编码与NCA-95交叉反应的单克隆抗CEA抗体（CEA 79）重链可变区（VH）和轻链可变区（VL）的cDNA的克隆与鉴定以及单链Fv分子（scFv）的产生

Cloning and characterization of cDNAs encoding VH and VL of a monoclonal anti-CEA antibody (CEA 79) cross-reactive with NCA-95 and generation of a single-chain Fv molecule (scFv).

作者信息

Chung J H, Choi S J, Kim H J, Kim I J, Choi I H, Lee S D, Yi K S, Suh P G, Ryu S H, Chung H K

机构信息

Department of Biochemistry, College of Medicine, Dongguk University, Kyungju, Korea.

出版信息

Mol Cells. 1997 Dec 31;7(6):816-9.

PMID:9509426

Abstract

We cloned complementary DNA (cDNA) encoding the variable regions of heavy chain (VH) and of light chain (VL) of a monoclonal anti-carcinoembryonic antigen (CEA) antibody cross-reactive with nonspecific cross-reacting antigen-95 (NCA-95), which had been previously prepared and designated as CEA 79 (gamma 2a, kappa). From these cDNAs, a phagemid expression vector for the CEA79 single chain variable fragment (scFv) was generated. Enzyme-linked immunosorbent assay (ELISA), competitive ELISAs, and Western blotting confirmed that the scFv displayed on the surface of the bacteriophage had retained affinity for CEA and NCA-95. We then determined the nucleotide sequences of the cloned cDNAs for VH and VL. The sequence analysis revealed that VH and VL of the CEA 79 antibody represent new members of the mouse heavy chain subgroup "miscellaneous" and the kappa light chain subgroup "V", respectively.

摘要

我们克隆了编码一种单克隆抗癌胚抗原（CEA）抗体重链可变区（VH）和轻链可变区（VL）的互补DNA（cDNA），该抗体与非特异性交叉反应抗原95（NCA-95）交叉反应，此抗体先前已制备并命名为CEA 79（γ2a，κ）。从这些cDNA中，构建了一个用于CEA79单链可变片段（scFv）的噬菌粒表达载体。酶联免疫吸附测定（ELISA）、竞争性ELISA和蛋白质印迹法证实，展示在噬菌体表面的scFv对CEA和NCA-95仍保持亲和力。然后我们测定了克隆的VH和VL cDNA的核苷酸序列。序列分析表明，CEA 79抗体的VH和VL分别代表小鼠重链“杂项”亚组和κ轻链“V”亚组的新成员。