Department of Immunology, DEPBIO, Faculty of Chemistry, Hygiene Institute, UDELAR, Av. A. Navarro 3051, 11600, Montevideo, Uruguay.
Parque Lecocq, IM, 12600, Montevideo, Uruguay.
N Biotechnol. 2021 Sep 25;64:9-16. doi: 10.1016/j.nbt.2021.05.002. Epub 2021 May 11.
Nanobodies are the smallest antibody fragments which bind to antigens with high affinity and specificity. Due to their outstanding physicochemical stability, simplicity and cost-effective production, nanobodies have become powerful agents in therapeutic and diagnostic applications. In this work, the advantages of nanobodies were exploited to develop generic and standardized anti-human IgM reagents for serology and IgM B-cell analysis. Selection of anti-IgM nanobodies was carried out by evaluating their yields, stability, binding kinetics and cross-reactivity with other Ig isotypes. High affinity nanobodies were selected with dissociation constants (KDs) in the nM range and high sensitivities for detection of total IgM by ELISA. The nanobodies also proved to be useful for capturing IgM in the serodiagnosis of an acute infection as demonstrated by detection of specific IgM in sera of dengue virus patients. Finally, due to the lack of an Fc region, the selected nanobodies do not require Fc receptor blocking steps, facilitating the immunophenotyping of IgM cells by flow cytometry, an important means of diagnosis of immunodeficiencies and B-cell lymphoproliferative disorders. This work describes versatile anti-IgM nanobodies that, due to their recombinant nature and ease of reproduction at low cost, may represent an advantageous alternative to conventional anti-IgM antibodies in research and diagnosis.
纳米抗体是最小的抗体片段，能以高亲和力和特异性结合抗原。由于其出色的物理化学稳定性、简单性和具有成本效益的生产方式，纳米抗体已成为治疗和诊断应用中的强大工具。在这项工作中，利用纳米抗体的优势，开发了用于血清学和 IgM B 细胞分析的通用和标准化抗人 IgM 试剂。通过评估其产量、稳定性、结合动力学和与其他 Ig 同种型的交叉反应性，对抗 IgM 纳米抗体进行了选择。选择了具有纳摩尔级解离常数 (KD) 和高灵敏度的高亲和力纳米抗体，可通过 ELISA 检测总 IgM。这些纳米抗体还被证明可用于急性感染的血清诊断中捕获 IgM，如通过检测登革热病毒患者血清中的特异性 IgM 来证明。最后，由于缺乏 Fc 区，所选纳米抗体不需要 Fc 受体阻断步骤，这有助于通过流式细胞术对 IgM 细胞进行免疫表型分析，这是诊断免疫缺陷和 B 细胞淋巴增生性疾病的重要手段。这项工作描述了多功能的抗 IgM 纳米抗体，由于其重组性质和低成本的可重复性，它们可能是研究和诊断中传统抗 IgM 抗体的有利替代品。
