Department of Environmental and Infectious Disease Sciences, American Registry of Pathology, and Armed Forces Institute of Pathology, Washington, DC 20306, USA.
J Immunol Methods. 2011 Sep 30;372(1-2):146-61. doi: 10.1016/j.jim.2011.07.009. Epub 2011 Jul 20.
To improve anti-Burkholderia monoclonal antibody (MAb) binding affinity, six single chain variable fragments (scFvs) constructed previously were used as scaffolds to construct large highly-diversified phage-displayed mouse scFv random and domain libraries. First, we employed random mutagenesis to introduce random point mutations into entire variable regions, generating six random libraries. Additionally, the oligonucleotide-directed mutagenesis was targeted on complementarity-determining region 3 (CDR3) from each variable region of heavy (VH) and light chains (VL) derived from six scFvs, and generated eighteen domain libraries including six VH CDR3, six VL CDR3, and six combined VH/VL CDR3 mutated domains, respectively. We collected high scFvs binders through panning experiment over the large (size ~1 × 10⁹) random and domain libraries. The quality of the libraries was validated by successful selection of high-affinity clones. Random mutagenesis generated many mutant scFv clones having more than one amino acid changes around framework regions, but not many in CDRs. Surprisingly, the resulting eight higher scFv binders were selected from CDR3 mutations, but not from random mutations. Six of them resulted from CDR3 mutations of light chain, except for two scFvs from heavy chain, showing both Burkholderia pseudomallei and Burkholderia mallei had preferentially influenced the VL CDR3. Furthermore, all eight higher scFvs converted to full format human IgG1 antibodies were expressed transiently in 293T cell line. Five chimeric MAbs showed improved higher binding activity, as much as 0.2-0.3 at O.D. 405 nm, than positive control MAbs. These libraries could be valuable sources for selection of anti-Burkholderia antibodies and discovery of the relevant epitope(s) for developing effective vaccines or therapeutics.
为了提高抗伯克霍尔德氏菌单克隆抗体（MAb）的结合亲和力，我们以前构建的六个单链可变片段（scFv）被用作支架，构建了大型高多样性的噬菌体展示鼠 scFv 随机和结构域文库。首先，我们采用随机诱变技术，在整个可变区引入随机点突变，生成了六个随机文库。此外，我们还针对来源于六个 scFv 的重链（VH）和轻链（VL）的可变区的互补决定区 3（CDR3）进行了寡核苷酸定向诱变，生成了包括六个 VH CDR3、六个 VL CDR3 和六个组合 VH/VL CDR3 突变结构域的 18 个结构域文库。我们通过对大型（大小约 1×10⁹）随机和结构域文库的淘选实验，收集了高 scFv 结合物。文库的质量通过成功选择高亲和力的克隆得到了验证。随机诱变产生了许多在框架区周围有一个以上氨基酸变化的突变 scFv 克隆，但在 CDR 中并没有那么多。令人惊讶的是，从 CDR3 突变中选择了八个更高亲和力的 scFv，而不是从随机突变中选择。其中六个来自轻链的 CDR3 突变，除了两个来自重链的 scFv，表明伯氏菌和类鼻疽伯氏菌都优先影响 VL CDR3。此外，所有八个更高亲和力的 scFv 都被转化为全长人 IgG1 抗体，在 293T 细胞系中瞬时表达。五个嵌合 MAb 表现出更高的结合活性，在 O.D.405nm 处高达 0.2-0.3，比阳性对照 MAb 更高。这些文库可以作为筛选抗伯克霍尔德氏菌抗体的有价值的来源，并发现相关的表位（s），以开发有效的疫苗或治疗方法。
