Department of Pharmaceutical Sciences, University of Michigan, Ann Arbor, Michigan, United States of America.
BioInterfaces Institute, University of Michigan, Ann Arbor, Michigan, United States of America.
PLoS One. 2021 Oct 11;16(10):e0252558. doi: 10.1371/journal.pone.0252558. eCollection 2021.
The identification of antibody variable regions in the heavy (VH) and light (VL) chains from hybridomas is necessary for the production of recombinant, sequence-defined monoclonal antibodies (mAbs) and antibody derivatives. This process has received renewed attention in light of recent reports of hybridomas having unintended specificities due to the production of non-antigen specific heavy and/or light chains for the intended antigen. Here we report a surprising finding and potential pitfall in variable domain sequencing of an anti-human CD63 hybridoma. We amplified multiple VL genes from the hybridoma cDNA, including the well-known aberrant Sp2/0 myeloma VK and a unique, full-length VL. After finding that the unique VL failed to yield a functional antibody, we discovered an additional full-length sequence with surprising similarity (~95% sequence identify) to the non-translated myeloma kappa chain but with a correction of its key frameshift mutation. Expression of the recombinant mAb confirmed that this highly homologous sequence is the antigen-specific light chain. Our results highlight the complexity of PCR-based cloning of antibody genes and strategies useful for identification of correct sequences.
鉴定杂交瘤中的重链（VH）和轻链（VL）抗体可变区对于生产重组、序列定义的单克隆抗体（mAbs）和抗体衍生物是必要的。由于产生了非抗原特异性的重链和/或轻链，最近有报道称杂交瘤具有意外的特异性，这一过程引起了人们的关注。在这里，我们报告了一个出人意料的发现，并指出了抗人 CD63 杂交瘤可变区测序中的潜在陷阱。我们从杂交瘤 cDNA 中扩增了多个 VL 基因，包括众所周知的异常 Sp2/0 骨髓瘤 VK 和独特的全长 VL。在发现独特的 VL 未能产生功能性抗体后，我们发现了一个额外的全长序列，与非翻译的骨髓瘤 κ 链具有惊人的相似性（~95%序列同一性），但纠正了其关键的移码突变。重组 mAb 的表达证实了这个高度同源的序列是抗原特异性的轻链。我们的结果强调了基于 PCR 的抗体基因克隆的复杂性，以及用于鉴定正确序列的有用策略。
