Kirchenbaum Greg A, Pawelec Graham, Lehmann Paul V
Research and Development, Cellular Technology Ltd. (CTL), Shaker Heights, OH 44122, USA.
Department of Immunology, University of Tübingen, D-72076 Tübingen, Germany.
Cells. 2025 Feb 5;14(3):223. doi: 10.3390/cells14030223.
Determining an individual's humoral immune reactivity to a pathogen, autoantigen, or environmental agent is traditionally accomplished through the assessment of specific antibody levels in blood. However, in many instances, titers of specific antibodies decline over time and thus do not faithfully reveal prior antigen exposure or establishment of immunological memory. To estimate an individual's humoral immune competence, it is therefore necessary to assess functional B cell memory. Here, we describe novel B cell ELISPOT and FluoroSpot assays (collectively referred to as ImmunoSpot) that can be rapidly developed and validated to characterize the memory B cell (B) repertoire specific for any desired antigen ex vivo and at single-cell resolution. Moreover, multiplexed variants of the B cell FluoroSpot assay enable high-throughput testing of antigen-specific B cells secreting distinct antibody classes and/or IgG subclasses, with minimal cell material requirements. B cell ImmunoSpot assays also enable measurement of affinity distributions within the antigen-specific B compartment and permit cross-reactivity measurements that can provide insights into B established against future pathogen variants. Collectively, the ImmunoSpot system presented here is highly reproducible, and can be readily validated for regulated tests. The newly gained ability to monitor the antigen-specific B compartment should catalyze a more comprehensive understanding of humoral immunity in health and disease.
传统上，通过评估血液中的特异性抗体水平来确定个体对病原体、自身抗原或环境因子的体液免疫反应性。然而，在许多情况下，特异性抗体的滴度会随时间下降，因此不能如实反映先前的抗原暴露或免疫记忆的建立。因此，为了评估个体的体液免疫能力，有必要评估功能性B细胞记忆。在此，我们描述了新型B细胞ELISPOT和FluoroSpot检测方法（统称为免疫斑点检测），这些方法可以快速开发和验证，以在体外以单细胞分辨率表征针对任何所需抗原的记忆B细胞（B细胞）库。此外，B细胞FluoroSpot检测的多重变体能够以最少的细胞材料需求，对抗原特异性B细胞分泌不同抗体类别和/或IgG亚类进行高通量检测。B细胞免疫斑点检测还能够测量抗原特异性B细胞区内的亲和力分布，并允许进行交叉反应性测量，从而为针对未来病原体变体建立的B细胞提供见解。总体而言，本文介绍的免疫斑点检测系统具有高度的可重复性，并且可以很容易地针对受监管的检测进行验证。新获得的监测抗原特异性B细胞区的能力应该会促进对健康和疾病中体液免疫的更全面理解。
