Persson Nina, Jansson Bo, Stuhr-Hansen Nicolai, Kovács András, Welinder Charlotte, Danielsson Lena, Blixt Ola
Chemical Glyco-Biology Laboratory, Department of Chemistry, Copenhagen University, Copenhagen, Denmark.
Division of Oncology and Pathology, Department of Clinical Sciences Lund, Lund University, Lund, Sweden.
PLoS One. 2016 Dec 21;11(12):e0168761. doi: 10.1371/journal.pone.0168761. eCollection 2016.
We have developed a combinatory antibody-antigen microarray for direct screening of multiple single-chain fragment variable (scFv) clones with no need for pre-purification or enrichment before screening. The straightforward workflow allows for early selection of binders to predefined peptide and glycopeptide targets. A capture antibody is contact printed on microarray slides, side by side with the antigens of interest. A large number of scFv clones, in supernatants, are printed on top of the capture antibody and the antigen in a "spot-on-spot" print. The printed scFv clones, which bind to the capture antibody, are detected using biotinylated antigen, while the binding of scFv clones to the printed antigen is detected through a mouse anti-tag antibody. Two different analyses are thus performed on the same slide, generating two kinds of information: one on the ability of an individual scFv clone to bind to the soluble form of the antigen, which may favour selection for higher affinity rather than avidity, while the other allows the identification of large numbers of clones, simultaneously, due to the binding of scFv clones to densely presented antigens, thus providing an overall increased hit rate. The functionality of the new screening approach was illustrated through the generation of antibodies against peptides from the chaperone complex Ku70/Ku80 and the GalNAcα-serine/threonine epitope on the IgA1 alpha chain hinge region. In total, 659 scFv clones were screened with a hit rate of approximately 20%. This approach allowed the identification of functional antibodies in both cases, illustrating the usefulness and capacity of this combinatory microarray screening technique for efficient analysis and validation of antibodies at an early stage of antibody generation.
我们开发了一种组合式抗体 - 抗原微阵列，用于直接筛选多个单链可变片段（scFv）克隆，筛选前无需预纯化或富集。这种简单直接的工作流程能够早期筛选出与预定义肽和糖肽靶标结合的抗体。将捕获抗体接触式打印在微阵列载玻片上，与感兴趣的抗原并排。大量处于上清液中的scFv克隆以“点上点”的方式打印在捕获抗体和抗原之上。与捕获抗体结合的打印scFv克隆，使用生物素化抗原进行检测，而scFv克隆与打印抗原的结合则通过小鼠抗标签抗体进行检测。因此，在同一张载玻片上进行两种不同的分析，产生两种信息：一种是关于单个scFv克隆与抗原可溶性形式结合的能力，这可能有利于选择高亲和力而非高亲合力的克隆；另一种则由于scFv克隆与高密度呈现的抗原结合，能够同时鉴定大量克隆，从而总体提高命中率。通过生成针对伴侣蛋白复合物Ku70/Ku80的肽段以及IgA1α链铰链区GalNAcα - 丝氨酸/苏氨酸表位的抗体，展示了这种新筛选方法的功能。总共筛选了659个scFv克隆，命中率约为20%。这种方法在两种情况下都能鉴定出功能性抗体，说明了这种组合式微阵列筛选技术在抗体生成早期阶段进行高效抗体分析和验证的实用性和能力。
