Name: quantifying myeloperoxidase-dna and neutrophil elastase-dna complexes from neutrophil extracellular traps by using a modified sandwich elisa
Uploaded: 2023-05-12T12:25:00
Description: Watch this Scientific Journal Video about Quantifying Myeloperoxidase-DNA and Neutrophil Elastase-DNA Complexes from Neutrophil Extracellular Traps by Using a Modified Sandwich ELISA at JoVE.com

作者感谢Huq Muhammad Aminul博士在审阅手稿时提供的帮助。

这项研究是根据《赫尔辛基宣言》进行的，并得到了爱知医科大学机构审查委员会的批准（2017-H341,2019-H137）。从每个参与者那里获得了书面知情同意。
1. 试剂制备
注意：为了进行夹心ELISA测定，试剂的制备如下所述。
<ol><li>包衣缓冲液：<ol><li>要制备0.1 mol/L碳酸氢盐缓冲液，称取10.6 g无水碳酸钠（分子量，106 g/mol）和8.4 g碳酸氢钠（分子量?...

<ol>
	<li>Sil, P., Yoo, D. G., Floyd, M., Gingerich, A., Rada, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+throughput+measurement+of+extracellular+DNA+release+and+quantitative+NET+formation+in+human+neutrophils+in+vitro.">High throughput measurement of extracellular DNA release and quantitative NET formation in human neutrophils in vitro.</a> Journal of Visualized Experiments. (112), e52779 (2016).</li><li>Yoo, D. G., Floyd, M., Winn, M., Moskowitz, S. M., Rada, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NET+formation+induced+by+Pseudomonas+aeruginosa+cystic+fibrosis+isolates+measured+as+release+of+myeloperoxidase-DNA+and+neutrophil+elastase-DNA+complexes.">NET formation induced by Pseudomonas aeruginosa cystic fibrosis isolates measured as release of myeloperoxidase-DNA and neutrophil elastase-DNA complexes.</a> Immunology Letters. 160 (2), 186-194 (2014).</li><li>Brinkmann, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+extracellular+traps+kill+bacteria.">Neutrophil extracellular traps kill bacteria.</a> Science. 303 (5663), 1532-1535 (2004).</li><li>Papayannopoulos, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+extracellular+traps+in+immunity+and+disease.">Neutrophil extracellular traps in immunity and disease.</a> Nature Reviews Immunology. 18, 134-147 (2018).</li><li>Chamardani, T. M., Amiritavassoli, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+NETosis+for+treatment+purposes:+Friend+or+foe.">Inhibition of NETosis for treatment purposes: Friend or foe.</a> Molecular and Cellular Biochemistry. 477 (3), 673-688 (2022).</li><li>Rao, A. N., Kazzaz, N. M., Knight, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Do+neutrophil+extracellular+traps+contribute+to+the+heightened+risk+of+thrombosis+in+inflammatory+diseases.">Do neutrophil extracellular traps contribute to the heightened risk of thrombosis in inflammatory diseases.</a> World Journal of Cardiology. 7 (12), 829-842 (2015).</li><li>S&#248;rensen, O. E., Borregaard, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+extracellular+traps+-+The+dark+side+of+neutrophils.">Neutrophil extracellular traps - The dark side of neutrophils.</a> Journal of Clinical Investigation. 126 (5), 1612-1620 (2016).</li><li>Abrams, S. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+assay+for+neutrophil+extracellular+traps+(NETs)+formation+independently+predicts+disseminated+intravascular+coagulation+and+mortality+in+critically+ill+patients.">A novel assay for neutrophil extracellular traps (NETs) formation independently predicts disseminated intravascular coagulation and mortality in critically ill patients.</a> American Journal of Respiratory and Critical Care Medicine. 200 (7), 869-880 (2019).</li><li>Brinkmann, V., Goosmann, C., K&#252;hn, L. I., Zychlinsky, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automatic+quantification+of+in+vitro+NET+formation.">Automatic quantification of in vitro NET formation.</a> Frontiers in Immunology. 3, 413 (2012).</li><li>Zhao, W., Fogg, D. K., Kaplan, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+image-based+quantitative+method+for+the+characterization+of+NETosis.">A novel image-based quantitative method for the characterization of NETosis.</a> Journal of Immunological Methods. 423, 104-110 (2015).</li><li>Masuda, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NETosis+markers:+Quest+for+specific,+objective,+and+quantitative+markers.">NETosis markers: Quest for specific, objective, and quantitative markers.</a> Clinica Chimica Acta. 459, 89-93 (2016).</li><li>Rada, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+extracellular+traps.">Neutrophil extracellular traps.</a> Methods in Molecular Biology. 1982, 517-528 (2019).</li><li>Kano, H., Huq, M. A., Tsuda, M., Noguchi, H., Takeyama, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sandwich+ELISA+for+circulating+myeloperoxidase-+and+neutrophil+elastase-DNA+complexes+released+from+neutrophil+extracellular+traps.">Sandwich ELISA for circulating myeloperoxidase- and neutrophil elastase-DNA complexes released from neutrophil extracellular traps.</a> Advanced Techniques in Biology &amp; Medicine. 5 (1), 1000196 (2016).</li><li>Prevel, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasma+markers+of+neutrophil+extracellular+trap+are+linked+to+survival+but+not+to+pulmonary+embolism+in+COVID-19-related+ARDS+patients.">Plasma markers of neutrophil extracellular trap are linked to survival but not to pulmonary embolism in COVID-19-related ARDS patients.</a> Frontiers in Immunology. 13, 851497 (2022).</li><li>Schechter, M. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=et+al.+extracellular+trap+(NET)+levels+in+human+plasma+are+associated+with+active+TB.">et al. extracellular trap (NET) levels in human plasma are associated with active TB.</a> PLoS One. 12, e0182587 (2017).</li><li>Papayannopoulos, V., Metzler, K. D., Hakkim, A., Zychlinsky, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+elastase+and+myeloperoxidase+regulate+the+formation+of+neutrophil+extracellular+traps.">Neutrophil elastase and myeloperoxidase regulate the formation of neutrophil extracellular traps.</a> Journal of Cell Biology. 191 (3), 677-691 (2010).</li><li>Gupta, S., Chan, W., Zaal, K. J., Kaplan, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-throughput+real-time+imaging+technique+to+quantify+NETosis+and+distinguish+mechanisms+of+cell+death+in+human+neutrophils.">A high-throughput real-time imaging technique to quantify NETosis and distinguish mechanisms of cell death in human neutrophils.</a> Journal of Immunology. 200 (2), 869-879 (2018).</li><li>Li, M., Lin, C., Leso, A., Nefedova, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+citrullinated+histone+H3+bound+DNA+for+detection+of+neutrophil+extracellular+traps.">Quantification of citrullinated histone H3 bound DNA for detection of neutrophil extracellular traps.</a> Cancers. 12 (11), 3424 (2020).</li><li>Th&#229;lin, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+citrullinated+histones:+Development+of+an+improved+assay+to+reliably+quantify+nucleosomal+H3Cit+in+human+plasma.">Quantification of citrullinated histones: Development of an improved assay to reliably quantify nucleosomal H3Cit in human plasma.</a> Journal of Thrombosis and Haemostasis. 18 (10), 2732-2743 (2020).</li><li>Li, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PAD4+is+essential+for+antibacterial+innate+immunity+mediated+by+neutrophil+extracellular+traps.">PAD4 is essential for antibacterial innate immunity mediated by neutrophil extracellular traps.</a> Journal of Experimental Medicine. 207 (9), 1853-1862 (2010).</li><li>Zuo, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+extracellular+traps+in+COVID-19.">Neutrophil extracellular traps in COVID-19.</a> JCI Insight. 5 (11), e138999 (2020).</li><li>Masso-Silva, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+peripheral+blood+neutrophil+activation+phenotypes+and+neutrophil+extracellular+trap+formation+in+critically+ill+Coronavirus+disease+2019+(COVID-19)+patients:+A+case+series+and+review+of+the+literature.">Increased peripheral blood neutrophil activation phenotypes and neutrophil extracellular trap formation in critically ill Coronavirus disease 2019 (COVID-19) patients: A case series and review of the literature.</a> Clinical Infectious Diseases. 74 (3), 479-489 (2022).</li><li>Gong, F. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+potential+biomarkers+and+immune+features+of+sepsis+using+bioinformatics+analysis.">Identification of potential biomarkers and immune features of sepsis using bioinformatics analysis.</a> Mediators of Inflammation. 2020, 3432587 (2020).</li><li>Almansa, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptomic+correlates+of+organ+failure+extent+in+sepsis.">Transcriptomic correlates of organ failure extent in sepsis.</a> Journal of Infection. 70 (5), 445-456 (2015).</li></ol>

我们已经描述了一种夹心ELISA方法，其中MPO或NE在含有MPO-DNA或NE-DNA复合物的样品的初始孵育期间与捕获抗体的一个位点结合。洗涤后，通过将样品与过氧化物酶相关的抗DNA单克隆抗体孵育来完成“三明治”。去除未结合的二抗后，通过添加显色ABTS过氧化物酶底物来检测结合的过氧化物酶偶联物，其产生可溶性终产物，可以在405nm处通过分光光度法读取。良好的线性、较高的测定间和测定内精密度?...

本文概述了一种定量生物体液中中性粒细胞细胞外捕获 （NET） 形成的方法，方法是使用夹心酶联免疫吸附测定法 （ELISA） 检测髓过氧化物酶 （MPO） 和中性粒细胞弹性蛋白酶 （NE） 与 DNA 1,2 的复合物。NET由装饰有源自中性粒细胞颗粒3,4的抗菌蛋白酶的DNA骨架组成。MPO-DNA和NE-DNA复合物都是NET的重要和特异性成分，并作为NETs的分解产物释放到细胞外空间3,4。
NETs除了在抗菌防御中起重要的生理作用3外，还具有多种病理作用4,5，包括促进血栓形成6 和脓毒症恶化7。因此，NET最近引起了人们的关注。然而，由于缺乏灵敏、可靠的定量测定方法，NET的 体内 定量已被证明具有挑战性。 
有几种方法可用，包括通过荧光显微镜8,9和流式细胞术10直接测量NET，以及间接测量循环游离DNA，核小体和瓜氨酸组蛋白H3，但每种方法都有自己的优点和局限性11。尽管免疫荧光显微镜方法特定于NETs，并且清楚地显示了NET的定位和形成程度，但样本仅限于活检组织和分泌物质。此外，该方法需要由熟练的研究人员进行，并且需要很长时间才能获得结果。通过流式细胞术测量NET相关成分的循环水平很容易，并且可以快速提供结果;但是，该方法并非特定于NETs 12。
我们13 和其他人 1,2 开发了一种高度灵敏且可靠的检测方法，用于使用改进的 ELISA 技术测量人血浆中循环的 NET 组分、MPO 偶联或 NE 偶联 DNA，该技术使用 MPO 或 NE 的特异性抗体作为捕获抗体和 DNA 特异性检测抗体。该测定法还可用于离体鉴定活化的中性粒细胞响应佛波醇 12-肉豆蔻酸 13-乙酸酯 （PMA） 刺激释放的细胞培养上清液中的 NET 组分。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1-Step Polymorphs</td>
			<td>Accurate Chemical and Scientific Corporation</td>
			<td>AN221725</td>
			<td>Isolation of PMN&#39;s from human blood.</td>
		</tr>
		<tr>
			<td>96-well microtiter plate</td>
			<td>Thermo Fisher Scientific</td>
			<td>467466</td>
			<td>flat bottom</td>
		</tr>
		<tr>
			<td>ABTS buffer solution</td>
			<td>Sigma-Aldrich Merck</td>
			<td>11 204 530 001</td>
			<td>Contains sodium perborate, citric acid, and disodium hydrogen phosphate.&#160;</td>
		</tr>
		<tr>
			<td>ABTS tablets</td>
			<td>Sigma-Aldrich Merck</td>
			<td>11 204 521 001&#160;</td>
			<td>Each tablet contains 5 mg ABTS substrate and 60 mg vehicle substances.</td>
		</tr>
		<tr>
			<td>Adhesive plastic cover, Axygen</td>
			<td>Thermo Fisher Scientific</td>
			<td>14222348</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-MPO antibody</td>
			<td>Sigma-Aldrich Merck</td>
			<td>&#160;07-496-I</td>
			<td>Store at 2-8 &#176;C. stable for 1 year. Host species is rabbit.</td>
		</tr>
		<tr>
			<td>Anti-NE antibody, clone AHN-10</td>
			<td>Sigma-Aldrich Merck</td>
			<td>MABS461</td>
			<td>Store at 2-8 &#176;C. stable for 1 year. Host species is mouse.</td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Biomedical Science</td>
			<td>BR-220700081</td>
			<td>Albumin from bovine fraction V. Store at 2&#8211;8 &#176;C. stable for 2 year.</td>
		</tr>
		<tr>
			<td>DNase I</td>
			<td>New England BioLabs</td>
			<td>M0303M</td>
			<td>Store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>IgG, rabbit, Isotype Control</td>
			<td>GENETEX, Inc.</td>
			<td>GTX35035</td>
			<td>Store as concentrated solution at 2&#8211;8 &#176;C.</td>
		</tr>
		<tr>
			<td>IgG1, mouse Isotype Control, clone Ci4</td>
			<td>Merck&#160;</td>
			<td>MABC002</td>
			<td>Store as concentrated solution at 2&#8211;8 &#176;C.</td>
		</tr>
		<tr>
			<td>Lithium heparin blood collection tube</td>
			<td>Becton Dickinson and Company</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate mixer</td>
			<td>As one corporation</td>
			<td>NS-P</td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate Reader</td>
			<td>Molecular Devices</td>
			<td>SpectraMax 190&#160;</td>
			<td>Any microplate plate reader capable of reading wavelengths from 405&#8211;490 nm can use.</td>
		</tr>
		<tr>
			<td>Microplate reader application</td>
			<td>Molecular Devices</td>
			<td>SoftMax pro</td>
			<td></td>
		</tr>
		<tr>
			<td>Peroxidase-conjugated anti-DNA antibody, Cell death Detection ELISA</td>
			<td>Roche Diagnostics</td>
			<td>1154467500</td>
			<td>&#160;bottle 2. Store at 2&#8211;8 &#176;C. stable for 1 year.</td>
		</tr>
		<tr>
			<td>Phorbol 12-myristate 13-acetate</td>
			<td>Sigma-Aldrich Merck</td>
			<td>P8139</td>
			<td>Activation of PMN&#39;s from human blood.</td>
		</tr>
		<tr>
			<td>Phosphate buffered solution</td>
			<td>Takara Bio</td>
			<td>T9181</td>
			<td>Store at room temperature. Stable for 6 months.</td>
		</tr>
		<tr>
			<td>SigmaPlot v14.5</td>
			<td>&#160;Systat Software Inc. San Jose, CA, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium azide</td>
			<td>Fujifilm Wako Chemicals</td>
			<td>190-14901</td>
			<td>Store at room temperature.</td>
		</tr>
		<tr>
			<td>t-Octylphenoxypolyethoxyethanol, Polyethylene glycol tert-octylphenyl ether</td>
			<td>Fujifilm Wako Chemicals</td>
			<td>9002-93-1</td>
			<td>Store at room temperature.</td>
		</tr>
	</tbody>
</table>

quantifying myeloperoxidase-dna and neutrophil elastase-dna complexes from neutrophil extracellular traps by using a modified sandwich elisa

某些刺激，如微生物，导致中性粒细胞释放中性粒细胞胞外陷阱（NET），其基本上是由DNA与颗粒蛋白组成的网状结构，如髓过氧化物酶（MPO）和中性粒细胞弹性蛋白（NE），以及细胞质和细胞骨架蛋白。尽管最近对NETs的兴趣有所增加，但没有灵敏，可靠的测定方法可用于在临床环境中测量NET。本文描述了一种改良的夹心酶联免疫吸附测定法，用于定量测量循环 NET 的两种成分，MPO-DNA 和 NE-DNA 复合物，它们是 NET 的特定成分，并作为 NET 的分解产物释放到细胞外空间。该测定使用用于MPO或NE的特异性单克隆抗体作为捕获抗体和DNA特异性检测抗体。MPO 或 NE 在含有 MPO-DNA 或 NE-DNA 复合物的样品初始孵育期间与捕获抗体的一个位点结合。该测定显示出良好的线性和高测定间和测定内精度。我们在16名伴有急性呼吸窘迫综合征的COVID-19患者中使用了它，发现MPO-DNA和NE-DNA的血浆浓度明显高于从健康对照获得的血浆。该检测法是研究人血浆和培养上清液中NET特征的可靠、高灵敏度和有用的方法。

该方法使用具有抗MPO、抗NE和抗DNA单克隆抗体的夹心ELISA来测量MPO相关和NE相关DNA（图1）。在该方法中，微量滴定板的孔涂有MPO特异性或NE特异性单克隆抗体，以捕获DNA相关的MPO和DNA相关的NE，以及非DNA相关的MPO和NE。为了计算测定内变异系数（CV），在同一板内对从COVID-19和健康对照患者收集的30个样本进行重复测量，并将%CV计算为重复测量的平均值;为了计算测定间CV（即板对...

Watch this Scientific Journal Video about Quantifying Myeloperoxidase-DNA and Neutrophil Elastase-DNA Complexes from Neutrophil Extracellular Traps by Using a Modified Sandwich ELISA at JoVE.com

Quantifying Myeloperoxidase-DNA and Neutrophil Elastase-DNA Complexes from Neutrophil Extracellular Traps by Using a Modified Sandwich ELISA

我们提出了一种改进的夹心酶联免疫吸附测定技术方案，以定量测量中性粒细胞细胞外捕获残留物的两种成分，髓过氧化物酶偶联-DNA 和中性粒细胞弹性蛋白酶偶联 DNA 复合物，来源于活化的中性粒细胞。

使用改良的夹心 ELISA 定量来自中性粒细胞胞外陷阱的髓过氧化物酶 DNA 和中性粒细胞弹性蛋白酶 DNA 复合物

Certain stimuli, such as microorganisms, cause neutrophils to release neutrophil extracellular traps (NETs), which are basically web-like structures ...

NETs最近引起了人们的关注，但事实证明，在体内量化NETs具有挑战性。该协议为研究临床环境中NET的特征提供了一种理想的，高度敏感且有价值的方法。该技术应扩展到评估脓毒症或脓毒性ARDS（急性呼吸窘迫综合征）的严重程度。人们普遍认为，调节倾倒或损伤相关分子模式是改善脓毒症临床状况的关键。该方法的最大优点包括在短时间内准确定量循环NET残余物，如髓过氧化物酶-DNA和中性粒细胞弹性蛋白酶-DNA。建议在测定前旋转样品并避免反复解冻。遵循方案中给出的孵育时间很重要。首先将新鲜分离的多形核中性粒细胞稀释至每毫升10， 000个细胞，加入不含酚红的RPMI 1640中。将它们筛入35毫米培养皿中。为了诱导中性粒细胞细胞外陷阱或NET，首先用25纳摩尔PMA刺激多形核中性粒细胞。然后通过加入每毫升0.6微克DNase I来部分消化细胞外陷阱，并将混合物在室温下孵育50分钟。现在，加入五毫摩尔EDTA以停止DNase活性，并收集含有合成NET的培养基。离心机去除细胞碎片。从四个健康对照中收集上清液。混合将它们存放在零下 80 摄氏度以备进一步使用。将含有0.05微克抗体的100微升稀释的抗髓过氧化物酶移液到ELISA板中。现在，用粘性塑料盖覆盖板以防止样品蒸发，并将样品在四摄氏度下孵育过夜，以允许捕获抗体结合。第二天，从孔中丢弃稀释的抗体溶液，并将300微升洗涤溶液移入每个孔中。在纸巾上将盘子擦干以去除多余的PBS。用200微升封闭缓冲液封闭板的每个孔。用粘性塑料盖盖住它，并通过孵育来阻塞孔。从孔中丢弃封闭溶液并清洗板三到四次后，在纸巾上将其敲干。接下来，将25微升血浆移液到除空白以外的所有孔中。并用75微升PBS稀释，使最终孔容积为100微升。然后将100微升PBS加入空白孔中。通过在室温下将板放在摇床上以250rpm振荡器10秒钟来混合样品。向所有孔中加入两微升100完全稀释的DNase I，并将密封板放在摇床上10秒钟以彻底混合样品。在室温下孵育15分钟。向每个孔中加入一微升0.5摩尔EDTA以停止DNase反应。然后将密封板放在摇床上15秒钟，以彻底混合样品。最后，将板在 4 摄氏度下孵育过夜，以使 NET 的蛋白质组分附着在捕获抗体上。第二天，丢弃井中的溶液。多次洗孔后，将100微升稀释的过氧化物酶偶联的抗DNA检测抗体移入每个孔中。将板孵育1 1/2小时，然后将溶液丢弃在孔中。洗涤孔三次后，将100微升ABTS底物溶液移液到每个孔中，并在黑暗中将密封板在摇床上孵育。通过加入50微升两摩尔硫酸来停止反应。通过小心敲击板侧面来混合孔内容物。接下来，将酶标仪连接到计算机并启动软件应用程序。在状态栏中，创建一个新实验并为其命名。然后通过选择吸光度作为读取类型，终点作为读取模式，两个作为波长来设置板读数参数。接下来，将λ一设置为405纳米，λ二设置为490纳米。现在，选择自动混合和消隐的关闭状态，同时将自动校准保持为开。然后选择条带下的读取整个板。最后，将列波长优先级设置为正常作为车速，然后在自动读取下选择关闭。将板充分放入仪器抽屉并关闭。单击读取，以便立即读取板。读取每个孔在405纳米处的吸光度，并从所有未知样品中自动减去测定介质吸光度。当吸光度值分别不超过0.93和0.9时，MPO-DNA和NE-DNA均获得了可靠的标准校准曲线。当施用0.6微克/毫升DNase I时，获得最高的OD。健康对照中复合物的测定间变异系数分别为1.871和0.987，而COVID-19患者的变异系数分别为2.532和2.010。MPO-DNA和NE-DNA的平均测定间变异系数分别为6.524和4.389。捕获抗体对MPO-DNA和NE-DNA复合物的特异性表明，同种型对照抗体对复合物的反应很小。相对于健康对照组，COVID-19患者血浆中这两种复合物的百分比更高。应优化脱氧核糖核酸酶消化时间，否则过度消化会降低吸光度。

Research

JoVE Journal

Immunology and Infection

Neutrophil Extracellular Traps: How to Generate and Visualize Them

嗜中性粒细胞胞外的陷阱：如何生成和可视化

Neutrophil granulocytes are the most abundant group of leukocytes in the peripheral blood. As professional phagocytes, they engulf bacteria and kill them ...

科研

免疫与感染

Simplified Human Neutrophil Extracellular Traps (NETs) Isolation and Handling

Simplified Human Neutrophil Extracellular Traps NETs Isolation and Handling

简化人力中性粒细胞胞外陷阱（英语教师）分离和处理

Neutrophil Extracellular Traps (NETs) have been recently identified as part of the neutrophil&#8217;s antimicrobial armamentarium. Apart from their role ...

Methods to Study Lipid Alterations in Neutrophils and the Subsequent Formation of Neutrophil Extracellular Traps

方法来研究中性粒细胞脂质的改变和中性粒细胞胞外陷阱随后形成

Lipid analysis performed by high performance thin layer chromatography (HPTLC) is a relatively simple, cost-effective method of analyzing a broad range of ...

A Simple Fluorescence Assay for Quantification of Canine Neutrophil Extracellular Trap Release

一个简单的荧光试验犬中性粒细胞胞外陷阱里放的量化

Neutrophil extracellular traps are networks of DNA, histones and neutrophil proteins released in response to infectious and inflammatory stimuli. Although ...

Automated Image-Based Quantification of Neutrophil Extracellular Traps Using NETQUANT

使用 NETQUANT 对中性粒细胞外陷阱的自动基于图像的定量

Neutrophil extracellular traps (NETs) are web-like antimicrobial structures consisting of DNA and granule derived antimicrobial proteins. ...

A High-throughput Assay to Assess and Quantify Neutrophil Extracellular Trap Formation

一种评估和定量中性粒细胞外细胞陷阱形成的高通量检测

Neutrophil extracellular traps (NETs) are immunogenic extracellular DNA structures that can be released by neutrophils upon a wide variety of triggers. ...

Identification of Neutrophil Extracellular Traps in Paraffin-Embedded Feline Arterial Thrombi using Immunofluorescence Microscopy

使用免疫荧光显微镜鉴定帕芬-嵌入式猫动脉血栓中中性粒细胞外陷阱

Neutrophil extracellular traps (NETs), composed of cell-free DNA (cfDNA) and proteins like histones and neutrophil elastase (NE), are released by ...

Morphological and Compositional Analysis of Neutrophil Extracellular Traps Induced by Microbial and Chemical Stimuli

微生物和化学刺激诱导的中性粒细胞胞外陷阱的形态学和组成分析

Neutrophils function as the first line of cellular defense in an innate immune response by employing diverse mechanisms, such as the formation of ...

Staining and Imaging to Visualize Neutrophil Extracellular Traps

Immunofluorescence Imaging of Neutrophil Extracellular Traps in Human and Mouse Tissues

Neutrophil Extracellular Traps Imaging in Human and Mouse Tissues (Video) | JoVE

人和小鼠组织中性粒细胞胞外陷阱的免疫荧光成像

Neutrophil extracellular traps (NETs) are released by neutrophils as a response to bacterial infection or traumatic tissue damage but also play a role in ...