辉瑞制药有限公司资助博士桑顿表示感谢。

1。血清和支气管肺泡灌洗液的收集，存储和制备<ol><li>收集血液凝固允许在4°C和分装血清于-20°C间商店使用前未经处理的血液样本血清。支气管肺泡灌洗液（BAL），也应在-20°C储存为等分注：存储血清和BAL等分限制为靶抗原的降解样品反复冻融过程中重复检测的潜力。 </li><li>解冻样品和血清和BAL样本彻底涡旋混合1分钟14,000转的离心机。 </li><li>对于常规检测人血清样品，稀释血清与组织培养基（中医）1:1（V / V）。中医组成的RPMI-1640培养基，10％（V / V）胎牛血清，1％（V / V）为200mm L-谷氨酰胺的解决方案，和叠氮化钠作为防腐剂（0.02％W / V）。中期提前做好准备，可在4°C保存几个月。适用于100μL的dilut编辑血清对LFD的。 </li><li>为人类灌洗液，从动物模型灌洗的液体，适用于100μL整齐的样品利丰，没有预先处理。 </li><li>从动物模型的血清，稀释1:2（V / V）的血清磷酸盐缓冲液含4％（W / V）EDTA在沸水浴中，在14000转离心5分钟，3分钟热，适用于100微升利丰设备整齐上清。 </li></ol> 2。血清和BAL的横向流动装置中的应用<ol><li>储存在室温（23℃）的横向流动装置。在此温度下，设备的稳定，为12个月。删除设备，从他们的邮袋和水平表面的地方。 </li><li>使用无菌枪头，适用于100微升血清或利落的BAL样本前处理设备释放端口。 </li><li>允许试验，运行15分钟，在室温下，当时的测试结果应记录。注：流体在几秒钟内将是SEEN迁移一直在观察窗的硝酸纤维素膜的毛细作用。 </li></ol> 3。 LFD的结果记录和解读<ol><li>利丰组成的内部控制线（塑料外壳上的字母C表示）和测试线（由字母T表示）。控制线应始终出现，不论曲霉菌抗原在血清或BAL样本。这表明，该法已正常运行。 </li><li> 曲霉抗原的血清或BAL样本中，检测线也将出现在15分钟的示例应用程序。因为测试线的强度是曲霉抗原目前在样品量成正比，检测线可出现弱阳性（+），中度阳性（+ +）或作为一个强大阳性（+） 。然而，任何积极的测试线，无论强度，表明曲霉样品中的抗原存在。在日E的黑曲霉抗原的情况下，没有检测线就会出现，并把结果为阴性（ - ）记录。 </li></ol> 4。代表结果代表性的例子是阴性，弱阳性，中度阳性，强阳性，BAL和血清样品LFD的结果如图1所示。 抗原阳性LFD的测试（强和弱）或急性髓细胞性白血病（AML）患者的诊断，根据EORTC /味精诊断标准灌洗流体的负面LFD的测试结果如表1所示。包括在本表中有相应的临床和真菌学（半乳甘露聚糖和文化）的数据，和曲霉菌特异性PCR在圣Bartholomews医院8开发的测试，为每一个病人的结果。对疾病的诊断是基于宿主因素（嗜中性白血球减少症，长期使用糖皮质激素治疗，与其他公认的T细胞immunosuppressants），临床标准和通用的灌洗阳性（这里定义为通用指标值&gt; 0.8）。根据2002年EORTC /味精指引10 12，13和16，患者被诊断为“可能”的宿主因素和临床标准或通用的积极性的基础上的保险业监督。根据修订（2008）EORTC /味精的指导方针，宿主因素和转基因阳性单独或宿主因素和临床标准将不会显示“可能”侵入性真菌感染，除非陪同下，分别从临床数据和真菌的证据支持。 6患者被诊断为“可能”保险业监督在2002年和2008年的指引，因为主机的因素，主要和次要的临床特点和转基因阳性。注，而利丰也不PCR检测既不是目前在EORTC /味精指引，有两个实验和商业半乳甘露聚糖试验表明曲霉抗原和核酸中存在的BAL样本之间的强有力的协议<html> 6例和12 S的。试验证明疗效的利丰和PCR检测诊断国际音标额外的结果，可以发现8约翰逊等人 。 <img alt="图1" src="/files/ftp_upload/3721/3721fig1.jpg" /> 图1负（仅控制线）和阳性（控制和测试线）利丰测试使用血清和BAL结果。测试线反应的强度是成正比的靶抗原在血清和BAL样本的浓度。反应通常从弱（+），通过适度的（+），以强（+）。无论测试线强度，所有三个血清阳性反应，表明侵袭性肺曲霉病，由于曲霉抗原在血液中循环的存在。灌洗反应阳性（弱，中度或强）表示可能在肺部的致病性菌丝萌发的孢子和发展。 pdflinebreak“&gt; <table border="1"><tbody><tr><td> 病人没有。 </td><td> 帕蒂耳鼻喉科信息</td><td> 1临床标准 </td><td> 灌洗文化2 </td><td> 通用汽车环境影响评估指数3 </td><td> EORTC / MSG（2002）4 </td><td> EORTC / MSG（2008）5 </td><td> 曲霉 PCR检测结果 </td><td> LFD的结果 </td></tr><tr><td> 6 </td><td> - </td><td>主要CT征象（结节和光环）和一个小</td><td>负</td><td> 0.9（正面） </td><td>可能</td><td>可能</td><td>积极</td><td>弱阳性（+）， </td></tr><tr><td> 12 </td><td>假定以前的真菌感染</td><td>没有大的，一个小</td><td> 光滑念珠菌 </td><td> 6.43（强阳性） </td><td>可能</td><td>没有</td><td>积极</td><td>强阳性（+ +） </td></tr><tr><td> 13 </td><td>凝固酶阴性葡萄球菌ylococcus和E。大肠杆菌在血液中</td><td>没有大的，两个未成年的</td><td>负</td><td> 0.25（阴性） </td><td>可能</td><td>没有</td><td>负</td><td>负（ - ） </td></tr><tr><td> 16 </td><td>肺部感染</td><td> 3次要标准，包括新的渗透，加上复数积液</td><td>负</td><td> 0.16（阴性） </td><td>可能</td><td>没有</td><td>负</td><td>负（ - ） </td></tr></tbody></table> 1结节或晕在电脑断层扫描，提示真菌感染 BALF中光滑念珠菌将被视为污染物，因为它是第二个最常见的酵母作为正常人体菌群的一部分分离 3指数&gt; 0.8在通用环境影响评估测试灌洗是曲霉菌感染的指标 4基于2002年EORTC /味精诊断标准“possiblE&#39;，&#39;可能&#39;或&#39;证明&#39;侵袭性真菌病10 5的基础上修订（2008）EORTC /味精诊断标准“可能”，“很可能”或“证明”侵袭性真菌病2 表1。BAL样本，可能近期行动计划“，并从控制AML患者（没有证据表明感染）急性髓细胞性白血病患者，LFD的测试和EORTC /味精感染的诊断结果。

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我们什么都没有透露。

“近期行动计划”明确鉴定，只有真正实现从活检标本的病原隔离，但在病得很重的患者往往是不可能恢复合适的样品和曲霉菌是从血很少可收回。而重大进展，已在使用胸部电脑断层扫描，在IPA诊断，提示是“光环”或“空气新月”的招牌，如肺“近期行动计划”的特点，是短暂的，或可以归结为呼吸文物真菌感染或其他11,12。因此，这些数据都辅以血清学技术，目标识别签名分子（GM和β-葡聚糖），真菌，病人的血清中循环，或者是在BAL液，痰液或尿液样本13。虽然这些测试显示令人满意的敏感性，他们缺乏足够的特异性或1,6一定的条件下遭受的干扰</sup&gt;。 在这里可以提出LFD的“近期行动计划”的检测测试点照顾&#39;IPA和攻击技术已用于迄今在测试中，检测病毒，细菌，寄生虫和毒素14-19最著名的诊断，怀孕在1988年首次推出单路径测试。在这里介绍的黑利丰， 曲霉菌单克隆抗体JF5固定多孔硝酸纤维素膜捕获区（测试线）。抗鼠免疫球蛋白固定在一个单独的区域膜担任内部控制（控制线）。血清或BALF中除了释放端口，单克隆抗体JF5胶体金释放垫共轭结合靶抗原和复杂的，然后沿通过毛细作用的多孔膜。单克隆抗体JF5固定在捕获区结合JF5-胶体金抗原复合物，造成红色测试线。任何绑定JF5-胶体金共轭结合的内部控制，表明该法已正常运行。这个结果在一个红色的控制线。 测试是快速，仅15分钟车程，到执行，是便宜的相比，基于对转基因和β-葡聚糖检测血清和BAL的测试，并且不需要昂贵的设备或广泛的实验室设施运行。此外，单克隆抗体JF5不交叉反应的药物，或已被证明的污染物，造成假阳性反应在通用汽车和β-葡聚糖试验1,4,6。一个额外的过流诊断测试的主要优势是LFDs能力检测活动，是曲霉菌的浸润性生长的指标。 在利丰过程中的一个关键步骤是需要读取结果，15分钟后血清或BAL样本中的应用设备。测试不应该被留时间超过15分钟，记录结果之前，今年5月偏置结果interpretati上。增强弱的反应将不会延长潜伏期。已被发现感染的动物模型血清热处理，以提高检测的灵敏度。测试的局限性在于它是定性的，并依赖于运营商的积极性的主观评价。根据血清和BAL样本（ 图1）抗原含量的检测线强度的变化。然而，任何积极的反应（通过比较已知的底片决定）表示曲霉抗原，因此感染的存在。要限制对LFD检测的主观性，手持设备可允许LFD的测试线强度的量化，使建立抗原检测的阈值20。 利丰未来的发展，包括它的商业化和多重利丰的发展，同时允许其他侵袭性真菌病原体检测使用高度特异性的单克隆抗体3。

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> 黑利丰</td><td>埃克塞特大学</td><td></td><td>可从相应的要求作者</td></tr><tr><td>的RPMI-1640 </td><td>西格玛</td><td> R0883 </td><td></td></tr><tr><td> L-谷氨酰胺</td><td>西格玛</td><td> G7513 </td><td></td></tr><tr><td>胎牛血清</td><td> biosera </td><td> S9100 </td><td>在中药制剂，可用于其他来源的胎牛血清</td></tr><tr><td> EDTA的</td><td> Fisher Scientific则</td><td> BPE120-1 </td><td></td></tr></tbody></table>

detection of invasive pulmonary aspergillosis in haematological malignancy patients by using lateral-flow technology

侵袭性肺曲霉病（IPA），是一种在血液系统恶性肿瘤患者造血干细胞移植1的发病率和死亡率的首要原因。检测的“近期行动计划”表示诊断的一项艰巨的挑战，并在一个&#39;黄金标准&#39;的情况下，结合临床资料和微生物学和病理学在可行的情况下依靠。 IPA的诊断必须符合欧洲组织的研究和治疗癌症和美国国立过敏和传染病真菌研究组（EORTC / MSG）的定义“证明”，“可能”，“可能”侵入真菌病害的共识研究所。目前，没有核酸为基础的测试外部被验证为“近期行动计划”的检测和聚合酶链反应（PCR），不包括在当前EORTC /味精的诊断标准。 曲霉鉴定组织切片是问题，因为西米在与其他侵袭性真菌病原体3，证明鉴定菌丝形态larities需要隔离在纯培养的病原体。依靠活检标本的可用性文化为基础的方法，但这些都不是生病的病人总是访问，并不总是可行的繁殖产生的文化时获得。 曲霉发病的一个重要特征是血管的入侵，提供机会跟踪真菌免疫试验检测血清和支气管肺泡灌洗（BAL）流体的特性抗原签名分子的一个特点。这导致的发展的Platelia酶免疫（GM-EIA）的检测曲霉半乳甘露聚糖和“泛真菌检测（Fungitell测试）检测保守的真菌细胞壁成分（1→3）-β-D葡聚糖，而不是在缺乏这种成分在细胞壁1,4毛霉菌。是起诉周围这些测试的准确性1,4-6已导致最近开发的新一代单克隆抗体（MAB）为基础的检测1,5感染的替代指标的检测。 桑顿5最近一个曲霉特异性抗体（JF5）的产生，利用杂交瘤技术，其开发利用免疫色谱横向流动设备，点照护（POC）诊断为“近期行动计划”（LFD）。一个LFD的主要优点是它能够探测到活动以来单克隆抗体JF5只有5木耳的积极增长过程中分泌到细胞外的糖蛋白抗原结合。这是一个重要的考虑因素，如肺灌洗诊断“近期行动计划”，自曲霉菌孢子的吸入空气的共同组成部分时，使用的液体。该设备在诊断“近期行动计划”的效用已被证实感染的动物模型，利丰显示改善的敏感性和规范ificity相比的Platelia通用和Fungitell（1→3）β-D-葡聚糖检测7。 在这里，我们提出了一个简单的LFD的过程曲霉抗原检测人体血清和BAL液。它的速度和准确性在血液系统恶性肿瘤患者的近期行动计划“的诊断提供了一种新的辅助测试点照顾。

Watch this Scientific Journal Video about Detection of Invasive Pulmonary Aspergillosis in Haematological Malignancy Patients by using Lateral-flow Technology at JoVE.com

Detection of Invasive Pulmonary Aspergillosis in Haematological Malignancy Patients by using Lateral-flow Technology

提出了一种快速，准确点保健侵袭性肺曲霉病的试验。侧流技术的优势，使用绑定到一个特定的单克隆抗体<em&gt;黑曲霉</em&gt;抗原分泌在肺部感染。检测血清和支气管肺泡是兼容的，代表了新型疾病诊断的辅助测试。

通过横向流技术检测在血液系统恶性肿瘤患者侵袭性肺曲菌病

Invasive pulmonary aspergillosis (IPA) is a leading cause of morbidity and mortality in haematological malignancy patients and hematopoietic stem cell ...

detection-invasive-pulmonary-aspergillosis-haematological-malignancy

Research

JoVE Journal

Immunology and Infection

27.8K 次观看.  University of Exeter. 提出了一种快速，准确点保健侵袭性肺曲霉病的试验。侧流技术的优势，使用绑定到一个特定的单克隆抗体黑曲霉抗原分泌在肺部感染。检测血清和支气管肺泡是兼容的，代表了新型疾病诊断的辅助测试。

视频: Detection of Invasive Pulmonary Aspergillosis in Haematological Malignancy Patients by using Lateral-flow Technology

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists. The value of 2P microscopy is that it provides rich spatiotemporal information regarding cell behaviors within intact tissues and in live mice. Leukocyte recruitment plays a significant role in host defense against infection and when unchecked, can contribute to inflammatory and autoimmune diseases. Studying leukocyte recruitment in vivo is technically challenging since cells are moving rapidly within vessels located deep within light scattering tissues. To date, most intravital imaging studies require surgical preparation to expose the blood vessels and tissues. To avoid the tissue damage and inflammation induced by surgery itself, here, we describe a non-invasive single-cell imaging approach that can be used to study leukocyte trafficking in the mouse footpad and phalanges. We discuss the technical aspects of our 2P imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Here, we describe a non-invasive two-photon (2P) microscopy approach to study leukocyte homing in the mouse footpad. We discuss the technical aspects of our tissue imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

白细胞的归位和迁移的非侵入性的成像在体内</em

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists.  The value of 2P microscopy is that it ...

科研

免疫与感染

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in situ hybridization (FISH) for rapid culture-independent detection of Salmonella spp. Cell-charged tapes can also be placed face-down on selective agar for solid-phase enrichment prior to detection. Alternatively, low-volume liquid enrichments (liquid surface miniculture) can be performed on the surface of the tape in non-selective broth, followed by FISH and analysis via flow cytometry. To begin, sterile adhesive tape is brought into contact with fresh produce, gentle pressure is applied, and the tape is removed, physically extracting microbes present on these surfaces. Tapes are mounted sticky-side up onto glass microscope slides and the sampled cells are fixed with 10% formalin (30 min) and dehydrated using a graded ethanol series (50, 80, and 95%; 3 min each concentration). Next, cell-charged tapes are spotted with buffer containing a Salmonella-targeted DNA probe cocktail and hybridized for 15 - 30 min at 55&deg;C, followed by a brief rinse in a washing buffer to remove unbound probe. Adherent, FISH-labeled cells are then counterstained with the DNA dye 4',6-diamidino-2-phenylindole (DAPI) and results are viewed using fluorescence microscopy. For solid-phase enrichment, cell-charged tapes are placed face-down on a suitable selective agar surface and incubated to allow in situ growth of Salmonella microcolonies, followed by FISH and microscopy as described above. For liquid surface miniculture, cell-charged tapes are placed sticky side up and a silicone perfusion chamber is applied so that the tape and microscope slide form the bottom of a water-tight chamber into which a small volume (&le; 500 &mu;L) of Trypticase Soy Broth (TSB) is introduced. The inlet ports are sealed and the chambers are incubated at 35 - 37&deg;C, allowing growth-based amplification of tape-extracted microbes. Following incubation, inlet ports are unsealed, cells are detached and mixed with vigorous back and forth pipetting, harvested via centrifugation and fixed in 10% neutral buffered formalin. Finally, samples are hybridized and examined via flow cytometry to reveal the presence of Salmonella spp. As described here, our "tape-FISH" approach can provide simple and rapid sampling and detection of Salmonella on tomato surfaces. We have also used this approach for sampling other types of fresh produce, including spinach and jalape&ntilde;o peppers.

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple adhesive-tape-based approach for sampling of tomato and other fresh produce surfaces, followed by rapid whole cell detection of Salmonella using fluorescence in situ hybridization (FISH).

胶粘剂基于磁带的采样和荧光组合原位杂交快速检测沙门氏菌新鲜农产品

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in ...

IgY Technology: Extraction of Chicken Antibodies from Egg Yolk by Polyethylene Glycol (PEG) Precipitation

Hens can be immunized by means of i.m. vaccination (Musculus pectoralis, left and right, injection volume 0.5-1.0 ml) or by means of Gene-Gun plasmid-immunization. Dependent on the immunogenicity of the antigen, high antibody-titres (up to 1:100,000 - 1:1,000,000) can be achieved after only one or 3 - 4 boost immunizations. Normally, a hen lays eggs continuously for about 72 weeks, thereafter the laying capacity decreases. This protocol describes the extraction of total IgY from egg yolk by means of a precipitation procedure (PEG. Polson et al. 1980). The method involves two important steps. The first one is the removal of lipids and the second is the precipitation of total IgY from the supernatant of step one. After dialysis against a buffer (normally PBS) the IgY-extract can be stored at -20&deg;C for more than a year. The purity of the extract is around 80 %, the total IgY per egg varies from 40-80 mg, dependent on the age of the laying hen. The total IgY content increases with the age of the hen from around 40 mg/egg up to 80 mg/egg (concerning PEG precipitation). The laying capacity of a hen per year is around 325 eggs. That means a total potential harvest of 20 g total IgY/year based on a mean IgY content of 60 mg total IgY/egg (see Table 1).

IgY Technology: Extraction of Chicken Antibodies from Egg Yolk by Polyethylene Glycol PEG Precipitation

This protocol describes in particular the extraction of total IgY from egg yolk by means of polyethylene glycol precipitation and gives general information about IgY technology.

IgY的技术：从蛋黄鸡抗体提取聚乙二醇（PEG）降水量

Hens can be immunized by means of  i.m. vaccination (Musculus pectoralis, left and right, injection volume  0.5-1.0 ml) or by means of Gene-Gun ...

In Vivo Imaging Systems (IVIS) Detection of a Neuro-Invasive Encephalitic Virus

Modern advancements in imaging technology encourage further development and refinement in the way viral research is accomplished. Initially proposed by Russel and Burch in Hume's 3Rs (replacement, reduction, refinement), the utilization of animal models in scientific research is under constant pressure to identify new methodologies to reduce animal usage while improving scientific accuracy and speed. A major challenge to Hume's principals however, is how to ensure the studies are statistically accurate while reducing animal disease morbidity and overall numbers. Vaccine efficacy studies currently require a large number of animals in order to be considered statistically significant and often result in high morbidity and mortality endpoints for identification of immune protection. We utilized in vivo imaging systems (IVIS) in conjunction with a firefly bioluminescent enzyme to progressively track the invasion of the central nervous system (CNS) by an encephalitic virus in a murine model. Typically, the disease progresses relatively slowly, however virus replication is rapid, especially within the CNS, and can lead to an often, lethal outcome. Following intranasal infection of the mice with TC83-Luc, an attenuated Venezuelan equine encephalitis virus strain modified to expresses a luciferase gene; we are able to visualize virus replication within the brain at least three days before the development of clinical disease symptoms. Utilizing CNS invasion as a key encephalitic disease development endpoint we are able to quickly identify therapeutic and vaccine protection against TC83-Luc infection before clinical symptoms develop. With IVIS technology we are able to demonstrate the rapid and accurate testing of drug therapeutics and vaccines while reducing animal numbers and morbidity.

In Vivo Imaging Systems IVIS Detection of a Neuro-Invasive Encephalitic Virus

Utilizing luciferase and in vivo imaging systems (IVIS) as a novel means to identify disease endpoints before clinical developments occur. IVIS has allowed us to visualize in real time the invasion of encephalitic viruses over multiple days, providing a more accurate disease model for future study. It has also allowed us to identify the potential protective features of antivirals and vaccines faster than currently utilized animal models. The capability to utilize individual animals over multiple time points ensures reduced animal requirements, costs, and overall morbidity to the animals utilized ensuring a more humane and more scientific means of disease study.

在活体成像系统（IVIS）的神经侵入性脑炎病毒的检测

Modern advancements in imaging technology encourage further development and refinement in the way viral research is accomplished. Initially proposed by ...

Detection of Human Leukocyte Antigen Biomarkers in Breast Cancer Utilizing Label-free Biosensor Technology

According to the American Cancer Society, more than 200,000 women will be diagnosed with invasive breast cancer each year and approximately 40,000 will die from the disease. The human leukocyte antigen (HLA) class I samples peptides derived from proteasomal degradation of cellular proteins and presents these fragments on the cell surface for interrogation by circulating cytotoxic T lymphocytes (CTL). Generation of T-cell receptor mimic (TCRm) monoclonal antibodies (mAbs) which recognize breast cancer specific peptide/HLA-A*02:01 complexes such as those derived from macrophage migration inhibitory factor (MIF19-27) and NY-ESO-1157-165 enable detection and destruction of breast cancer cells in the absence of an effective anti-tumor CTL response. Intact class I HLA/peptide complexes are shed by breast cancer cells and represent potentially relevant cancer biomarkers. In this work, a breakthrough biomarker screening system for cancer diagnostics incorporating T-cell receptor mimic monoclonal antibodies combined with a novel, label-free biosensor utilizing guided-mode resonance (GMR) sensor technology is presented. Detection of shed MIF/HLA-A*02:01 complexes in MDA-MB-231 cell supernatants, spiked human serum, and patient plasma is demonstrated. The impact of this work could revolutionize personalized medicine through development of companion disease diagnostics for targeted immunotherapies.

Intact class I HLA/peptide complexes are shed by cancer cells, representing a potential relevant cancer biomarker. Utilizing label-free sensor technology and T-cell receptor mimicking monoclonal antibodies, detection of shed MIF/HLA-A*02:01 complexes in MDA-MB-231 cell supernatants, spiked human serum, and patient plasma is demonstrated, enabling development of a novel cancer diagnostic platform.

在乳腺癌检测人类白细胞抗原生物标志物的利用无标记生物传感技术

According to the American Cancer Society, more than 200,000 women will be diagnosed with invasive breast cancer each year and approximately 40,000 will ...

High-throughput Detection of Respiratory Pathogens in Animal Specimens by Nanoscale PCR

Nanoliter scale real-time PCR uses spatial multiplexing to allow multiple assays to be run in parallel on a single plate without the typical drawbacks of combining reactions together. We designed and evaluated a panel based on this principle to rapidly identify the presence of common disease agents in dogs and horses with acute respiratory illness. This manuscript describes a nanoscale diagnostic PCR workflow for sample preparation, amplification, and analysis of target pathogen sequences, focusing on procedures that are different from microliter scale reactions. In the respiratory panel presented, 18 assays were each set up in triplicate, accommodating up to 48 samples per plate. A universal extraction and pre-amplification workflow was optimized for high-throughput sample preparation to accommodate multiple matrices and DNA and RNA based pathogens. Representative data are presented for one RNA target (influenza A matrix) and one DNA target (equine herpesvirus 1). The ability to quickly and accurately test for a comprehensive, syndrome-based group of pathogens is a valuable tool for improving efficiency and ergonomics of diagnostic testing and for acute respiratory disease diagnosis and management.

High-throughput testing of DNA and RNA based pathogens by nanoscale PCR is described using a syndromic canine and equine respiratory PCR panel.

在动物标本呼吸道病原体的高通量检测通过纳米PCR

Nanoliter scale real-time PCR uses spatial multiplexing to allow multiple assays to be run in parallel on a single plate without the typical drawbacks of ...

Generation of Murine Monoclonal Antibodies by Hybridoma Technology

Monoclonal antibodies are universal binding molecules and are widely used in biomedicine and research. Nevertheless, the generation of these binding molecules is time-consuming and laborious due to the complicated handling and lack of alternatives. The aim of this protocol is to provide one standard method for the generation of monoclonal antibodies using hybridoma technology. This technology combines two steps. Step 1 is an appropriate immunization of the animal and step 2 is the fusion of B lymphocytes with immortal myeloma cells in order to generate hybrids possessing both parental functions, such as the production of antibody molecules and immortality. The generated hybridoma cells were then recloned and diluted to obtain stable monoclonal cell cultures secreting the desired monoclonal antibody in the culture supernatant. The supernatants were tested in enzyme-linked immunosorbent assays (ELISA) for antigen specificity. After the selection of appropriate cell clones, the cells were transferred to mass cultivation in order to produce the desired antibody molecule in large amounts. The purification of the antibodies is routinely performed by affinity chromatography. After purification, the antibody molecule can be characterized and validated for the final test application. The whole process takes 8 to 12 months of development, and there is a high risk that the antibody will not work in the desired test system.

An optimized protocol is presented for the generation of monoclonal antibodies based on the hybridoma technology. Mice were immunized with an immunoconjugate. Spleen cells were fused by PEG and an electric impulse with immortal myeloma cells. Antibody-producing hybridoma cells were selected by HAT and antigen-specific ELISA screening.

鼠单克隆抗体的产生杂交瘤技术

Monoclonal antibodies are universal binding molecules and are widely used in biomedicine and research. Nevertheless, the generation of these binding ...

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

Enterohemorrhagic E. coli (EHEC) O157:H7, which is a foodborne pathogen that causesdiarrhea, hemorrhagic colitis (HS), and hemolytic uremic syndrome (HUS), colonize to the intestinal tract of humans. To study the detailed mechanism of EHEC colonization in vivo, it is essential to have animal models to monitor and quantify EHEC colonization. We demonstrate here a mouse-EHEC colonization model by transforming the bioluminescent expressing plasmid to EHEC to monitor and quantify EHEC colonization in living hosts. Animals inoculated with bioluminescence-labeled EHEC show intense bioluminescent signals in mice by detection with a non-invasive in vivo imaging system. After 1 and 2 days post infection, bioluminescent signals could still be detected in infected animals, which suggests that EHEC colonize in hosts for at least 2 days. We also demonstrate that these bioluminescent EHEC locate to mouse intestine, specifically in the cecum and colon, from ex vivo images. This mouse-EHEC colonization model may serve as a tool to advance the current knowledge of the EHEC colonization mechanism.

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

A detailed protocol of a mouse model for enterohemorrhagic E. coli (EHEC) colonization by using bioluminescence-labeled bacteria is presented. The detection of these bioluminescent bacteria by a non-invasive in vivo imaging system in live animals can advance our current understanding of EHEC colonization.

非侵入性 体内生物发光系统检测小鼠宿主肠出血性大肠杆菌的定植

Enterohemorrhagic E. coli (EHEC) O157:H7, which is a foodborne pathogen that causesdiarrhea, hemorrhagic colitis (HS), and hemolytic uremic syndrome ...

Histological Quantification to Determine Lung Fungal Burden in Experimental Aspergillosis

The quantification of lung fungal burden is critical for the determination of the relative levels of immune protection and fungal virulence in mouse models of pulmonary fungal infection. Although multiple methods are used to assess fungal burden, quantitative polymerase chain reaction (qPCR) of fungal DNA has emerged as a technique with several advantages over previous culture-based methods. Currently, a comprehensive assessment of lung pathology, leukocyte recruitment, fungal burden, and gene expression in mice with invasive aspergillosis (IA) necessitates the use of a significant number of experimental and control animals. Here the quantification of lung histological staining to determine fungal burden using a reduced number of animals was examined in detail. Lung sections were stained to identify fungal structures with Gomori&#39;s modified methanamine silver (GMS) staining. Images were taken from the GMS-stained sections from 4 discrete fields of each formalin-fixed paraffin-embedded lung. The GMS stained areas within each image were quantified using an image analysis program, and from this quantification, the mean percentage of stained area was determined for each sample. Using this strategy, eosinophil-deficient mice exhibited decreased fungal burden and disease with caspofungin therapy, while wild-type mice with IA did not improve with caspofungin. Similarly, fungal burden in mice lacking &#947;&#948; T cells were also improved by caspofungin, as measured by qPCR and GMS quantification. GMS quantification is therefore introduced as a method for the determination of relative lung fungal burden that may ultimately reduce the quantity of experimental animals required for comprehensive studies of invasive aspergillosis.

Here we describe a protocol to determine pulmonary fungal burden in mice with invasive aspergillosis by quantification of Gomori&#39;s modified methanamine silver staining in histological sections. Use of this method resulted in comparable results with less animals compared to assessment of fungal burden by quantitative PCR of lung fungal DNA.

实验性曲霉病肺真菌负担的组织学定量测定

The quantification of lung fungal burden is critical for the determination of the relative levels of immune protection and fungal virulence in mouse ...

Ultrasensitive Detection of Biomarkers by Using a Molecular Imprinting Based Capacitive Biosensor

The ability to detect and quantitate biomolecules in complex solutions has always been highly sought-after within natural science; being used for the detection of biomarkers, contaminants, and other molecules of interest. A commonly used technique for this purpose is the Enzyme-linked Immunosorbent Assay (ELISA), where often one antibody is directed towards a specific target molecule, and a second labeled antibody is used for the detection of the primary antibody, allowing for the absolute quantification of the biomolecule under study. However, the usage of antibodies as recognition elements limits the robustness of the method; as does the need of using labeled molecules. To overcome these limitations, molecular imprinting has been implemented, creating artificial recognition sites complementary to the template molecule, and obsoleting the necessity of using antibodies for initial binding. Further, for even higher sensitivity, the secondary labeled antibody can be replaced by biosensors relying on the capacitance for the quantification of the target molecule. In this protocol, we describe a method to rapidly and label-free detect and quantitate low-abundant biomolecules (proteins and viruses) in complex samples, with a sensitivity that is significantly better than commonly used detection systems such as the ELISA. This is all mediated by molecular imprinting in combination with a capacitance biosensor.

Here, we present a protocol for the detection and quantification of low abundant molecules in complex solutions using molecular imprinting in combination with a capacitance biosensor.

基于分子印迹的电容生物传感器超灵敏检测生物标志物

The ability to detect and quantitate biomolecules in complex solutions has always been highly sought-after within natural science; being used for the ...