Name: antigen-capture enzyme-linked immunosorbent assay for specific detection of mycoplasma pneumoniae
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这项研究由突尼斯卫生部和突尼斯高等教育和科学研究部资助。

本研究是根据突尼斯巴斯德研究所伦理委员会制定的伦理问题进行的。
1. 预 ELISA 步骤：先决条件和预处理
<ol><li>细菌菌株和生长培养基 注意：本研究中使用的柔膜菌种类和壁细菌及其生长培养基列于 表1中。<ol><li>柔膜菌纲物种的生长<ol><li>从每种物质的甘油原液中接种 200 μL 到 1，800 μL 培养基中。</li>
			<li>在37°C和5%CO2 下静态生长柔膜...

<ol>
	<li>Razin, S., Yogev, D., Naot, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+biology+and+pathogenicity+of+mycoplasmas.">Molecular biology and pathogenicity of mycoplasmas.</a> Microbiology and Molecular Biology Reviews. 62 (4), 1094-1156 (1998).</li><li>Chanock, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mycoplasma+infections+of+man.">Mycoplasma infections of man.</a> The New England Journal of Medicine. 273 (22), 1199-1206 (1965).</li><li>Braun, G. S., Wagner, K. S., Huttner, B. 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本文介绍了一种内部 ELISA 的一般描述，主要用于确保特定筛选 M. pneumoniae 感染。提供了有关ELISA测定本身的方案以及一些预处理和后处理步骤的详细信息。吸附技术确保了该测定的特异性。该程序先前在为诊断人类和禽而开发的ELISA测试中有所描述 Mycoplasmas17,24.它还用于 ELISA 测定，用于诊断其他细菌感染，如莱姆病25.在?...

支原体 是已知最小和最简单的原核生物之一。它们与其他细菌的主要区别在于缺乏细胞壁结构。因此， 支原体 被归类为一个名为柔膜菌纲1的独立类别。细胞壁缺陷赋予这些微生物对某些抗菌剂的内在抗性，并且是其多态性的主要原因。 支原体 基因组小，体积缩小，这限制了它们的代谢和生物合成能力，并解释了它们的寄生和腐生性质1。
肺炎支原体是感染人类的支原体之一，被认为是毒性最强的支原体2。肺炎支原体定植于上呼吸道，导致儿童和年轻成人的非典型肺炎。肺炎支原体感染引起的临床症状为流感样，伴有头痛、发热和咳嗽3。肺炎支原体对宿主细胞的细胞粘附由附着细胞器介导，包括P1主要粘附和几种辅助蛋白4，5。由于肺炎支原体与气道粘膜的紧密粘附导致局部炎症和宿主免疫系统刺激，可能会出现更多的临床表现6。虽然肺炎是肺炎支原体感染的标志，但已经发现这种细菌的感染也可能是不同解剖部位（如中枢神经系统、心脏、皮肤和关节）中广泛非肺部表现的原因7。
对于所有支原体属，肺炎支原体的诊断具有挑战性。引起支原体病的临床症状大多不明显且不典型8。由于仅依靠临床表现和症状很难诊断肺炎支原体感染，因此实验室筛查特别令人感兴趣9。通过培养检测肺炎支原体菌落是正确诊断的金标准方法。然而，苛刻的生长要求和提供明确结果（1-2周）所需的时间使培养复杂化，因此意味着它很少用于常规诊断10。核酸扩增技术在速度和效率方面得到了验证，但由于其成本相对较高且在某些医疗机构中不可用，因此这些分子技术不被视为一线诊断测试。诚然，商业PCR检测被广泛用于诊断肺炎支原体感染，但它们仍然不能取代血清学。此外，假阴性和假阳性结果的频繁发生限制了PCR9的使用。常规情况下，血清学仍然是实验室中诊断肺炎支原体感染最常用的方法。几十年来已经报道了几种血清学方法，例如冷血凝素、补体固定试验 11、间接血凝试验12、免疫荧光13 和 ELISA 技术，该技术于 1980 年代初首次应用于支原体血清学 14，15，16。在对肺炎支原体感染进行ELISA血清学诊断时遇到的主要问题之一是交叉反应，这大大降低了该技术的特异性。以前报道过人血清与肺炎支原体抗原的非特异性吸附;事实上，由于肺炎支原体与一些细菌18，19和一些动物和人体组织20的共性，ELISA在人血清中检测到的许多抗体可能并不总是与支原体抗原17结合。
由于在实验室中实施的常规ELISA测试中观察到的高背景读数，结果的解释通常很复杂，因此提供正确的肺炎支原体诊断是一项艰巨的任务。在面对这个问题时，我们选择通过去除肺炎支原体抗原与待测试抗体的非特异性反应来改善肺炎支原体 ELISA。为此，我们使用吸附技术研究了非特异性肺炎支原体抗原的选择性去除。事实上，抗原捕获ELISA的主要目标是特异性检测人血清样本中的肺炎支原体免疫球蛋白（Ig）G。该ELISA的概念主要包括在添加人血清样品之前选择性捕获肺炎支原体特异性抗原。这种选择性是通过将肺炎支原体粗抗原与实验室中兔子中产生的肺炎支原体多克隆抗血清孵育来保证的，并通过吸附一组异源细菌（属于或不属于柔膜菌类）使其具有物种特异性，与肺炎支原体共享抗原 属和/或已知在呼吸道定植。吸附过程重复三次，并通过免疫印迹测试其消除交叉反应性的效率。开发的 ELISA 测定是夹心和间接 ELISA 的组合。简而言之，首先用肺炎支原体特异性多克隆抗血清包被ELISA板的孔。然后，加入肺炎支原体抗原并将其捕获在抗血清和待测血清样品中存在的抗体之间。形成的免疫复合物由二级酶偶联抗体（过氧化物酶偶联IgG）检测。通过加入显色底物来观察反应，并通过分光光度法测量吸光度。该内部 ELISA 示意图如图 1 所示。自制的ELISA被证明在特异性检测肺炎支原体感染方面是有效的，目前是常规诊断活动中最常用的测试之一。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>4-chloro-1-naphtol</td>
			<td>Sigma-Aldrich</td>
			<td>C6788-50 TAB</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto peptone</td>
			<td>BD</td>
			<td>211677</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto tryptone</td>
			<td>BD</td>
			<td>211705</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A9647-50 G</td>
			<td></td>
		</tr>
		<tr>
			<td>Carbonate bicarbonate buffer</td>
			<td>Sigma-Aldrich</td>
			<td>C3041-50 Cap</td>
			<td></td>
		</tr>
		<tr>
			<td>Casein</td>
			<td>Sigma-Aldrich</td>
			<td>C7078-1 KG</td>
			<td></td>
		</tr>
		<tr>
			<td>CMRL1066&#160;</td>
			<td>VWR</td>
			<td>P0058-N1L</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G7528-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Difco PPLO Broth</td>
			<td>BD</td>
			<td>255420</td>
			<td>Frey media</td>
		</tr>
		<tr>
			<td>ELISA plate-Reader</td>
			<td>MULTISKAN GO, Thermo Scientific</td>
			<td>Ref: 51119200</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Capricorn Scientific</td>
			<td>FBS-12A</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat peroxidase-conjugated anti-human IgG</td>
			<td>Abcam</td>
			<td>ab6759</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat peroxidase-conjugated anti-rabbit IgG</td>
			<td>Life Technologies</td>
			<td>656120</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric Acid 37%</td>
			<td>Prolab</td>
			<td>2025.290</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrogen peroxide (H2O2), solution 30%</td>
			<td>Scharlau</td>
			<td>HI01361000</td>
			<td></td>
		</tr>
		<tr>
			<td>L-arginin</td>
			<td>Sigma-Aldrich</td>
			<td>A5006-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>LB broth</td>
			<td>Prepared in Pasteur Institute of Tunis</td>
			<td></td>
			<td>Provided by the laboratory of bacteriology of the Pasteur Institute of Tunis</td>
		</tr>
		<tr>
			<td>Mycoplasma pneumoniae polyclonal antiserum produced in rabbit</td>
			<td>Produced in Pasteur Institute of Tunis</td>
			<td></td>
			<td>Serum was produced by rabbit immunization at the Pasteur Institute of Tunis</td>
		</tr>
		<tr>
			<td>Nicotinamide adenine dinucleotide</td>
			<td>Sigma-Aldrich</td>
			<td>N7004-10G</td>
			<td></td>
		</tr>
		<tr>
			<td>Nunc Maxisorp flat-bottom 96-well microtiter plate&#160;</td>
			<td>Invitrogen</td>
			<td>44-2404-21</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin G sodium (1 MIU)</td>
			<td>PANPHARMA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Phenol red</td>
			<td>fluka chemika</td>
			<td>77660</td>
			<td></td>
		</tr>
		<tr>
			<td>Skim milk</td>
			<td>MP Biomedicals</td>
			<td>902887</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium bicarbonate&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>S6297-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium carbonate</td>
			<td>Sigma-Aldrich</td>
			<td>S7795-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride (NaCl)</td>
			<td>Novachim</td>
			<td>PS02805</td>
			<td></td>
		</tr>
		<tr>
			<td>Sulfuric acid (H2SO4) 95-97%</td>
			<td>Merck</td>
			<td>1007311011</td>
			<td></td>
		</tr>
		<tr>
			<td>Thimerosal</td>
			<td>USB</td>
			<td>22215</td>
			<td></td>
		</tr>
		<tr>
			<td>TMB substrate (3,3&#8217;, 5,5&#8217; TetraMethyl-Benzidine solution)</td>
			<td>Abcam</td>
			<td>ab142042</td>
			<td></td>
		</tr>
		<tr>
			<td>Trizma base</td>
			<td>Sigma-Aldrich</td>
			<td>T6791-1 KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Sigma-Aldrich</td>
			<td>1379-500 ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast extract, powder, Ultrapure</td>
			<td>Thermo Scientific&#160;</td>
			<td>J23547&#160;</td>
			<td></td>
		</tr>
	</tbody>
</table>

antigen-capture enzyme-linked immunosorbent assay for specific detection of mycoplasma pneumoniae

肺炎支原体是一种细胞壁缺陷的原核生物，主要在人类呼吸道定植并流行，每 6 年流行一次，在年龄较大的儿童和年轻人中流行一次。肺炎支原体的诊断具有挑战性，因为病原体的苛刻性和无症状携带的可能性。基于患者血清样本中抗体滴定的肺炎支原体感染的实验室诊断仍然是最常用的方法。由于使用多克隆血清治疗肺炎支原体存在免疫交叉反应的潜在问题，因此开发了抗原捕获酶联免疫吸附测定 （ELISA） 以提高血清学诊断的特异性。ELISA平板涂有肺炎支原体多克隆抗体，在兔子中饲养，并在吸附一组与肺炎支原体属属共享抗原和/或已知在呼吸道定植的异源细菌后具有特异性。然后，反应的肺炎支原体同源抗原被血清样品中的相应抗体特异性识别。抗原捕获ELISA所经历的物理化学参数的进一步优化导致了高度特异性，灵敏和可重复的ELISA。

非吸附多克隆肺炎支原体抗血清对异源细菌的免疫印迹活性 交叉反应性确实存在，如免疫印迹结果所示（图2），与阳性对照（泳道1）相比，一些肺炎支原体抗原与筛选的细菌共享。这些交叉反应的强度是可变的。例如，败血症支原体和模仿支原体抗原体抗血清（泳道9和10）的反应性最强，除了是禽支原...

Watch this Scientific Journal Video about Antigen-Capture Enzyme-Linked Immunosorbent Assay for Specific Detection of Mycoplasma pneumoniae at JoVE.com

Antigen-Capture Enzyme-Linked Immunosorbent Assay for Specific Detection of Mycoplasma pneumoniae

在 肺炎支原体 感染中，血清学检测可产生良好的结果，但由于免疫学交叉反应，特异性较低。本文所述的内部抗原捕获ELISA保证了高物种特异性，并已被证明是准确诊断 肺炎支原体的可靠筛查测试。

抗原捕获酶联免疫吸附测定用于肺炎支原体特异性检测

Mycoplasma pneumoniae is a cell wall-deficient prokaryote, mainly known to colonize the human respiratory tract and to be endemic, with epidemic peaks ...

至于所有支原体患者，由于交叉反应，他们对肺炎支原体的诊断具有挑战性。我们提出的ELISA基于使用吸附技术选择性去除非特异性肺炎支原体抗原。本视频中描述的内部抗原捕获 ELISA 保证了高广的特异性。实际上，它已被证明是准确诊断肺炎支原体感染的可靠筛查测试。展示ELISA前步骤的将是博士生Imen Chniba。ELISA步骤将是Nadine Khadraoui，我们实验室的技术员。要开始抗原吸附程序，将12种异源细菌抗原拉入微量离心管中，并调整对应于要吸附的抗血清一半的蛋白质浓度。接下来，将抗原混合物在14， 000 G下在4摄氏度下离心10分钟以收集沉淀。然后，用纯化的多克隆抗肺炎支原体IgG重新悬浮沉淀，并在37摄氏度下缓慢搅拌孵育两个小时。在孵育结束时，在4摄氏度下再次以14， 000G离心悬浮液10分钟，并回收对应于特定多克隆肺炎支原体抗血清的上清液。要用捕获抗体包被 96 孔 ELISA 板，请将抗体稀释至 pH 9.6 下每毫升 10 μg 和 0.1 摩尔碳酸氢盐缓冲液。然后，向微孔板的每个孔中加入100微升稀释的抗体，并在4摄氏度下孵育过夜。第二天，除去涂层溶液并用洗涤缓冲液洗涤板五次。接下来，通过向每个孔中添加 100 微升封闭溶液来封闭涂层孔的未结合表面。在室温下孵育板一小时后，用移液管除去封闭溶液并如前所述洗涤板。以每孔10纳克的浓度加入等量的肺炎支原体全蛋白，并使抗原抗体反应在室温下发生两小时。孵育后，去除多余的抗原并如图所示洗涤平板。接下来，稀释人血清检测样品并参考阳性和阴性血清对照。然后，将100微升稀释的测试样品和对照添加到适当的孔中并重复。将既不含抗原也不含有血清的孔标记为空白。在室温下孵育板90分钟后，除去血清溶液并用洗涤缓冲液洗涤板五次。将100微升适当的稀释酶偶联检测抗体移液到每个孔中，并在黑暗中在37摄氏度下孵育一小时。取出未结合的检测体并清洗板后，加入100微升TMB色原溶液以可视化抗原 - 抗体结合。让板在室温下显影 30 分钟。然后，通过加入100微升7.5%硫酸来停止反应。最后，使用酶标仪读取450纳米处的吸光度，并根据阳性指数的计算对结果进行排序。肺炎支原体非吸附多克隆抗血清和异源细菌抗原之间的免疫印迹分析显示交叉反应性，但强度不同。例如，败血症支原体和拟支原体抗原尽管是禽支原体，但表现出最强的反应性。相比之下，解脲支原体的两种人类临床分离株均未显示任何反应。然而，剩余的人生殖器支原体物种的抗原和其他细菌抗原与抗血清产生了相当大的交叉反应。免疫印迹分析证实了肺炎支原体多克隆抗血清吸附异源细菌抗原的效率。吸附程序消除了所有交叉反应，从而使肺炎支原体抗血清对所有后续血清学和免疫印迹测试具有特异性。此外，使用本研究中开发的抗原捕获ELISA测试的人血清集被证明对肺炎支原体IgG呈阳性，具有良好的特异性。这种ELISA的特殊性主要由吸附技术保证，因为仔细执行这一关键步骤并重复至少两次或三次是最基本的。该技术的原理可用于诊断其他支原体物种或其他细菌。然而，选择用于吸附的细菌抗原应该是无关紧要和充分的。

Research

JoVE Journal

Biology

Antigen-Capture Enzyme-Linked Immunosorbent Assay for Specific Detection of Mycoplasma pneumoniae

Detection of Protein Ubiquitination

蛋白泛素化的检测

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of ...

科研

生物学

ELIME (Enzyme Linked Immuno Magnetic Electrochemical) Method for Mycotoxin Detection

ELIME Enzyme Linked Immuno Magnetic Electrochemical Method for Mycotoxin Detection

ELIME霉菌毒素的检测方法（酶联免疫磁电化学）

Immunoassays are a valid alternative to the more expensive and time consuming quantitative HPLC or GC1, 2 methods for the screening detection of hazardous ...

An Allele-specific Gene Expression Assay to Test the Functional Basis of Genetic Associations

等位基因特异性基因表达检测，测试的遗传协会的功能基础

The number of significant genetic associations with common complex traits is constantly increasing. However, most of these associations have not been ...

Chromatin Immunoprecipitation Assay for Tissue-specific Genes using Early-stage Mouse Embryos

使用早期的小鼠胚胎组织特异性基因的染色质免疫沉淀检测

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells 1-3. This ...

Optimized PCR-based Detection of Mycoplasma

优化的基于PCR的检测支原体

The maintenance of contamination-free cell lines is essential to cell-based research. Among the biggest contaminant concerns are mycoplasma contamination. ...

A Rapid and Specific Microplate Assay for the Determination of Intra- and Extracellular Ascorbate in Cultured Cells

快速，特异微孔板检测为内和细胞外抗坏血酸在培养细胞的测定

Vitamin C (ascorbate) plays numerous important roles in cellular metabolism, many of which have only come to light in recent years. For instance, within ...

A Multi-detection Assay for Malaria Transmitting Mosquitoes

一种多检测试验为疟疾蚊子发射

The Anopheles gambiae species complex includes the major malaria transmitting mosquitoes in Africa. Because these species are of such medical importance, ...

Detection of Intracellular Gene Expression in Live Cells of Murine, Human and Porcine Origin Using Fluorescence-labeled Nanoparticles

利用荧光标记的纳米粒子检测的小鼠，人与猪源活细胞内的基因表达

The reprogramming of somatic cells to induced pluripotent stem cells (iPS) has successfully been performed in different mammalian species including mouse, ...

Parallel High Throughput Single Molecule Kinetic Assay for Site-Specific DNA Cleavage

平行高通量单分子动力学测定，用于现场特异性DNA裂解

Site-specific DNA cleavage (SSDC) is a key step in many cellular processes, and it is crucial to gene editing. This work describes a kinetic assay capable ...