This work was funded by The MITRE Corporation through the MITRE Innovation Program (Project 25MSR621-CA). The authors would like to thank Mark Maybury, Ph.D., Richard Games, Ph.D., James Patton, Ellen Mac Garrigle, Ph.D., Carl Picconatto, Ph.D., and Caroline Gary for their support and review of this work.

1.细菌和肽基片的制备注意:全细胞细菌底物和纯化的肽聚糖被用作在这两个的MicroSlide扩散法和染料释放测定酶的底物。这些基板需要之前在进行酶反应制备。以下方案描述了它们的制备方法。 <ol><li>整个细菌细胞基质<ol><li>接种500毫升营养肉汤的枯草芽孢杆菌 168的2ml的过夜培养产生细菌电池基板（美国典型培养物保藏中心; ATCC 23857）。在30℃孵育直到培养物达到生长的指数相，其定义为导致细菌培养的倍增快速增长阶段振荡（125转）。对于沙门氏菌亚种的培育。 肠道 （ATCC 10708），在37℃，使用营养肉汤作为培养基振荡（125转）。 </li&gt; <li>通过在121℃下3个大气压压力高压灭菌10分钟，热自相残杀文化。 </li><li>收获离心热灭活细菌衬底在5000×g下20分钟。与第一类型水洗涤沉淀三次，并在水的最小量重新悬浮。在这项研究中，暂停基板在1200微升。 </li><li>等分300μl的细菌细胞基板至1.5 ml离心管并储存在20℃。 </li></ol></li><li>纯化的肽聚糖底<ol><li>从目标基板细菌7-10净化肽或从供应商获取（见材料和设备 表 ）。从净化配件细胞壁聚合物肽粗制品。 </li></ol></li></ol> 2.定性的MicroSlide扩散分析[从Lachica改性等人 11] 注意:的MicroSlide扩散测定法是用于检测样品中的抗微生物酶的存在的定性方法。作为酶扩散通过琼脂糖含有矩阵基板，结算的区域发展作为酶水解的底物。以下方案描述了的MicroSlide扩散测定法的用于定性检测蛋白质抗微生物剂的存在下制备和性能。 <ol><li>确定要使用根据制造商的协议的二喹啉甲酸（BCA）蛋白分析试剂盒进行评估的抗微生物酶的浓度。 </li><li>利用磷酸盐缓冲盐水（PBS）作为酶缓冲;然而，根据经验确定该缓冲器适合于给定的酶。 </li><li>执行使用磷酸缓冲盐水（PBS）作为反应缓冲液中的抗微生物酶的系列稀释。在这些的MicroSlide扩散测定中使用的连续稀释生成0皮克，10微克，100皮克，1毫微克，10毫微克，100纳克，1微克，和1的最终测定蛋白质量每反应体积0微克。 </li><li>蛋白质分装成群众个人离心管和调节蛋白质卷20微升，准备用PBS中除了反应的MicroSlide井。 </li><li>使由0.25克琼脂糖溶解在50ml PBS中0.5％的琼脂糖溶液。加热该溶液到沸腾，直到琼脂糖完全溶解。重量确定熔化过程失去水和添加回到溶液中。 </li><li>在水中加入50μl的10％叠氮化钠的琼脂糖溶液，并调整在水浴的溶液至50℃的温度。的叠氮化钠加入到抑制的MicroSlide扩散试验的温育过程中的污染细菌的生长。如果存活细胞被用作衬底，省略从琼脂糖溶液中的叠氮化钠。 </li><li>在冰上解冻的热灭活细菌衬底。 </li><li>重悬于12ml琼脂糖溶液的约细菌衬底匹配2.0 MCF的浊度蓝德等价标准（见材料表 ）。通过可视化通过解白色背景上的黑线，直至近似浊度达到增加细菌衬底琼脂糖比较溶液的浊度。 </li><li>立即吸管3毫升琼脂糖基质溶液到每个的MicroSlide（25×75×1毫米3）。 </li><li>幻灯片固化后，用打孔器（个直径为4.8 MM）每个的MicroSlide的琼脂糖基质层中打孔三口井。加入20μl的每个调节蛋白系列稀释的各自的孔中。 </li><li>在阴性对照使用的PBS（20微升）或牛血清白蛋白（5微克于20μl的PBS）阱。使用适合的基材已知溶菌酶，如溶菌酶，作为阳性对照。 </li><li>孵育在37℃下或在湿度室中滑动的酶的最佳活性温度。孵化的长度取决于浓度和抗微生物酶的比活性。一个典型的反应时间过夜（约16小时）。 </li><li>培养后，使用数字单镜头反光照相机在约30cm的焦距为60毫米的镜头观察下显影测定的间接光，并捕获图像的幻灯片。 注:蛋白质抗微生物对给定底物的酶的活性是用水解的明确区域，作为酶扩散入琼脂糖形成围绕井的发展密切相关。 </li><li>为符合资格的酶活性，观察周围的每一个形成良好的区域的大小。高的酶活性定性涉及较大区域，而低的酶活性定性涉及较小区域。 </li></ol> 3.基材标识-雷马素马斯亮蓝R染料标记[从周12加减] 注意:在染料释放测定中，底物是共价林克d可雷马素艳蓝色R染料。以下方案描述了染色酶底物的制备。 <ol><li>通过在99毫升I型水1克NaOH溶解使250mM的氢氧化钠（NaOH）溶液用于制备200mM的雷玛唑亮蓝R染料（RBB）的解决方案。 </li><li>使98.75毫升±1毫升新鲜的250毫米氢氧化钠溶液（3.1步），由1.25克RBB溶解在200毫米RBB的解决方案。 </li><li>重悬热灭活的细菌细胞在30毫升的RBB溶液0.5克湿重的浓度。用于纯化肽，重新悬浮在0.3克湿重的浓度在30毫升的RBB溶液的肽聚糖。 </li><li>孵育6小时，在37℃下温和混合旋转平台上在锥形烧瓶内的反应混合物。 </li><li>将反应混合物转移到4℃培养箱，孵育温和混合另外12小时。 </li><li>孵育后，收获离心的染色基板在3000 XG30分钟。倒出从衬底粒料染料溶液。 </li><li>通过用I型水中，随后离心洗涤该染料标记的细胞或肽重复（大约3-5次洗涤）从基片的非共价连接的水溶性染料。与每个加水，彻底重新悬浮沉淀。 注意:当未结合的，水溶性染料不再离心后水洗涤可见。衬底应给予一个附加的洗涤。注意，衬底将保持蓝色，而在最后洗涤物的上清液将会清楚。 </li><li>存储悬浮在水中的最小量在-20℃以备后用的染色基板。 </li></ol> 4.定量染料释放试验注意:在染料释放测定，RBB染色基板的在染色产品进入反应上清液中的酶反应释放的水解。释放的染料的量的比色测定表示摩UNT样品对于给定的酶中存在的酶活性。定量方法允许跨基材不同的酶的比较，并允许对影响酶反应（ 如 ，温度，盐度和pH下）的环境条件的变化。以下方案描述了染料释放测定的定量检测的蛋白质抗微生物的酶活性的制备和性能。 <ol><li>对于染料释放测定制剂<ol><li>允许冷冻染色的衬底，返回到室温并用测定缓冲液（PBS）中，这是根据经验对给定酶测定洗两次。 </li><li>计算由将要执行由染料释放测定的数目200微升反应体积乘以所需底物悬浮液的体积。 </li><li>通过加入染色底物的分析缓冲液的体积，在4.1.2确定准备基板悬浮液，直到光密度（的2.0 OD）用分光光度计595nm处实现。留在分光光度计的功能限制内，测量2.0的OD 595 1.0对于1:浓缩液1稀释。使用测定缓冲液作为空白。 注意:对于该反应的底物的浊度可以超出2.0提高到符合高效的酶的活性水平。 </li><li>暂停该蛋白质的抗微生物以在测定缓冲液以1毫克/毫升的估计浓度进行评估。 </li><li>使用根据制造商的协议的BCA蛋白质测定试剂盒的微板测定中，确定待测定蛋白质样品的实际浓度。 /调节纸浆悬浮液的浓度为1毫克毫升用测定缓冲液。 </li></ol></li><li>使用Dye释放测定确定为蛋白质抗菌最优培养条件<ol><li>稀释股票抗菌蛋白停牌至100毫微克/微升。这种集中使自动对焦的每单位体积（10微升）的蛋白质的1微克INAL反应物料加入到测定反应。 </li><li>确定热范围被评定为溶菌蛋白。热范围在这些测定中包括5℃，15℃，25℃，35℃，45℃，55℃，和65℃。 </li><li>在0.5ml微量离心管进行反应实验。对于每一个热条件下，加入10微升股票蛋白的200微升准备好的底物悬浮液中。 </li><li>孵育在热循环仪8小时以颠倒每小时一次混合。 </li><li>孵育后，加入25微升乙醇逮捕反应。 </li><li>在3000×g离心2分钟，除去离心未消化，不溶性基底，并转移150微升反应上清液的各反应混合物到96孔平底微量培养板，注意不要扰乱未消化衬底的颗粒。 </li><li>测量蛋白质antimicrob的酶活性通过确定可溶性RBB染料的量IAL对于给定的基片从酶促水解后的基板分离。量化的酶活性，使用微板分光光度计测量上清液的吸光度在595nm处。通过从标记的底物释放到上清液中的可溶性染料增加吸光度酶活性的定量测量。 注意:在吸光度的变化是由活动单位（AU）表示，其中1 AU导致在595纳米的0.01增加的染料释放反应上清液的光密度。空白反应，与除酶所有反应组分孵育，用于减去释放从RBB标记的底物，由于培养的任何染料。最佳酶解温度提供了活动高峰计量单位。 </li><li> 4.2.1通过4.2.7使用各种孵化缓冲剂，以确定所述抗微生物酶的最佳缓冲液条件重复步骤。在THESE测定，用PBS。 </li></ol></li><li>使用Dye释放测定确定在最佳培养温度极小活性成分浓度为蛋白质抗菌<ol><li>执行使用测定缓冲液存量抗微生物蛋白悬浮液的系列稀释。稀释系列范围将与抗微生物酶的活性而变化。在这些试验中使用的连续稀释产生0 PG，PG 10，PG 100，1毫微克，10毫微克，100纳克，1微克和10微克的最终测定蛋白质的量。 </li><li>在48孔平底微孔板执行每个反应检测。对于每一个实验条件下，加入10微升各自的蛋白质悬浮于200微升准备好的底物悬浮液中。调节加入到反应使用的反应缓冲液10微升的蛋白质溶液的体积的任何变化。 </li><li>用密封板密封膜的微孔，以避免因蒸发量的变化。孵育在摇床培养箱中于在所确定的最佳cubation温度为给定的蛋白质的抗微生物振摇16小时。 </li><li>孵育后，通过加入25微升的乙醇每个逮捕反应良好，并通过离心以3000 xg离心除去未消化的底物2分钟。 </li><li>转移150微升反应上清液的各反应混合物到96孔平底微量培养板，小心不要破坏未消化衬底的颗粒。 </li><li>量化的酶活性，使用微板分光光度计测量上清液的吸光度在595nm处。空白反应，与除酶所有反应组分孵育，用于减去释放从RBB标记的底物，由于培养的任何染料。通过从标记的底物释放到上清液中的可溶性染料增加吸光度提供的定量测量到的酶活性。 注意:在吸光度的变化是由活动单位（AU），其中1 AU结果表示在595纳米的0.01增加的染料释放反应上清液的光密度。 </li></ol></li></ol>

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作者宣称，他们没有竞争的经济利益。 MITRE公司是一个不以营利为目的的公司，经营多个联邦政府资助的研究和开发中心（FFRDCs）。

该扩散的MicroSlide法和染料释放法检测和鉴定以前未知的蛋白质抗菌药物提供的优势。这些快速和灵敏的方法允许之前投入更大研究资源感兴趣的酶的鉴定有机体源库的高通量筛选。作为高调靶酶被识别，检测分析的变化可以用来快速检测酶或确定酶的生化特性。例如，一个多蛋白质纯化方案中，所述的MicroSlide扩散分析能够快速检测含有目的酶的级分。另外，对于酶反应最适可通过改变反应缓冲液和培养温度在染料释放测定法来确定。 用于检测在所述的MicroSlide扩散分析所需的酶物质的范围是酶characteri的函数STICS。该测定的检测限制一般要求使用较多量的酶比染料释放测定的。在这项研究中，所述的MicroSlide扩散分析（ 图1）的染料释放测定（ 图4）的检测限的比较示出，该的MicroSlide扩散试验比染料释放测定较不敏感的技术。当酶浓度不是一个问题，该法的MicroSlide允许用于生产抗菌蛋白对低劳动力和设备的需求目标底物文库的快速初步筛选。尽管需要更多的努力和设备，该染料释放测定法更灵敏，可重复的，得到定量结果，允许对于给定的基片相同或不同的酶的染料释放测定的比较。这两种方法都容易在大多数微生物实验室，无需高度专业化的仪器进行的。在的代理问题略去测定法，对照酶，α胰凝乳蛋白酶，在量低至使用的染料释放测定100纳克（ 图5）检测到的，而在的MicroSlide扩散分析检测出的最小的量为1微克（数据未示出）。 提供了可用作抗菌酶定性检测测定，一些扩散试验的变体已经报道11,13。在审查这些试验中，扩散的MicroSlide测定其相对较高的灵敏度作为初始屏幕，因为它容易反应遵守的开发。从Lachica 等人 11的工作，其中的琼脂糖覆盖被用于由金黄色葡萄球菌的菌株以检测生产核酸的修饰，所述的MicroSlide扩散试验的操作类似于径向免疫扩散法14作为蛋白质从井扩散到琼脂糖含有底物。径向immunodiffu当系统达到琼脂糖​​内漫射抗原和抗体之间的复合物形成的平衡锡永方法停止免疫沉淀的区的扩展。与此相反，的MicroSlide扩散分析的衬底最初由漫射蛋白抗微生物在包围井裂解的不断扩大区消化。区域的增大的直径继续，直到酶失去活性或衬底被耗尽。生产裂解的区的速率加快作为纯度，因而比活性，酶的或加入到孔中增加酶的浓度。 用于定量评估蛋白质抗微生物剂的抗热灭活B的酶活性枯草芽孢杆菌 ，被选择的染料释放测定。使用雷马素马斯亮蓝R染料（RBB）比色法标记的底物会使蛋白质抗菌活性状况的通用性和敏感性评价TY。 RBB标记的底物的结果中，可以用分光光度计在595nm处容易地测得的可溶性蓝色产品释放的酶水解。的RBB染料释放测定的几个变型中，开发了以表征其他蛋白质的抗菌酶，表明此测定应用与其它基材的灵活性。例如，在确定溶葡球菌酶溶菌活性的影响，周等人 ，报告了使用RBB-染色葡萄球菌细胞和葡萄球菌肽聚糖作为底12的染料释放测定。在此RBB染料释放测定的应用的变型，RBB标记藤黄微球菌衬底提出用作一个快速和灵敏的方法来诊断和筛选的疾病例如细菌感染和恶性肿瘤的存在，它可以在血液血清15相关，以人溶菌酶表达水平。此外，RBB标记细菌细胞衬底具有一个LSO在用于检测溶菌酶16的酶谱法中使用。 我们证明降低的检出限，方便和染料释放测定的再现性，从而允许快速文库筛选产酶。该测定的修饰允许下游定量生化表征测定法，包括温度，pH值，和盐度的影响以及对抗微生物蛋白6的活性的其它蛋白水解酶的影响。此外，定量的染料释放测定可用于多种酶的活性比较对于给定的衬底上。的RBB染料释放测定已报道用抗除表征和蛋白的抗微生物剂检测的多糖酶活性效用。卡尔 - 佩特森和埃里克森报告使用纤维素，木聚糖，甘露聚糖，以及保单的无定形，RBB染色多糖珠子检测内聚糖酶的活性的测定17。在这些研究结果的扩展，用于检测由苏云金芽孢杆菌的Bt-107产生的几丁质酶的酶的一个灵敏的方法使用标记的RBB 18胶体几丁质被开发。从这些和其他研究，RBB染色基板的市售来源已经出现在检测糖酵解活性，包括对角蛋白，支链淀粉，直链淀粉，糖原，海带多糖，D-木聚糖，偶氮大麦葡聚糖和淀粉的用途。 在这项研究中，我们证明在微量格式的染料释放测定的效用，降低以产生比色结果所需RBB标记的底物和酶的体积。如在图4和图5所示，该微板染料释放测定提供了比酶活性检测的MicroSlide扩散分析检测下限。对于快速的使用，节约劳动力，以及便于解释，麦克风roslide扩散分析提供了抗微生物活性的存在，以及抗微生物蛋白存在的下游蛋白质的分离和纯化步骤的指示初始的高通量筛选效用。这两项试验的组合，新的蛋白质抗菌药物初步筛选，并最终鉴定相得益彰

Antimicrobial agents play an essential role in targeting a wide range of microorganisms such as viruses, fungi, and bacteria. The antimicrobial activities produced by various bacteria range in target specificity from broad-spectrum to species-specific, including clinically relevant bacterial pathogens 1. In nature, protein antimicrobial compounds like bacteriocins and lysins represent a microbial arsenal of tools for self-defense, replication, and nutrient acquisition 2,3. Serving as mediators of population dynamics within microbial communities, protein antimicrobials provide a competitive advantage to the producing organism over nonproducing, sensitive species 4. As recombinant enzymes and probiotics, these proteins also have an economic and societal importance as the need for new sources of pathogen control increases with each new expansion of antimicrobial resistance within the bacterial population 5.
The first evaluations of newly discovered protein antimicrobials include determining the basic physical characteristics that are inherent to the enzyme. Methods for rapid screening of potential antimicrobials allow selection of candidates with hydrolytic activities against the desired target substrates. The microslide diffusion assay and the quantitative dye-release assay allow for the use of a variety of substrates in the detection of enzymatic hydrolysis. For protein antimicrobial enzymes with activity against the bacterial cell wall, the versatility of these assays allow rapid and effective, qualitative and quantitative screening against viable bacterial cells (microslide assay only), heat-killed whole bacterial cell substrates, or purified peptidoglycan from the target bacterium. These assays have utility in antimicrobial enzyme discovery for use in fields such as alternative therapeutics, food supplements, and inhibitors of biofilm production.
The microslide diffusion assay and the dye-release assay are demonstrated methods for detection and characterization of novel protein antimicrobials. The microslide diffusion assay provides a relative (qualitative) estimate of antimicrobial activity and allows for minimal inference of enzyme concentration and activity. Using this method, activity is readily visualized as the development of a zone of lysis with the absence of activity resulting in a lack of zone formation. The rate of zone development and the zone diameter are indicative of the amount and purity of the enzyme present in the sample; however, this rate and the quality of the substrate clearing within the zone can also be affected by the presence of contaminants, the completeness of the hydrolysis reaction, and the type of substrate. Interfering contaminants can affect the rate of enzyme diffusion from the well, while incomplete hydrolysis or variation of the substrate type can result in a turbid zone of lysis 6.
The dye-release assay quantitatively assesses the amount of covalently-linked Remazol brilliant blue R dye products released into the reaction supernatant from the enzymatically hydrolyzed substrate. The method has only slight variability associated with a difference in substrate, thus allowing greater sensitivity for detection of hydrolysis as compared to the microslide diffusion assay. These properties allow the method to have utility in the characterization of biochemical properties of the enzyme and in the standardization of the assay for comparison between different antimicrobial enzymes.
In this protocol, we provide methods to perform the basic microslide diffusion assay and dye-release assay, while demonstrating the scale of detection limits and variability for each. The assays are used to perform basic evaluations of an unknown protein antimicrobial purified from the culture supernatant of an uncharacterized soil bacterial isolate. Observed activities against bacterial cell substrates within the assays allow comparison of reaction activities between a previously uncharacterized protein antimicrobial and &#945;-chymotrypsin, a control proteolytic enzymes. These protocols provide a foundation for the application of the two techniques that can be expanded and modified to accommodate varied applications.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>48-well flat-bottom microplate with low evaporation lid</td>
			<td>Becton Dickinson</td>
			<td>3078</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well flat-bottom microplate (Costar 3595)</td>
			<td>Corning, Inc.</td>
			<td>3595</td>
			<td></td>
		</tr>
		<tr>
			<td>agarose</td>
			<td>Fisher Scientific</td>
			<td>BP162-100</td>
			<td></td>
		</tr>
		<tr>
			<td>&#945;-chymotrypsin</td>
			<td>MP Biomedicals</td>
			<td>215227205</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacillus subtilis 168</td>
			<td>American Type Culture Collection</td>
			<td>23857</td>
			<td></td>
		</tr>
		<tr>
			<td>C1000 Touch Thermal Cycler</td>
			<td>Bio-Rad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>cork borer</td>
			<td>Humboldt</td>
			<td>H-9661</td>
			<td></td>
		</tr>
		<tr>
			<td>lysozyme</td>
			<td>Sigma-Aldrich</td>
			<td>L6876-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>McFarland equivalence standard (2.0)</td>
			<td>Fisher Scientific</td>
			<td>R20412</td>
			<td></td>
		</tr>
		<tr>
			<td>microslides</td>
			<td>Fisher Scientific</td>
			<td>22-38-103</td>
			<td></td>
		</tr>
		<tr>
			<td>nutrient broth</td>
			<td>Fisher Scientific</td>
			<td>S25959B</td>
			<td></td>
		</tr>
		<tr>
			<td>peptidoglycan of Bacillus subtilis 168</td>
			<td>Sigma-Aldrich</td>
			<td>69554-10MG-F</td>
			<td></td>
		</tr>
		<tr>
			<td>phosphate-buffered saline (1&#215;)</td>
			<td>Teknova</td>
			<td>P0200</td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce BCA Protein Assay Kit</td>
			<td>Life Technologies</td>
			<td>23227</td>
			<td></td>
		</tr>
		<tr>
			<td>Synergy HT microplate spectrophotometer</td>
			<td>BioTek Instruments, Inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Remazol brilliant blue R dye (RBB)</td>
			<td>Sigma-Aldrich</td>
			<td>R8001</td>
			<td></td>
		</tr>
		<tr>
			<td>Salmonella enterica subsp. enterica</td>
			<td>American Type Culture Collection</td>
			<td>10708</td>
			<td></td>
		</tr>
		<tr>
			<td>sodium azide</td>
			<td>Fisher Scientific</td>
			<td>S227-100</td>
			<td></td>
		</tr>
		<tr>
			<td>sodium hydroxide (NaOH)</td>
			<td>Fisher Scientific</td>
			<td>S318-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Titer Tops pre-cut adhesive polyethylene film</td>
			<td>Diversified Biotechnology</td>
			<td>T-TOPS-50</td>
			<td></td>
		</tr>
		<tr>
			<td>UltraPure distilled water</td>
			<td>Invitrogen</td>
			<td>10977-015</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Solution</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>agarose solution 
			50 ml PBS 
			0.25 g agarose</td>
			<td></td>
			<td></td>
			<td>Add 10% sodium azide to the molten PBS/agarose solution</td>
		</tr>
		<tr>
			<td>nutrient broth medium 
			4 g nutrient broth 
			500 ml Type I water</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>250mM sodium hydroxide (NaOH) solution 
			1 g NaOH 
			99 ml Type I water</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>200mM Remazol brilliant blue R dye (RBB) 
			1.25 g RBB 
			98.75 ml 250 mM NaOH solution</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

qualitative and quantitative assays for detection and characterization of protein antimicrobials

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods to screen for target enzymes prior to investing greater research effort and resources. The goal of this protocol is to demonstrate two complementary assays for conducting these initial evaluations. The microslide diffusion assay provides an initial or simple detection screen to enable the qualitative and rapid assessment of proteolytic activity against an array of both viable and heat-killed bacterial target substrates. As a counterpart, the increased sensitivity and reproducibility of the dye-release assay provides a quantitative platform for evaluating and comparing environmental influences affecting the hydrolytic activity of protein antimicrobials. The ability to label specific heat-killed cell culture substrates with Remazol brilliant blue R dye expands this capability to tailor the dye-release assay to characterize enzymatic activity of interest.

该扩散的MicroSlide法和定量染料释放试验是用于筛选和测量的新的蛋白质抗菌药物，初步调查的有效方法。每个实验都有其优点和局限性;然而，在结合实施时，它们允许快速的初始筛选和抗微生物的基本特征。 该扩散的MicroSlide检测可以有效微生物库的快速筛选，生产抗菌蛋白。当酶浓度水平是令人关注的，检测的限制灵敏度可能会限制该测定，要求被加入到反应孔比染料释放测定酶的量更大。正如图1所示，测定的定性性质允许观察者进行比较在每个井本相对酶量。 图1A中所示的（25酶微克），该区域的前缘显示基板的混浊或不完全溶解，而最靠近孔的区显示一个更完整的基底裂解。这种现象，在前缘扩散酶的递减浓度的产物，该区域的直径的精确测量变得复杂。如在图1中观察到的孔B（15微克），C（10微克），D（5微克），E（1.0微克），和F（0.1微克），完全裂解的区的直径消散灭绝相关以酶的量减少。对于条件F（0.1微克），在琼脂糖内的酶浓度低于这将允许移动通过琼脂糖，水解基板而被可视化漫射酶限制。该扩散的MicroSlide测定也跑用纯化的枯草芽孢杆菌 peptidoglycan作为底物（ 图2）。而该区域是比那些全细胞沙门氏菌观察较少定义由于肽聚糖的磁阻在琼脂糖均匀暂停，肽聚糖由未知的抗菌酶水解是显而易见的。 染料释放测定比的MicroSlide扩散分析更灵敏的和通用的测定法，允许影响酶反应环境因素检测下限和变化。中的代表性测定中，温度是变化的，以确定所述抗微生物酶的最佳温度，确定为35℃的PBS中（ 图3）。这个最佳在反应上清液清楚地看到作为蓝色（ 图3B），以及在从吸光度测量在595nm（ 图3A）衍生的活性单位表示的量的增加。该染料释放测定的多功能性允许研究者变化不仅温度，而且在反应缓冲液和缓冲液组分，以迅速地确定对于给定的酶的最佳温育条件。 未知的抗菌酶（ 图4）和α-糜蛋白酶控制酶（ 图5）的活性水平是在35℃下在PBS中所确定的最佳培养温度对RBB标记枯草杆菌热灭活底物测定。从图4和图5的结果的比较表明，对未知的抗菌酶具有几乎两倍为B的亲和力枯草基板。此外，α胰凝乳蛋白酶的控制并没有完全消化热灭活B. （数据未显示）阱内枯草衬底。 α-糜蛋白酶控制的活动开始到板盟周围0.3微克相比，在活性单位持续上升的所有酶的量未知的抗菌酶（ 图4和图5），为。这可能表明该未知酶具有更大的持续活性或有切割位点的提供给B内的酶的较大数目枯草基板。 <img alt="图1" src="/files/ftp_upload/53819/53819fig1.jpg" /> 图 1:抗 沙门氏菌 全细胞底物 酶活性的的MicroSlide扩散测定用于定性评价对热灭活肠道沙门氏菌亚种未知蛋白的抗微生物活性的肠道 （ATCC 10708）。悬浮于磷酸盐缓冲盐水（PBS）的未知的抗微生物加入到各孔中该蛋白质量幻灯片包含25微克（A井），15微克（井B），10微克（以及C），5微克（井D），1微克（以及E）和0.​​1微克（以及F）。单独PBS中用于测定的阴性对照。裂解区的6小时培养后成像。 <a href="https://www.jove.com/files/ftp_upload/53819/53819fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/53819/53819fig2.jpg" /> 图 2:酶活性抗 枯草杆菌 肽聚糖细胞壁被使用的的MicroSlide扩散分析来定性评估对枯草芽孢杆菌 168的肽聚糖的未知蛋白的抗微生物的活性。在20微升的PBS，未知抗菌剂10微克暂停加入一个良好的microslides的。单独PBS用作阴性控制试验（井B）。裂解的区域在37℃，6小时培养后成像。 <a href="https://www.jove.com/files/ftp_upload/53819/53819fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/53819/53819fig3.jpg" /> 图3:最适反应温度为蛋白质抗菌染料释放测定的多功能性允许的物理和化学参数，例如培养温度和缓冲液的变化，以确定在感兴趣6的酶的活性的影响。如该图所示，未知蛋白的抗微生物（1微克）的活性在PBS下的范围内孵化温度的评价针对RBB标记热灭活枯草杆菌 。显示为活动单位（AU）的（A），RBB-B的吸收ound水解使用微板分光光度计在595nm处测量后的产品释放到上清液中。在各温度条件下，与没有添加酶反应被用来减去源于孵育在各种温度下的任何释放的染料的影响。对应于每个孵化温度，反应上清之前测定吸光度（B）的成像。 <a href="https://www.jove.com/files/ftp_upload/53819/53819fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/53819/53819fig4.jpg" /> 图4:对于蛋白质活性范围抗菌未知蛋白的抗微生物活性范围是为0，100，200，300，400，500，600，700，800，900，以及1000纳克过夜孵育后测得的活性单位如通过BCA PROT测EIN测定（A）。对于这些反应中，RBB标记B.枯草衬底水平升高在595nm得到的5.0的光密度，以确保与酶（1000纳克）的最高量，反应并不完全水解的反应潜伏期内的所有可用的衬底。与没有添加酶的对照反应被用来减去引起单独孵育任何释放的染料的影响。对应于每个酶用量，反应上清之前测量吸光度（B）的成像。 <a href="https://www.jove.com/files/ftp_upload/53819/53819fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/53819/53819fig5.jpg" /> 图5:对α胰凝乳蛋白酶的活性范围为α胰凝乳蛋白酶活性范围测量为ACTIV性单位过夜孵化后0，100，200，300，400，500，600，700，800，900，1000纳克酶（A）的。对于这些反应中，RBB标记B.枯草衬底水平升高在595nm得到的5.0的光密度，以确保与酶（1000纳克）的最高量，反应并不完全水解的反应潜伏期内的所有可用的衬底。与没有添加酶的对照反应被用来减去引起单独孵育任何释放的染料的影响。对应于每个酶用量，反应上清之前测量吸光度（B）的成像。 <a href="https://www.jove.com/files/ftp_upload/53819/53819fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Qualitative and Quantitative Assays for Detection and Characterization of Protein Antimicrobials at JoVE.com

Qualitative and Quantitative Assays for Detection and Characterization of Protein Antimicrobials

定性和定量测定为检测和蛋白质抗菌药物的表征

Here, we present protocols to rapidly screen and characterize enzymes for antimicrobial activity. The microslide diffusion assay and the dye-release assay utilize target bacterial substrates for qualitative and quantitative enzymatic activity evaluation.

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods ...

qualitative-quantitative-assays-for-detection-characterization

Research

JoVE Journal

Immunology and Infection

18.4K 次观看.  The MITRE Corporation. Here, we present protocols to rapidly screen and characterize enzymes for antimicrobial activity. The microslide diffusion assay and the dye-release assay utilize target bacterial substrates for qualitative and quantitative enzymatic activity evaluation.

视频: Qualitative and Quantitative Assays for Detection and Characterization of Protein Antimicrobials

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 ...

科研

免疫与感染

Protocol for Recombinant RBD-based SARS Vaccines: Protein Preparation, Animal Vaccination and Neutralization Detection

Based on their safety profile and ability to induce potent immune responses against infections, subunit vaccines have been used as candidates for a wide variety of pathogens 1-3. Since the mammalian cell system is capable of post-translational modification, thus forming properly folded and glycosylated proteins, recombinant proteins expressed in mammalian cells have shown the greatest potential to maintain high antigenicity and immunogenicity 4-6.
Although no new cases of SARS have been reported since 2004, future outbreaks are a constant threat; therefore, the development of vaccines against SARS-CoV is a prudent preventive step and should be carried out. The RBD of SARS-CoV S protein plays important roles in receptor binding and induction of specific neutralizing antibodies against virus infection 7-9. Therefore, in this protocol, we describe novel methods for developing a RBD-based subunit vaccine against SARS. Briefly, the recombinant RBD protein (rRBD) was expressed in culture supernatant of mammalian 293T cells to obtain a correctly folded protein with proper conformation and high immunogenicity 6. The transfection of the recombinant plasmid encoding RBD to the cells was then performed using a calcium phosphate transfection method 6,10 with some modifications. Compared with the lipid transfection method 11,12, this modified calcium phosphate transfection method is cheaper, easier to handle, and has the potential to reach high efficacy once a transfection complex with suitable size and shape is formed 13,14. Finally, a SARS pseudovirus neutralization assay was introduced in the protocol and used to detect the neutralizing activity of sera of mice vaccinated with rRBD protein. This assay is relatively safe, does not involve an infectious SARS-CoV, and can be performed without the requirement of a biosafety-3 laboratory 15.
The protocol described here can also be used to design and study recombinant subunit vaccines against other viruses with class I fusion proteins, for example, HIV, respiratory syncytial virus (RSV), Ebola virus, influenza virus, as well as Nipah and Handra viruses. In addition, the methods for generating a pseudovirus and subsequently establishing a pseudovirus neutralization assay can be applied to all these viruses.

This protocol describes a general procedure for studying recombinant receptor-binding domain (RBD)-based subunit vaccines against SARS. It includes methods for transfection and expression of RBD protein in 293T cells, immunization of mice with RBD and detection of neutralization activity of mouse sera using an established SARS pseudovirus neutralization assay.

协议RBD - SARS疫苗的重组蛋白的制备，动物疫苗接种和中检测

Based on their safety profile and ability to induce potent immune responses against infections, subunit vaccines have been used as candidates for a wide ...

Quantitative Analyses of all Influenza Type A Viral Hemagglutinins and Neuraminidases using Universal Antibodies in Simple Slot Blot Assays

Hemagglutinin (HA) and neuraminidase (NA) are two surface proteins of influenza viruses which are known to play important roles in the viral life cycle and the induction of protective immune responses1,2. As the main target for neutralizing antibodies, HA is currently used as the influenza vaccine potency marker and is measured by single radial immunodiffusion (SRID)3. However, the dependence of SRID on the availability of the corresponding subtype-specific antisera causes a minimum of 2-3 months delay for the release of every new vaccine. Moreover, despite evidence that NA also induces protective immunity4, the amount of NA in influenza vaccines is not yet standardized due to a lack of appropriate reagents or analytical method5. Thus, simple alternative methods capable of quantifying HA and NA antigens are desirable for rapid release and better quality control of influenza vaccines.
Universally conserved regions in all available influenza A HA and NA sequences were identified by bioinformatics analyses6-7. One sequence (designated as Uni-1) was identified in the only universally conserved epitope of HA, the fusion peptide6, while two conserved sequences were identified in neuraminidases, one close to the enzymatic active site (designated as HCA-2) and the other close to the N-terminus (designated as HCA-3)7. Peptides with these amino acid sequences were synthesized and used to immunize rabbits for the production of antibodies. The antibody against the Uni-1 epitope of HA was able to bind to 13 subtypes of influenza A HA (H1-H13) while the antibodies against the HCA-2 and HCA-3 regions of NA were capable of binding all 9 NA subtypes. All antibodies showed remarkable specificity against the viral sequences as evidenced by the observation that no cross-reactivity to allantoic proteins was detected. These universal antibodies were then used to develop slot blot assays to quantify HA and NA in influenza A vaccines without the need for specific antisera7,8. Vaccine samples were applied onto a PVDF membrane using a slot blot apparatus along with reference standards diluted to various concentrations. For the detection of HA, samples and standard were first diluted in Tris-buffered saline (TBS) containing 4M urea while for the measurement of NA they were diluted in TBS containing 0.01% Zwittergent as these conditions significantly improved the detection sensitivity. Following the detection of the HA and NA antigens by immunoblotting with their respective universal antibodies, signal intensities were quantified by densitometry. Amounts of HA and NA in the vaccines were then calculated using a standard curve established with the signal intensities of the various concentrations of the references used. 
Given that these antibodies bind to universal epitopes in HA or NA, interested investigators could use them as research tools in immunoassays other than the slot blot only.

A simple slot blot method was developed for the quantification of influenza viral hemagglutinin and neuraminidase using universal antibodies targeting their most conserved sequences identified through bioinformatics analyses. This innovative approach may provide a useful alternative to quantitative determination of all viral hemagglutinin and neuraminidase.

所有流感的定量分析类型使用通用抗体的一种病毒Hemagglutinins和Neuraminidases，在简单的狭缝杂交分析

Hemagglutinin (HA) and neuraminidase (NA) are two surface proteins of influenza viruses which are known to play important roles in the viral life cycle ...

Assays for the Identification of Novel Antivirals against Bluetongue Virus

To identify potential antivirals against BTV, we have developed, optimized and validated three assays presented here. The CPE-based assay was the first assay developed to evaluate whether a compound showed any antiviral efficacy and have been used to screen large compound library. Meanwhile, cytotoxicity of antivirals could also be evaluated using the CPE-based assay. The dose-response assay was designed to determine the range of efficacy for the selected antiviral, i.e. 50% inhibitory concentration (IC50) or effective concentration (EC50), as well as its range of cytotoxicity (CC50). The ToA assay was employed for the initial MoA study to determine the underlying mechanism of the novel antivirals during BTV viral lifecycle or the possible effect on host cellular machinery. These assays are vital for the evaluation of antiviral efficacy in cell culture system, and have been used for our recent researches leading to the identification of a number of novel antivirals against BTV.

Three assays, including the cytopathic effect (CPE)-based assay, dose-response assay and Time-of-Addition (ToA) assay have been developed, optimized, validated and utilized to identify novel antivirals against Bluetongue virus (BTV), as well as to determine the possible Mechanism-of-Action (MoA) for newly identified antivirals.

为检验新型抗病毒药物的鉴定对蓝舌病病毒

To identify potential antivirals against BTV, we have  developed, optimized and validated three assays presented here. The CPE-based  assay was the first ...

Methods for Quantitative Detection of Antibody-induced Complement Activation on Red Blood Cells

Antibodies against red blood cells (RBCs) can lead to complement activation resulting in an accelerated clearance via complement receptors in the liver (extravascular hemolysis) or leading to intravascular lysis of RBCs. Alloantibodies (e.g. ABO) or autoantibodies to RBC antigens (as seen in autoimmune hemolytic anemia, AIHA) leading to complement activation are potentially harmful and can be - especially when leading to intravascular lysis - fatal1. Currently, complement activation due to (auto)-antibodies on RBCs is assessed in vitro by using the Coombs test reflecting complement deposition on RBC or by a nonquantitative hemolytic assay reflecting RBC lysis1-4. However, to assess the efficacy of complement inhibitors, it is mandatory to have quantitative techniques. Here we describe two such techniques. First, an assay to detect C3 and C4 deposition on red blood cells that is induced by antibodies in patient serum is presented. For this, FACS analysis is used with fluorescently labeled anti-C3 or anti-C4 antibodies. Next, a quantitative hemolytic assay is described. In this assay, complement-mediated hemolysis induced by patient serum is measured making use of spectrophotometric&#160;detection of the released hemoglobin. Both of these assays are very reproducible and quantitative, facilitating studies of antibody-induced complement activation.

Here we describe two assays for measuring complement activation induced by antibodies against red blood cells. The major advantage over the current assays is their quantitative and easy-to-interpret nature.

定量检测抗体诱导补体激活的方法对红血细胞

Antibodies against red blood cells (RBCs) can lead to complement activation resulting in an accelerated clearance via complement receptors in the liver ...

Loop-mediated Isothermal Amplification (LAMP) Assays for the Species-specific Detection of Eimeria that Infect Chickens

Eimeria species parasites, protozoa which cause the enteric disease coccidiosis, pose a serious threat to the production and welfare of chickens. In the absence of effective control clinical coccidiosis can be devastating. Resistance to the chemoprophylactics frequently used to control Eimeria is common and sub-clinical infection is widespread, influencing feed conversion ratios and susceptibility to other pathogens such as Clostridium perfringens. Despite the availability of polymerase chain reaction (PCR)-based tools, diagnosis of Eimeria infection still relies almost entirely on traditional approaches such as lesion scoring and oocyst morphology, but neither is straightforward. Limitations of the existing molecular tools include the requirement for specialist equipment and difficulties accessing DNA as template. In response a simple field DNA preparation protocol and a panel of species-specific loop-mediated isothermal amplification (LAMP) assays have been developed for the seven Eimeria recognised to infect the chicken. We now provide a detailed protocol describing the preparation of genomic DNA from intestinal tissue collected post-mortem, followed by setup and readout of the LAMP assays. Eimeria species-specific LAMP can be used to monitor parasite occurrence, assessing the efficacy of a farm&#8217;s anticoccidial strategy, and to diagnose sub-clinical infection or clinical disease with particular value when expert surveillance is unavailable.

Loop-mediated Isothermal Amplification LAMP Assays for the Species-specific Detection of Eimeria that Infect Chickens

Diagnosis of Eimeria infection in chickens remains demanding. Parasite morphology- and host pathology-led approaches are commonly inconclusive, while molecular approaches based on PCR have proven demanding in cost and expertise. The aim of this protocol is to establish loop-mediated isothermal amplification (LAMP) as a straightforward molecular diagnostic for eimerian infection.

环介导等温扩增（LAMP）法进行的物种特异性检测<em&gt;艾美耳球虫</em&gt;可感染鸡

Eimeria species parasites, protozoa which cause the enteric disease coccidiosis, pose a serious threat to the production and welfare of chickens. In the ...

Qualitative and Quantitative Analysis of the Immune Synapse in the Human System Using Imaging Flow Cytometry

The immune synapse is the area of communication between T cells and antigen-presenting cells (APCs). T cells polarize surface receptors and proteins towards the immune synapse to assure a stable binding and signal exchange. Classical confocal, TIRF, or super-resolution microscopy have been used to study the immune synapse. Since these methods require manual image acquisition and time-consuming quantification, the imaging of rare events is challenging. Here, we describe a workflow that enables the morphological analysis of tens of thousands of cells. Immune synapses are induced between primary human T cells in pan-leukocyte preparations and Staphylococcus aureus enterotoxin B (SEB)-loaded Raji cells as APCs. Image acquisition is performed with imaging flow cytometry, also called In-Flow microscopy, which combines features of a flow cytometer and a fluorescence microscope. A complete gating strategy for identifying T cell/APC couples and analyzing the immune synapses is provided. As this workflow allows the analysis of immune synapses in unpurified pan-leukocyte preparations and hence requires only a small volume of blood (i.e., 1 mL), it can be applied to samples from patients. Importantly, several samples can be prepared, measured, and analyzed in parallel.

Here, we describe a complete workflow for the qualitative and quantitative analysis of immune synapses between primary human T cells and antigen-presenting cells. The method is based on imaging flow cytometry, which allows the acquisition and evaluation of several thousand cell images within a relatively short period of time.

影像性流式细胞仪对人体免疫突触的定性和定量分析

The immune synapse is the area of communication between T cells and antigen-presenting cells (APCs). T cells polarize surface receptors and proteins ...

Isolation of Peritoneum-derived Mast Cells and Their Functional Characterization with Ca2+-imaging and Degranulation Assays

Mast cells (MCs), as a part of the immune system, play a key role in defending the host against several pathogens and in the initiation of the allergic immune response. The activation of MCs via the cross-linking of surface IgE bound to high affinity IgE receptor (Fc&#949;RI), as well as through the stimulation of several other receptors, initiates the rise of the free intracellular Ca2+ level ([Ca2+]i) that promotes the release of inflammatory and allergic mediators. The identification of molecular constituents involved in these signaling pathways is crucial for understanding the regulation of MC function. In this article, we describe a protocol for the isolation of murine connective tissue type MCs by peritoneal lavage and cultivation of peritoneal MCs (PMCs). Cultures of MCs from various knockout mouse models by this methodology represent a useful approach to the identification of proteins involved in MC signaling pathways. In addition, we also describe a protocol for single cell Fura-2 imaging as an important technique for the quantification of Ca2+ signaling in MCs. Fluorescence-based monitoring of [Ca2+]i is a reliable and commonly used approach to study Ca2+ signaling events, including store-operated calcium entry, which is of utmost importance for MC activation. For the analysis of MC degranulation, we describe a &#946;-hexosaminidase release assay. The amount of &#946;-hexosaminidase released into the culture medium is considered as a degranulation marker for all three different secretory subsets described in MCs. &#946;-hexosaminidase can easily be quantified by its reaction with a colorigenic substrate in a microtiter plate colorimetric assay. This highly reproducible technique is cost-effective and requires no specialized equipment. Overall, the provided protocol demonstrates a high yield of MCs expressing typical MC surface markers, displaying typical morphological and phenotypic features of MCs, and demonstrating highly reproducible responses to secretagogues in Ca2+-imaging and degranulation assays.

Isolation of Peritoneum-derived Mast Cells and Their Functional Characterization with Ca2+-imaging and Degranulation Assays

Here, we present a protocol to isolate and cultivate murine peritoneal mast cells. We also describe two protocols for their functional characterization: a fluorescent imaging of intracellular free Ca2+ concentration and a degranulation assay based on colorimetric quantification of the released &#946;-hexosaminidase.

腹膜源性肥大细胞的分离及其功能特征的 Ca2 +成像和脱粒测定

Mast cells (MCs), as a part of the immune system, play a key role in defending the host against several pathogens and in the initiation of the allergic ...

Electrochemiluminescence Assays for Human Islet Autoantibodies

Pinpointing islet autoantibodies associated with type 1 diabetes (T1D) leads the way to project and deter this disease in the general population. A novel ECL assay is a nonradioactive fluid phase assay for islet autoantibodies with higher sensitivity and specificity than the current 'gold' standard radio-binding assay (RBA). ECL assays can more precisely define the onset of presymptomatic T1D by distinguishing the high-risk, high-affinity autoantibodies from the low-risk, low-affinity autoantibodies generated in RBAs, and conventional enzyme-linked immunosorbent assays (ELISA). The antigen protein used in this ECL assay is labeled with Sulfo-tag and Biotin, respectively. Each ECL autoantibody assay that uses a particular antigen protein needs an optimization step before it can be used for laboratory application. This step is especially vital in determining the requirements for serum acid treatments, concentrations, and ratios of the two different antigens labeled with Sulfo-tag and Biotin. To perform the assay, serum samples are mixed with Sulfo-tag-conjugated and biotinylated capture antigen protein in phosphate buffered solution (PBS), containing 5% Bovine Serum Albumin (BSA). Afterwards, the samples are incubated overnight at 4 &#176;C. The same day, a streptavidin-coated plate is prepared with blocker buffer and incubated overnight at 4 &#176;C. On the second day, wash the streptavidin plate and transfer the serum-antigen mixture onto the plate. Place the plate on the plate shaker, set it at low speed, and incubate at room temperature for 1 h. Subsequently, the plate is washed again, and reader buffer is added. The plate is then counted on the plate reader machine. The results are conveyed through an index, which is generated from internal standard positive and negative control serum samples.

Here, we presented the methods to detect human islet autoantibodies using electrochemiluminescence (ECL) assays. The protocol, used to predict type 1 diabetes, can be expanded upon to detect autoantibodies for other autoimmune diseases.

人胰岛自身抗体的电化学发光测定

Pinpointing islet autoantibodies associated with type 1 diabetes (T1D) leads the way to project and deter this disease in the general population. A novel ...

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella spp./Shigella spp. The gold standard method for the detection of Salmonella spp./Shigella spp. is direct culture but this is labor-intensive and time-consuming. Here, we describe a high-throughput platform for Salmonella spp./Shigella spp. screening, using real-time polymerase chain reaction (PCR) combined with guided culture. There are two major stages: real-time PCR and the guided culture. For the first stage (real-time PCR), we explain each step of the method: sample collection, pre-enrichment, DNA extraction and real-time PCR. If the real-time PCR result is positive, then the second stage (guided culture) is performed: selective culture, biochemical identification and serological characterization. We also illustrate representative results generated from it. The protocol described here would be a valuable platform for the rapid, specific, sensitive and high-throughput screening of Salmonella spp./Shigella spp.

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Salmonella spp./Shigella spp. are common pathogens attributed to diarrhea. Here, we describe a high-throughput platform for the screening of Salmonella spp./Shigella spp. using real-time PCR combined with guided culture.

沙门氏菌筛选的高通量平台.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella ...