A rare antibody that is able to tolerate physio-chemical factors is preferred and highly demanded in diagnosis and therapy. Rabbit monoclonal antibodies (RmAbs) are distinguished owing to their high affinity and stability. However, the efficiency and availability of traditional methods for RmAb discovery are limited, particularly for small molecules. Here, we present an indirect competitive screening method in nanowells, named CSMN, for single rabbit antibody-secreting cells (ASCs) selection with 20.6 h and propose an efficient platform for RmAb production against small molecules within 5.8 days for the first time. Chloramphenicol (CAP) as an antibacterial agent poses a great threat to public health. We applied CSMN to select CAP-specific ASCs and produced one high-affinity RmAb, surprisingly showed extremely halophilic properties with an IC₅₀ of 0.08 ng mL⁻¹ in the saturated salt solution, which has not yet been seen for other antibodies. The molecular dynamic simulation showed that the negatively charged surface improved the stability of the RmAb structure with additional disulfide bonds compared with mouse antibodies. Moreover, the reduced solvent accessible surface area of the binding pocket increased the interactions of RmAb with CAP in a saturated salt solution. Furthermore, RmAb was used to develop an immunoassay for the detection of CAP in real biological samples with simple pretreatment, shorter assay time, and higher sensitivity. The results demonstrated that the practical and efficient CSMN is suitable for rare RmAb discovery against small molecules.

中文翻译：

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基于单个半抗原特异性兔抗体分泌细胞的间接竞争筛选的罕见单克隆抗体发现

能够耐受理化因素的稀有抗体在诊断和治疗中是首选和高度需要的。兔单克隆抗体 (RmAb) 因其高亲和力和稳定性而著称。然而，用于 RmAb 发现的传统方法的效率和可用性是有限的，特别是对于小分子。在这里，我们提出了一种名为 CSMN 的纳米孔间接竞争筛选方法，用于在 20.6 小时内选择单个兔抗体分泌细胞 (ASC)，并首次提出了一个在 5.8 天内针对小分子生产 RmAb 的有效平台。氯霉素（CAP）作为一种抗菌剂对公众健康构成了巨大威胁。我们应用 CSMN 来选择 CAP 特异性 ASC，并产生了一种高亲和力的 RmAb，令人惊讶的是，它在 IC 上表现出极其嗜盐的特性饱和盐溶液中 0.08 ng mL ^-1的₅₀倍，这在其他抗体中尚未见到。分子动力学模拟表明，与小鼠抗体相比，带负电荷的表面通过额外的二硫键提高了 RmAb 结构的稳定性。此外，结合袋的溶剂可及表面积减少，增加了 RmAb 与 CAP 在饱和盐溶液中的相互作用。此外，RmAb 被用于开发一种用于检测真实生物样品中 CAP 的免疫测定法，该方法具有简单的预处理、更短的测定时间和更高的灵敏度。结果表明，实用且高效的 CSMN 适用于针对小分子的稀有 RmAb 发现。