Identification of human antibody fragment clones specific for tetanus toxoid in a bacteriophage lambda immunoexpression library.
Identification of human antibody fragment clones specific for tetanus toxoid in a bacteriophage lambda immunoexpression library.
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噬菌体 lambda 免疫表达文库中破伤风类毒素特异性人抗体片段克隆的鉴定。
DOI：
10.1073/pnas.87.20.8095
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发表时间：
1990
影响因子：
11.1
通讯作者：
Short,JM
中科院分区：
文献类型：
--
作者：
Mullinax,RL;Gross,EA;Amberg,JR;Hay,BN;Hogrefe,HH;Kubitz,MM;Greener,A;Alting-Mees,M;Ardourel,D;Short,JM
我们应用分子生物学方法鉴定人单克隆抗体。将人外周血淋巴细胞mRNA转化为cDNA，并通过聚合酶链反应扩增选择的子集。这些产品，含有许多免疫球蛋白重链和κ轻链可变区和恒定区结构域的编码序列，被插入到修饰的噬菌体λ表达载体中，并通过感染引入大肠杆菌，以产生组合免疫表达文库。通过与放射性标记抗原的过滤杂交鉴定具有破伤风类毒素结合活性的克隆，并以0.2%的频率出现在文库中。这些人抗原结合片段由共价连接至轻链的重链片段组成，显示出与破伤风类毒素结合的高亲和力，平衡常数在纳摩尔范围内，但不与测试的其他蛋白质交叉反应。我们估计这个人免疫表达文库包含20，000个对我们选择的抗原具有高亲和力和特异性的克隆。
We have applied a molecular biology approach to the identification of human monoclonal antibodies. Human peripheral blood lymphocyte mRNA was converted to cDNA and a select subset was amplified by the polymerase chain reaction. These products, containing coding sequences for numerous immunoglobulin heavy- and kappa light-chain variable and constant region domains, were inserted into modified bacteriophage lambda expression vectors and introduced into Escherichia coli by infection to yield a combinatorial immunoexpression library. Clones with binding activity to tetanus toxoid were identified by filter hybridization with radiolabeled antigen and appeared at a frequency of 0.2% in the library. These human antigen binding fragments, consisting of a heavy-chain fragment covalently linked to a light chain, displayed high affinity of binding to tetanus toxoid with equilibrium constants in the nanomolar range but did not cross-react with other proteins tested. We estimate that this human immunoexpression library contains 20,000 clones with high affinity and specificity to our chosen antigen.