Optimisation of the cultured ELISpot/Fluorospot technique for the selective investigation of SARS-CoV-2 reactive central memory T cells

埃利斯波特 启动(农业) 记忆T细胞 免疫学 T细胞 抗原 背景(考古学) 生物 表位 免疫系统 医学 植物 发芽 古生物学
作者
Jack R. Jerome,Kirsty Wilson,Joshuah Fialho,Georgia Goodchild,Monica D. Prakash,Charlie McLeod,Peter Richmond,Vasso Apostolopoulos,Katie L. Flanagan,Magdalena Plebanski
出处
期刊:Frontiers in Immunology [Frontiers Media]
卷期号:16
标识
DOI:10.3389/fimmu.2025.1547220
摘要

Introduction This study presents an optimised cultured ELISpot protocol for detecting central memory T-cell interferon gamma (IFNγ) responses against SARS-CoV-2 peptides following an initial priming with either peptides, or whole spike protein. Methods Key variations optimised include the culture length, timing of exogenous survival signals (IL-2), and endpoint analysis modality and cell density to enhance assay sensitivity without compromising specificity for central memory T-cell IFNγ recall responses to cognate antigen. Results We noted a culture duration of 10 days, combined with a delayed IL-2 administration on day 5 to enhance assay sensitivity while maintaining response specificity towards cognate antigen when compared with shorter culture periods or earlier exogenous survival signal provision. With regards to lower-frequency T-cell interactions, as we observed with our donor SARS-CoV-2 epitope responses, our findings suggest Fluorospot to be preferable to the chromogenic ELISpot modality, and an immediate cell washing after culture collection to better facilitate cognate antigen responses. Fluorospot enabled a higher cell density while minimising the generation of visual artefacts, meanwhile immediate cell washing was critical for improving endpoint assay sensitivity. CCR7+ cell depletion was used to demonstrate our optimised protocol to selectively demonstrate central memory T-cell responses. Lastly, we provide evidence for the capacity of our assay to delineate individual responding peptides following peptide pool priming, and to explore cross-reactivity between viral variant peptides. Conclusion This work advances the methodology for investigating T-cell immunity, particularly in the context of SARS-CoV-2, and emphasises the balance between enhancing specific cognate central memory responses while limiting non-specific activation.

科研通智能强力驱动
Strongly Powered by AbleSci AI
更新
PDF的下载单位、IP信息已删除 (2025-6-4)

科研通是完全免费的文献互助平台,具备全网最快的应助速度,最高的求助完成率。 对每一个文献求助,科研通都将尽心尽力,给求助人一个满意的交代。
实时播报
磨人的老妖精完成签到,获得积分10
刚刚
yw完成签到,获得积分10
2秒前
H-China完成签到,获得积分20
2秒前
木木发布了新的文献求助10
2秒前
开心完成签到 ,获得积分10
3秒前
小怪兽完成签到,获得积分10
3秒前
火星上白风完成签到,获得积分10
4秒前
asheng98完成签到 ,获得积分10
4秒前
5秒前
77发布了新的文献求助10
5秒前
clm完成签到 ,获得积分10
6秒前
小野菌完成签到,获得积分10
6秒前
董晴完成签到,获得积分20
6秒前
6秒前
7秒前
丘比特应助tuzhihong采纳,获得10
7秒前
Winks完成签到,获得积分10
8秒前
8秒前
8秒前
storm完成签到,获得积分10
9秒前
xuzj应助KK采纳,获得10
9秒前
9秒前
10秒前
张德兆发布了新的文献求助30
10秒前
善学以致用应助yang采纳,获得20
11秒前
CodeCraft应助顶级牛马采纳,获得10
11秒前
小土豆完成签到,获得积分10
11秒前
12秒前
12秒前
12秒前
sb完成签到,获得积分10
13秒前
Stageruner完成签到,获得积分10
13秒前
腼腆的耷完成签到,获得积分10
13秒前
银河唯一的秘密完成签到,获得积分10
13秒前
14秒前
billevans完成签到,获得积分10
14秒前
王存臻关注了科研通微信公众号
14秒前
小魏哥哥完成签到,获得积分10
14秒前
DJDJ发布了新的文献求助10
14秒前
CipherSage应助THEEVE采纳,获得10
15秒前
高分求助中
Picture Books with Same-sex Parented Families: Unintentional Censorship 1000
A new approach to the extrapolation of accelerated life test data 1000
ACSM’s Guidelines for Exercise Testing and Prescription, 12th edition 500
Nucleophilic substitution in azasydnone-modified dinitroanisoles 500
不知道标题是什么 500
Indomethacinのヒトにおける経皮吸収 400
Phylogenetic study of the order Polydesmida (Myriapoda: Diplopoda) 370
热门求助领域 (近24小时)
化学 材料科学 医学 生物 工程类 有机化学 生物化学 物理 内科学 纳米技术 计算机科学 化学工程 复合材料 遗传学 基因 物理化学 催化作用 冶金 细胞生物学 免疫学
热门帖子
关注 科研通微信公众号,转发送积分 3977208
求助须知:如何正确求助?哪些是违规求助? 3521439
关于积分的说明 11207911
捐赠科研通 3258380
什么是DOI,文献DOI怎么找? 1799052
邀请新用户注册赠送积分活动 878108
科研通“疑难数据库(出版商)”最低求助积分说明 806764