4):265-72. doi: 10.1089/027245701753179857.
Eight monoclonal antibodies (MAbs) against carcinoembryonic antigen (CEA) were characterized. Five clones are IgG(1), two clones are IgM and one clone is IgG(2b); all have kappa light chain. The affinities are in the range of 1.1 x 10(-7) approximately 2.4 x 10(-9) M; the affinities of two IgM clones could not be estimated because of their low enzyme-linked immunoadsorbent assay (ELISA) signal. Each clone was constructed as single-chain Fv (scFv) and expression was performed in E. coli. Four clones out of 8 could express scFv soluble to culture media and the expression was confirmed further by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. The nucleotide and amino acid sequences of V(H) and V(L) of four scFvs were deduced and their family and subgroup were analyzed. We found that the clones that do not express the scFv have aberrant kappa chain (incorrect V/J recombination or stop codon); in contrast, their heavy chain sequences proved correct. The E. coli-expressed scFvs showed 1.5 x 3.4-fold lower affinities (2.8 x 10(-8) approximately 3.6 x 10(-9) M) than those of hybridoma-derived parental antibodies except the one clone (C5), which exhibited approximately 10(-6) M of affinity.
对8种抗癌胚抗原（CEA）的单克隆抗体（MAb）进行了特性分析。5个克隆为IgG(1)，2个克隆为IgM，1个克隆为IgG(2b)；所有抗体均具有κ轻链。亲和力范围为1.1×10(-7)至约2.4×10(-9) M；由于两种IgM克隆的酶联免疫吸附测定（ELISA）信号较低，其亲和力无法估计。每个克隆均构建为单链Fv（scFv）并在大肠杆菌中表达。8个克隆中有4个可将scFv分泌到培养基中，通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）和免疫印迹进一步证实了表达情况。推导了4种scFv的V(H)和V(L)的核苷酸和氨基酸序列，并分析了它们的家族和亚组。我们发现不表达scFv的克隆具有异常的κ链（V/J重排错误或存在终止密码子）；相比之下，它们的重链序列是正确的。除了一个亲和力约为10(-6) M的克隆（C5）外，大肠杆菌表达的scFv的亲和力比杂交瘤来源的亲本抗体低1.5至3.4倍（2.8×10(-8)至约3.6×10(-9) M）。
