Rasooly Reuven, Do Paula, He Xiaohua, Hernlem Bradley
Foodborne Toxin Detection & Prevention Research Unit, Western Regional Research Center, Agricultural Research Service, United States Department of Agriculture, Albany, CA 94710, USA.
Toxins (Basel). 2021 Apr 23;13(5):300. doi: 10.3390/toxins13050300.
Staphylococcal enterotoxin type B (SEB) is associated with food poisoning. Current methods for the detection of biologically active SEB rely upon its ability to cause emesis when administered to live kittens or monkeys. This technique suffers from poor reproducibility and low sensitivity and is ethically disfavored over concerns for the welfare of laboratory animals. The data presented here show the first successful implementation of an alternative method to live animal testing that utilizes SEB super-antigenic activity to induce cytokine production for specific novel cell-based assays for quantifiable detection of active SEB. Rather than using or sacrificing live animals, we found that SEB can bind to the major histocompatibility complex (MHC) class II molecules on Raji B-cells. We presented this SEB-MHC class II complex to specific Vβ5.3 regions of the human T-cell line HPB-ALL, which led to a dose-dependent secretion of IL-2 that is capable of being quantified and can further detect 10 pg/mL of SEB. This new assay is 100,000 times more sensitive than the ex vivo murine splenocyte method that achieved a detection limit of 1 µg/mL. The data presented here also demonstrate that SEB induced proliferation in a dose-dependent manner for cells obtained by three different selection methods: by splenocyte cells containing 22% of CD4 T-cells, by CD4 T-cells enriched to >90% purity by negative selection methods, and by CD4 T-cells enriched to >95% purity by positive selection methods. The highly enriched and positively isolated CD4 T-cells with the lowest concentration of antigen-presenting cells (APC) (below 5%) provided higher cell proliferation than the splenocyte cells containing the highest concentration of APC cells.
B型葡萄球菌肠毒素（SEB）与食物中毒有关。目前检测生物活性SEB的方法依赖于将其注射给活小猫或猴子时引发呕吐的能力。该技术存在重复性差和灵敏度低的问题，并且出于对实验动物福利的担忧，在伦理上不受青睐。本文所呈现的数据表明，首次成功实施了一种替代活体动物检测的方法，该方法利用SEB的超抗原活性来诱导细胞因子产生，用于基于特定新型细胞的活性SEB定量检测分析。我们发现SEB可以与Raji B细胞上的主要组织相容性复合体（MHC）II类分子结合，而不是使用或牺牲活体动物。我们将这种SEB-MHC II类复合体呈递给人T细胞系HPB-ALL的特定Vβ5.3区域，这导致了IL-2的剂量依赖性分泌，该分泌能够被定量，并且能够进一步检测到10 pg/mL的SEB。这种新的检测方法比离体小鼠脾细胞方法灵敏100,000倍，后者的检测限为1 μg/mL。本文所呈现的数据还表明，SEB对通过三种不同筛选方法获得的细胞以剂量依赖性方式诱导增殖：通过含有22% CD4 T细胞的脾细胞、通过阴性筛选方法富集至纯度>90%的CD4 T细胞以及通过阳性筛选方法富集至纯度>95%的CD4 T细胞。与含有最高浓度抗原呈递细胞（APC）的脾细胞相比，具有最低浓度APC（低于5%）的高度富集且经阳性分离的CD4 T细胞提供了更高的细胞增殖。
