Monoclonal antibodies are commonly assumed to be monospecific, but anecdotal studies have reported genetic diversity in antibody heavy chain and light chain genes found within individual hybridomas. As the prevalence of such diversity has never been explored, we analyzed 185 random hybridomas, in a large multicenter dataset. The hybridomas analyzed were not biased towards those with cloning difficulties or known to have additional chains. Of the hybridomas we evaluated, 126 (68.1%) contained no additional productive chains, while the remaining 59 (31.9%) contained one or more additional productive heavy or light chains. The expression of additional chains degraded properties of the antibodies, including specificity, binding signal and/or signal-to-noise ratio, as determined by enzyme-linked immunosorbent assay and immunohistochemistry. The most abundant mRNA transcripts found in a hybridoma cell line did not necessarily encode the antibody chains providing the correct specificity. Consequently, when cloning antibody genes, functional validation of all possible VH and VL combinations is required to identify those with the highest affinity and lowest cross-reactivity. These findings, reflecting the current state of hybridomas used in research, reiterate the importance of using sequence-defined recombinant antibodies for research or diagnostic use.

通常认为单克隆抗体是单特异性的，但是传闻研究已经报道了在单个杂交瘤中发现的抗体重链和轻链基因的遗传多样性。由于从未研究过这种多样性的普遍性，因此我们在大型多中心数据集中分析了185个随机杂交瘤。分析的杂交瘤不会偏向克隆困难或已知具有其他链的杂交瘤。在我们评估的杂交瘤中，有126个（68.1％）不包含其他生产链，而其余59个（31.9％）包含一个或多个其他生产性重链或轻链。通过酶联免疫吸附测定和免疫组织化学测定，其他链的表达降低了抗体的特性，包括特异性，结合信号和/或信噪比。在杂交瘤细胞系中发现的最丰富的mRNA转录物不一定编码提供正确特异性的抗体链。因此，克隆抗体基因时，需要对所有可能的VH和VL组合进行功能验证，以鉴定具有最高亲和力和最低交叉反应性的抗体。这些发现反映了用于研究的杂交瘤的当前状态，重申了将序列确定的重组抗体用于研究或诊断用途的重要性。