<meta charset="utf8"></head><body>用于分析真菌和病毒免疫的优化全血免疫检测

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>7-AAD Solution</td>
			<td>Miltenyi Biotec</td>
			<td>130-111-568</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-IFN-g-PE, human, REA600, 100 tests</td>
			<td>Miltenyi Biotec</td>
			<td>130-113-498</td>
			<td></td>
		</tr>
		<tr>
			<td>Aspergillus fumigatus lysate</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Kindly provided by the Hans-Knoell Institute Jena, Germany</td>
		</tr>
		<tr>
			<td>Brilliant Violet 650 anti-human CD197 (CCR7) Antibody</td>
			<td>BioLegend</td>
			<td>353234</td>
			<td></td>
		</tr>
		<tr>
			<td>Buffer EL</td>
			<td>Qiagen</td>
			<td>79217</td>
			<td>Erythrocyte lysis buffer</td>
		</tr>
		<tr>
			<td>CD154-PE-Vio770, human, REA238, 100 tests</td>
			<td>Miltenyi Biotec</td>
			<td>130-113-614</td>
			<td></td>
		</tr>
		<tr>
			<td>CD279 (PD1)-VioBright 515, human, 100 tests</td>
			<td>Miltenyi Biotec</td>
			<td>130-120-386</td>
			<td></td>
		</tr>
		<tr>
			<td>CD28 pure, human - functional grade</td>
			<td>Miltenyi Biotec</td>
			<td>130-093-375</td>
			<td></td>
		</tr>
		<tr>
			<td>CD3-VioBright R720, human, REA613, 100 tests</td>
			<td>Miltenyi Biotec</td>
			<td>130-127-377</td>
			<td></td>
		</tr>
		<tr>
			<td>CD45RO-APC-Vio770, human, REA611, 100 tests</td>
			<td>Miltenyi Biotec</td>
			<td>130-113-557</td>
			<td></td>
		</tr>
		<tr>
			<td>CD49d pure, human</td>
			<td>Miltenyi Biotec</td>
			<td>130-093-279</td>
			<td></td>
		</tr>
		<tr>
			<td>CD4-VioBlue, human, REA623, 100 tests</td>
			<td>Miltenyi Biotec</td>
			<td>130-114-534</td>
			<td></td>
		</tr>
		<tr>
			<td>CD69-PE-Vio615, human, REA824, 100 tests</td>
			<td>Miltenyi Biotec</td>
			<td>130-112-617</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 Incubator</td>
			<td>PHCbi</td>
			<td>MCO-170AICD-PE</td>
			<td></td>
		</tr>
		<tr>
			<td>CPI Positive Control Solution</td>
			<td>ImmunoSpot</td>
			<td>CTL-CPI-001</td>
			<td></td>
		</tr>
		<tr>
			<td>CRX-527</td>
			<td>Invivogen</td>
			<td>tlrl-crx527</td>
			<td></td>
		</tr>
		<tr>
			<td>CytExpert Acquisition and Analysis Software</td>
			<td>Beckman Coulter</td>
			<td>Version 2.4</td>
			<td>Flow cytometer operating softwware</td>
		</tr>
		<tr>
			<td>CytoFlex S B2-R3-V4-Y4</td>
			<td>Beckman Coulter</td>
			<td>B75408</td>
			<td>Flow cytometer</td>
		</tr>
		<tr>
			<td>GraphPad Prism</td>
			<td>GraphPad</td>
			<td>Version 10.1.0</td>
			<td></td>
		</tr>
		<tr>
			<td>Herpes simplex virus 1 lysate</td>
			<td>AID Autoimmun Diagnostika</td>
			<td>ELSP 5916K</td>
			<td></td>
		</tr>
		<tr>
			<td>HU IFN G Uncoated ELISA 2X96T PLT</td>
			<td>Thermo Fisher Scientific</td>
			<td>88-7316-22</td>
			<td></td>
		</tr>
		<tr>
			<td>HydroFLex Microplate Washer</td>
			<td>Tecan</td>
			<td>30220085</td>
			<td></td>
		</tr>
		<tr>
			<td>Infinite M Plex</td>
			<td>Tecan</td>
			<td>30213614</td>
			<td>Multimode Microplate Reader</td>
		</tr>
		<tr>
			<td>Kaluza Analysis Software</td>
			<td>Beckman Coulter</td>
			<td>Version 2.1.00003.20057</td>
			<td>Flow cytometric data analysis software</td>
		</tr>
		<tr>
			<td>MACS Inside Stain Kit</td>
			<td>Miltenyi Biotec</td>
			<td>130-090-477</td>
			<td></td>
		</tr>
		<tr>
			<td>Mastercycler X50l</td>
			<td>Eppendorf</td>
			<td>6303000010</td>
			<td></td>
		</tr>
		<tr>
			<td>Nanodrop One</td>
			<td>Thermo Fisher Scientific</td>
			<td>ND-ONE-W</td>
			<td></td>
		</tr>
		<tr>
			<td>nCounter Sprint Cartridge</td>
			<td>Nanostring</td>
			<td>100078</td>
			<td></td>
		</tr>
		<tr>
			<td>nCounter Sprint Profiler</td>
			<td>Nanostring</td>
			<td>100170</td>
			<td></td>
		</tr>
		<tr>
			<td>nCounter Sprint Reagent Pack</td>
			<td>Nanostring</td>
			<td>100077</td>
			<td></td>
		</tr>
		<tr>
			<td>nSolver</td>
			<td>Nanostring</td>
			<td>Version 4.0</td>
			<td>Nanostring nCounter data analysis software</td>
		</tr>
		<tr>
			<td>Octeniderm farblos 250 ml FL</td>
			<td>Schuelke</td>
			<td>118211</td>
			<td></td>
		</tr>
		<tr>
			<td>Omnifix F Solo, 1 ml, ohne Kan&#252;le, 3-teilig</td>
			<td>Braun</td>
			<td>9161406V</td>
			<td>Syringes</td>
		</tr>
		<tr>
			<td>PepTivator CMV pp65, human, 60nmol</td>
			<td>Miltenyi Biotec</td>
			<td>130-093-435</td>
			<td></td>
		</tr>
		<tr>
			<td>PepTivator SARS-CoV-2 Prot_S, research grade, for stimulation of 1&#215;108 cells</td>
			<td>Miltenyi Biotec</td>
			<td>130-126-700</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAshredder</td>
			<td>Qiagen</td>
			<td>79654</td>
			<td></td>
		</tr>
		<tr>
			<td>RNA Protect</td>
			<td>Qiagen</td>
			<td>76526</td>
			<td></td>
		</tr>
		<tr>
			<td>RNeasy Plus Mini Kit</td>
			<td>Qiagen</td>
			<td>74134</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640 Medium, GlutaMAX Supplement, HEPES</td>
			<td>Thermo Fisher Scientific</td>
			<td>72400047</td>
			<td></td>
		</tr>
		<tr>
			<td>Safe 2020 1.5 Microbiological Safety Cabinet</td>
			<td>Thermo Fisher Scientific</td>
			<td>51026959</td>
			<td></td>
		</tr>
		<tr>
			<td>S-Monovette lithium-heparin, 4.9 ml</td>
			<td>Sarstedt</td>
			<td>04.1939.001</td>
			<td>Blood collection tubes</td>
		</tr>
		<tr>
			<td>S-Monovette neutral, 2.7 ml</td>
			<td>Sarstedt</td>
			<td>05.1729</td>
			<td>Stimulation environment tubes</td>
		</tr>
		<tr>
			<td>Sterican Safety G 19 x 1 1/2&#39;&#39; 1,1 x 40 mm</td>
			<td>Braun</td>
			<td>4670052S-01</td>
			<td>Needles</td>
		</tr>
		<tr>
			<td>Stop Solution, 100 ML</td>
			<td>Thermo Fisher Scientific</td>
			<td>BMS409.0100</td>
			<td></td>
		</tr>
		<tr>
			<td>Wash Buffer 20X, 500 ML</td>
			<td>Thermo Fisher Scientific</td>
			<td>BMS408.0500</td>
			<td></td>
		</tr>
		<tr>
			<td>Water, 1 l</td>
			<td>Carl Roth</td>
			<td>3478.1</td>
			<td></td>
		</tr>
		<tr>
			<td>XT Hs Exhaustion CSO</td>
			<td>Nanostring</td>
			<td>115000466</td>
			<td>Nanostring Immune Exhaustion Panel</td>
		</tr>
	</tbody>
</table>

whole blood assay with dual co-stimulation for antigen-specific analysis of host immunity to fungal and viral pathogens

Quantification and characterization of antigen-specific immunity, especially specific T-cell responses, is pivotal for immunobiology and vaccination research, as well as some diagnostic tests. Traditionally, antigen-specific immunoassays commonly relied on isolated peripheral blood mononuclear cells (PBMCs). However, isolation of these cells is time-consuming and resource-intensive and often requires relatively large blood volumes. Additionally, to prevent granulocyte activation and subsequent T-cell disturbance during pre-analytic storage1 rapid processing of the samples is paramount, which is often not feasible in clinical practice. These limitations hamper the practicability of antigen-specific immunoassays in high-throughput research scenarios and clinical routines. Therefore, the development of easy-to-use and potentially automatable whole blood-based approaches in recent years has opened new areas of immunoassay applications. However, current commercially available systems usually lack optimal co-stimulatory environments for T-cells and are susceptible to pre-analytic delays. For instance, a widely used whole blood-based IFN-&#947; release assay has a 19% positive to negative reversion rate after 6 h of pre-analytic blood storage2. Optimized protocols with dual anti-CD28 and anti-CD49d co-stimulation have been developed to overcome these limitations3,4,5,6.
The protocol presented here allows for accurate and reproducible quantification and characterization of antigen-specific T-cells, assessment of antigen-induced cytokine responses, and other (flow cytometric or transcriptional) functional immune markers from minimal blood volume, i.e., 500 &#181;L of blood per stimulation tube. Further advantages of this protocol include low hands-on time, high resilience to pre-analytic confounders, and preservation of functional inter-cellular interactions in a relatively physiological ex vivo environment. The comparability of whole blood-based flow cytometric antigen-specific&#160;T-cell characterization with data generated from traditional PBMC-based assays has been previously shown in the context of mold-specific T-cell quantification6. Furthermore, direct stimulation of the subjects&#39; blood abrogates the need for supplementation with autologous, allogenic, or even xenogeneic serum that is commonly required for optimal PBMC stimulation. Omission of cell isolation also reduces shear and temperature stress, thereby improving cell viability. Most importantly, whole blood-based assays preserve granulocyte populations that are lost during gradient centrifugation for isolation of PBMCs7. Thereby, this assay setup preserves and captures functional interaction loops between granulocytes and mononuclear cells4.
Of note, this protocol requires only minimal modifications to accommodate different readout modalities and even allows for dual analysis of cytokine release and transcriptional responses from the same stimulation tube. Specifically, while cytokines are analyzed from the culture supernatant after stimulation, the cell pellet can be used for RNA isolation with subsequent transcriptomic analysis. The general workflow for the various readout modalities is summarized in Figure 1.
In recent years, an increasing number of whole blood-based assays has been developed for pathogen-reactive immune monitoring in research and clinical settings, e.g., for Mycobacterium tuberculosis8,9, Bordetella pertussis3, Orientia tsutsugamushi10, and SARS-CoV-25,11,12. For instance, a previously established system has been used for multiple antigens, including M. tuberculosis, Influenza A virus, and SARS-CoV-2, but does not use co-stimulatory factors optimized for T-helper (Th) cell stimulation13,14,15. Even though the blood volume required for these assays is already significantly lower than that used for traditional PBMC-based assays or commercially available whole blood stimulation kits, an even smaller sample volume might be warranted for applications in pediatrics, neonatology, patients in the intensive care unit (ICU), and preclinical research in small animal models. For instance, even terminal blood sampling from mice (e.g., by cardiac puncture) commonly yields a maximum of 0.7-1 mL of blood. Thus, the possibility to further downscale previously established whole blood-based immunoassay protocols4,6 for precise quantification and characterization of antigen-reactive T-cell responses from 250 &#181;L of blood volume per stimulation tube has been evaluated as part of this protocol.

<meta charset="utf8"></head><body>作者聚焦：使用全血检测推进重症监护患者的免疫监测

具有双重共刺激的全血测定，用于宿主对真菌和病毒病原体免疫的抗原特异性分析

Our research focuses on the immune monitoring of patients in the intensive care unit. Some patients develop severe viral pneumonia during mechanical ventilation. We isolate the cause of the viruses, but also characterize the local and systemic immune response using the protocol presented in this video to identify immunological risk factors.Traditional antigen-specific T-cell assays that are based on isolated mononuclear cells are highly susceptible to pre-analytical confounders, time-and resource-intensive, and require large amounts of blood. This poses severe limitations to the implementation of antigen-specific T-cell assays in the clinical research or clinical routine use as prognostic or diagnostic biomarkers. We present a versatile immunoassay protocol, which accommodates various downstream readouts from as little as 250 micros blood per stimulus.This enables applications, even when that volume is limited or several antigens need to be screened. Furthermore, our assay is easy to perform and amenable to several pathogens. In addition to its versatility and the low blood volume required, our protocol used as stimulation environment that was thoroughly and specifically optimized for antigen-induced T-helper cell responses.This was achieved through dual core stimulation, which also makes the protocol more robust towards confounders, such as immunosuppressive pharmacotherapy or pre-analytical delays. We are using this protocol for immune monitoring of patients with ventilation-associated viral pneumonia. Our collaborators in Wuerzburg and Houston are working on a clinical translation of cytomegalovirus-specific immunoassays.Furthermore, they're using this protocol for studies on fungal immunopathogenesis and preclinical immunotherapy studies in mouse models for opportunistic infections.

Rapid and resource-efficient sample processing, high throughput, and high robustness are critical for effective scientific and clinical application of ...

whole-blood-assay-with-dual-co-stimulation-for-antigen-specific

Research

JoVE Journal

Immunology and Infection

Whole Blood Assay with Dual Co-Stimulation for Antigen-Specific Analysis of Host Immunity to Fungal and Viral Pathogens

421 次观看.  University Hospital Augsburg. 我们的研究重点是重症监护病房患者的免疫监测。一些患者在机械通气期间发展为严重的病毒性肺炎。我们隔离了病毒的原因，但也使用本视频中介绍的方案来表征局部和全身免疫反应，以确定免疫学风险因素。基于分离的单核细胞的传统抗原特异性 T 细胞检测极易受到分析前混杂因素的影响，耗费时间和资源，并且需要大量血液。这严重限制...