开发和优化用于检测免疫功能的稀释全血 ELISpot 检测方法。

IF 1.6 4区 医学 Q4 BIOCHEMICAL RESEARCH METHODS
Ricardo F. Ungaro , Julie Xu , Tamara A. Kucaba , Mahil Rao , Christian B. Bergmann , Scott C. Brakenridge , Philip A. Efron , Michael D. Goodman , Robert W. Gould , Richard S. Hotchkiss , Muxuan Liang , Monty B. Mazer , Patrick W. McGonagill , Lyle L. Moldawer , Kenneth E. Remy , Isaiah R. Turnbull , Charles C. Caldwell , Vladimir P. Badovinac , Thomas S. Griffith
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引用次数: 0

摘要

败血症仍是全球死亡的主要原因之一,但目前尚无行之有效的免疫调节疗法。脓毒症患者免疫内型分层('SPIES')是一项前瞻性多中心观察性研究,目的是测试 ELISpot 作为一种功能性生物测定的实用性,专门测量细胞因子刺激后产生的细胞,以确定免疫抑制内型,预测脓毒症患者的临床结果,并测试潜在的临床开发免疫刺激剂。大多数 ELISpot 检测方案都要求在将 PBMC 纳入检测前对其进行分离。与此相反,我们开发了一种稀释全血(DWB)ELISpot 方案,该方案已在多个实验室得到验证。我们从健康献血者和脓毒症患者身上采集了肝素化全血,并在不同的刺激条件下进行了测试,以评估血液稀释度、刺激物浓度、血液储存和刺激时间对 ELISpot 检测的体内外 IFNγ 和 TNFα 产生的影响。我们展示了全血稀释度的动态范围,这些稀释度能使体内外细胞因子对刺激产生强有力的反应。此外,还可利用各种浓度的刺激物来诱导细胞因子的产生。进一步的修改表明,抗凝全血可在室温下保存 24 小时而不会失去明显的功能。最后,我们还证明体内外刺激可短至 4 小时,从而大大缩短了处理时间。这些数据证明了在各种刺激条件下使用 ELISpot 检测 DWB 内细胞功能能力的可行性,从而让临床医生了解脓毒症患者免疫失调的程度。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Development and optimization of a diluted whole blood ELISpot assay to test immune function

Sepsis remains a leading cause of death worldwide with no proven immunomodulatory therapies. Stratifying Patient Immune Endotypes in Sepsis (‘SPIES’) is a prospective, multicenter observational study testing the utility of ELISpot as a functional bioassay specifically measuring cytokine-producing cells after stimulation to identify the immunosuppressed endotype, predict clinical outcomes in septic patients, and test potential immune stimulants for clinical development. Most ELISpot protocols call for the isolation of PBMC prior to their inclusion in the assay. In contrast, we developed a diluted whole blood (DWB) ELISpot protocol that has been validated across multiple laboratories. Heparinized whole blood was collected from healthy donors and septic patients and tested under different stimulation conditions to evaluate the impact of blood dilution, stimulant concentration, blood storage, and length of stimulation on ex vivo IFNγ and TNFα production as measured by ELISpot. We demonstrate a dynamic range of whole blood dilutions that give a robust ex vivo cytokine response to stimuli. Additionally, a wide range of stimulant concentrations can be utilized to induce cytokine production. Further modifications demonstrate anticoagulated whole blood can be stored up to 24 h at room temperature without losing significant functionality. Finally, we show ex vivo stimulation can be as brief as 4 h allowing for a substantial decrease in processing time. The data demonstrate the feasibility of using ELISpot to measure the functional capacity of cells within DWB under a variety of stimulation conditions to inform clinicians on the extent of immune dysregulation in septic patients.

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来源期刊
CiteScore
4.10
自引率
0.00%
发文量
120
审稿时长
3 months
期刊介绍: The Journal of Immunological Methods is devoted to covering techniques for: (1) Quantitating and detecting antibodies and/or antigens. (2) Purifying immunoglobulins, lymphokines and other molecules of the immune system. (3) Isolating antigens and other substances important in immunological processes. (4) Labelling antigens and antibodies. (5) Localizing antigens and/or antibodies in tissues and cells. (6) Detecting, and fractionating immunocompetent cells. (7) Assaying for cellular immunity. (8) Documenting cell-cell interactions. (9) Initiating immunity and unresponsiveness. (10) Transplanting tissues. (11) Studying items closely related to immunity such as complement, reticuloendothelial system and others. (12) Molecular techniques for studying immune cells and their receptors. (13) Imaging of the immune system. (14) Methods for production or their fragments in eukaryotic and prokaryotic cells. In addition the journal will publish articles on novel methods for analysing the organization, structure and expression of genes for immunologically important molecules such as immunoglobulins, T cell receptors and accessory molecules involved in antigen recognition, processing and presentation. Submitted full length manuscripts should describe new methods of broad applicability to immunology and not simply the application of an established method to a particular substance - although papers describing such applications may be considered for publication as a short Technical Note. Review articles will also be published by the Journal of Immunological Methods. In general these manuscripts are by solicitation however anyone interested in submitting a review can contact the Reviews Editor and provide an outline of the proposed review.
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