State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-Products, Ningbo University, Ningbo, China.
Laboratory of Biochemistry and Molecular Biology, School of Marine Sciences, Ningbo University, Ningbo, China.
Front Immunol. 2021 Nov 16;12:771231. doi: 10.3389/fimmu.2021.771231. eCollection 2021.
Akin to their mammalian counterparts, teleost fish possess a complex assortment of highly specialized immune cells that are capable of unleashing potent innate immune responses to eradicate or mitigate incoming pathogens, and also differentiate into memory lymphocytes to provide long-term protection. Investigations into specific roles and functions of fish immune cells depend on the precise separation of each cell type. Commonly used techniques, for example, density gradient centrifugation, rely on immune cells to have differing sizes or densities and thus fail to separate between similar cell types (e.g. T and B lymphocytes). Furthermore, a continuously growing database of teleost genomic information has revealed an inventory of cellular markers, indicating the possible presence of immune cell subsets in teleost fish. This further complicates the interpretation of results if subsets of immune cells are not properly separated. Consequently, monoclonal antibodies (mAbs) against specific cellular markers are required to precisely identify and separate novel subsets of immune cells in fish. In the field of fish immunology, mAbs are largely generated using the hybridoma technology, resulting in the development of mAbs against specific cellular markers in different fish species. Nevertheless, this technology suffers from being labour-intensive, time-consuming and most importantly, the inevitable loss of diversities of antibodies during the fusion of antibody-expressing B lymphocytes and myeloma cells. In light of this, the focus of this review is to discuss the potential applications of fluorescence-activated cell sorting and droplet-based microfluidics, two emerging technologies capable of screening and identifying antigen-specific B lymphocytes in a high-throughput manner, in promoting the development of valuable reagents for fish immunology studies. Our main goal is to encourage the incorporation of alternative technologies into the field of fish immunology to promote the production of specific antibodies in a high-throughput and cost-effective way, which could better allow for the precise separation of fish immune cells and also facilitate the identification of novel immune cell subsets in teleost fish.
类似于哺乳动物，硬骨鱼拥有复杂多样的高度特化免疫细胞，这些细胞能够引发强大的固有免疫反应来清除或减轻入侵病原体，还能分化为记忆淋巴细胞提供长期保护。鱼类免疫细胞的特定功能和作用的研究依赖于每种细胞类型的精确分离。常用技术，例如密度梯度离心，依赖于免疫细胞具有不同的大小或密度，因此无法分离相似的细胞类型（例如 T 和 B 淋巴细胞）。此外，硬骨鱼基因组信息的不断增长的数据库揭示了细胞标记物的清单，表明硬骨鱼中可能存在免疫细胞亚群。如果免疫细胞亚群没有得到适当分离，这将使结果的解释更加复杂。因此，需要针对特定细胞标记物的单克隆抗体（mAb）来精确识别和分离鱼类中新的免疫细胞亚群。在鱼类免疫学领域，mAb 主要使用杂交瘤技术产生，导致针对不同鱼类物种的特定细胞标记物的 mAb 的发展。然而，该技术存在劳动强度大、耗时且最重要的是，在表达抗体的 B 淋巴细胞和骨髓瘤细胞融合过程中抗体多样性不可避免的损失等缺点。有鉴于此，本文的重点是讨论荧光激活细胞分选和基于液滴的微流控这两种新技术在促进鱼类免疫学研究有价值试剂的发展方面的潜在应用，这两种技术能够以高通量的方式筛选和鉴定抗原特异性 B 淋巴细胞。我们的主要目标是鼓励将替代技术纳入鱼类免疫学领域，以高通量且具有成本效益的方式生产特异性抗体，这将更好地允许鱼类免疫细胞的精确分离，并有助于鉴定硬骨鱼中的新型免疫细胞亚群。
