Name: analysis of iophenoxic acid analogues in small indian mongoose (herpestes auropunctatus) sera for use as an oral rabies vaccination biological marker
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Description: Watch this Scientific Journal Video about Analysis of Iophenoxic Acid Analogues in Small Indian Mongoose Herpestes Auropunctatus Sera for Use as an Oral Rabies Vaccination Biological Marker at JoVE.co

这项研究部分得到了美国农业部、动植物卫生检验局、野生动物服务局、国家狂犬病管理项目和IDT Biologika(德国德绍-罗斯劳)的校内研究计划的支持。

所有程序都通过美国农业部国家野生动物研究中心的机构动物护理和使用委员会根据批准的研究协议QA-2597批准。
注:以下协议描述了检测蒙古血清中甲基-硫磷酸的分析过程。该方法是迭代过程的最终版本,从分析蒙古血清中的乙酰-硫磷酸开始。在初步评估乙基-硫磷酸时,对方法进行了细微的修改,最终形成了如下协议。代表性的结果包括两次迭代期间获得的结果。
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为研究开发的LC-MS/MS方法利用多反应监测的选择性,准确量化了ms-IPA和et-IPA中的驼鹿血清。MS/MS 检测的选择性还允许一个简单的清理方案,仅依靠醋酸酯在分析前从血清中沉淀蛋白质。
硫磷酸可溶于ACN,但几乎不溶于水。为了排除ACN萃取中的水,添加了氯化钠以迫使清水:ACN相分离,通过增加水(血清)相的离子强度。还添加了挥发性酸三氟化酸(TFA),以确保在提取过程中将硫磷酸进行质?...

狂犬病病毒(RABV)是一种负感单搁浅赖萨病毒,在食肉动物和奇罗普特拉的指令内的各种野生植物群中传播。多种驼鹿是RABV的储藏库,而小印度驼鹿是波多黎各和西半球其他加勒比岛屿中唯一的水库。1、2、3.控制野生生物水库的RABV循环通常是通过口服狂犬病疫苗接种(ORV)策略实现的。在美国(美国),这一管理活动由美国农业部/APHIS/野生动物服务局国家狂犬病管理计划(NRMP)4协调。目前波多黎各不存在野生动物ORV项目。对狂犬病疫苗和各种诱饵类型的研究已经进行,有希望的结果表明,一个ORV计划为驼鹿是可能的5,6,7,8。
监测ORV的影响依赖于估计目标物种的诱饵吸收,这通常涉及评估RV抗体血清患病率的变化。然而,在野生动物或病毒项目活跃的地区,或者在RV是动物的、RABV中和抗体(RVNA)的背景水平存在于储层物种的地区,这一策略可能具有挑战性。在这种情况下,诱饵或外部诱饵基质中包含的生物标志物可能很有用。
各种生物标记已经被用来监测诱饵的摄入在众多物种,包括熊(普罗西翁乐透)9,10, stoats(Mustela ermine)11,12,欧洲 (梅莱斯·梅莱斯)13、野猪(苏斯·斯克鲁瓦)14只,印度小猩猩15只,草原犬(西诺米卢斯多维丘斯)16只,17只,等等。在美国,操作的ORV诱饵通常包括1%四环素生物标志物在诱饵基质监测诱饵的捕杀18,19。然而,使用四环素的缺点包括越来越担心抗生素在环境中的分布,四环素的检测通常是侵入性的,需要拔牙或破坏动物才能获得骨骼样本20.罗丹胺B已被评估为各种组织中的标记物,可以用紫外线(UV)光和荧光在头发和胡须10、21中检测出来。
硫磷酸 (IPA) 是一种白色,结晶粉末,已用于评估在土狼(Canis latrans)22的诱饵消费, 北极狐 (Vulpes lagopus)23,红狐 (Vulpes vulpes)24,熊9,25、野猪14只、红鹿(塞弗乌斯·拉普胡斯)26只、欧洲12只野猪和雪铁龙(M.furo)27只,以及其他几种哺乳动物。 IPA的保留时间因物种而异,从一些动物不到两周28,29,到至少26周,在未顾胶26和超过52周的家庭狗(卡尼斯狼疮熟悉)30。保留时间也可能与剂量相关31。硫磷酸与血清白蛋白强结合,历史上通过测量血碘水平32来检测。这种间接方法被高性能液相色谱 (HPLC) 方法所取代,用 UV 检测33直接测量硫磷酸浓度,并最终使用液相色谱和质谱法 (LCMS)34,35.在这项研究中,开发了一种高度敏感和选择性的液相色谱与串联质谱法(LC-MS/MS)方法,利用多反应监测(MRM)来量化两种类似物的硫磷酸。我们的目标是使用此 LC-MS/MS 方法评估 2-(3-羟基-2,4,6-三硫多苯基)丙酸(甲基-IPA 或 me-IPA)和 2-(3-羟基-2、4、6-三硫多苯基)丁酸(乙基-IPA 或 et-IPA)的标记能力,并在 ORV 中交付诱饵圈养的蒙古人。
蒙古人被关在网箱陷阱中,诱饵上装有商业上可用的熏香肠和鱼油。蒙古被安置在单独的60厘米x60厘米x40厘米不锈钢笼子,并喂养每天口粮约50克商业干猫食品,每周两次补充与商业可用的鸡大腿。水是可用的。 我们向安慰剂或V诱饵中的圈养的驼鹿交付了IPA的两种衍生物,乙基IPA和甲基IPA。所有诱饵均由28毫米x20毫米x9毫米箔泡包组成,带有外部涂层(下同"诱饵基质"),含有粉状鸡蛋和明胶(材料表)。诱饵含有0.7 mL的水或IPA衍生物,重约3克,其中±2克为外部诱饵基质。
我们提供16个圈养的蒙古和IPA在三种浓度: 0.14% (2.8毫克和IPA在+2 g诱饵基质; 3男性[m],3雌性[f]),0.4%(0.7 mL水泡组体积为2.8毫克和IPA;3m,3f),和1.0%(0.7 mL起泡包体积为7.0毫克乙酰IPA;2m,2f)。总剂量为2.8毫克,相当于5毫克/千克27,36的剂量率,以波多黎各560克的平均驼鹿体重为基础。我们选择1%作为最高浓度,因为研究表明,某些生物标志物的味觉厌恶在某些物种37中可能发生浓度>1%。我们只提供水泡包中的1%剂量,因为絮凝剂防止溶质溶解在溶剂中充分,以均匀地融入诱饵基质。一个对照组(2米,1f)收到装满无菌水的诱饵,没有IPA。在喂食其日常维持配给期间或之前,我们于上午(上午 8 点)向驼鹿提供诱饵。大约24小时后,诱饵遗骸被移走。我们在治疗前采集血液样本,治疗后一天,治疗后每周8周。我们通过吸入亚福兰毒气来麻醉驼鹿,并通过颅面静脉穿刺收集高达1.0 mL的全血,如雪铁龙38所述。我们离心全血样本,将血清转移到冷冻液中,并将其储存在-80°C,直到分析。并非所有动物都对所有时间段进行取样,以尽量减少重复采血对动物健康的影响。对照动物在治疗后第0天进行采样,然后每周进行8周。
我们在三种浓度中交付了 me-IPA:0.035%(0.7 毫克)、0.07%(1.4 毫克)和 0.14% (2.8 毫克),全部纳入诱饵基质,每个治疗组有 2 名男性和 2 名女性。两名男性和两名女性接受充满无菌水的诱饵,没有IPA。提供时间和蒙古麻醉的诱饵如上所述。我们在治疗前第1天采集血液样本,然后在治疗后每周4周。
我们测试了血清浓度数据的正常性,并估计了不同治疗组血清IPA浓度的方法。我们使用线性混合模型来比较平均血清和IPA浓度汇集在个人之间。诱饵类型(矩阵/水泡包)是一个固定的效果,除了实验日,而动物ID是一个随机效应。所有程序均使用通用统计软件(材料表)运行,显著性在 ±0.05 处进行评估。

<table border="0" cellpadding="0" cellspacing="0" style="width:1036px;" width="1036">
	<tbody>
		<tr height="25">
			<td height="25" style="height:25px;width:380px;">Acetonitrile, Optima grade</td>
			<td style="width:193px;">Fisher</td>
			<td style="width:304px;">A996</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:380px;">Analytical balance</td>
			<td style="width:193px;">Mettler Toledo</td>
			<td style="width:304px;">XS204</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:380px;">C18 column, 2.1 x 50 mm, 2.5-&#181;m particle size</td>
			<td style="width:193px;">Waters Corp.</td>
			<td style="width:304px;">186003085</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:380px;">ESI Source</td>
			<td style="width:193px;">Agilent&#160;</td>
			<td style="width:304px;">G1958-65138</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:380px;">Ethyl-iophenoxic acid, 97 %</td>
			<td style="width:193px;">Sigma Aldrich</td>
			<td style="width:304px;">N/A</td>
			<td style="width:159px;">Lot MKBP5399V</td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:380px;">Formic acid, LC/MS grade</td>
			<td style="width:193px;">Fisher</td>
			<td style="width:304px;">A117</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:380px;">LCMS software</td>
			<td style="width:193px;">Agilent</td>
			<td style="width:304px;">MassHunter Data Acquisition and Quantitative Analysis</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:380px;">Methyl-iophenoxic acid, 97 % (w/w)</td>
			<td style="width:193px;">PR EuroChem Ltd.</td>
			<td style="width:304px;">N/A</td>
			<td style="width:159px;">Lot PR0709514717</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:380px;">Microanalytical balance</td>
			<td style="width:193px;">Mettler Toledo</td>
			<td style="width:304px;">XP6U</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:380px;">Microcentrifuge</td>
			<td style="width:193px;">Eppendorf</td>
			<td style="width:304px;">5415C</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:380px;">MS/MS</td>
			<td style="width:193px;">Agilent</td>
			<td style="width:304px;">G6470A</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:380px;">N-Evap</td>
			<td style="width:193px;">Organomation</td>
			<td style="width:304px;">115</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:380px;">Oral Rabies Vaccine Baits</td>
			<td style="width:193px;">IDT Biologika, Dessau Rossleau, Germany</td>
			<td style="width:304px;">N/A</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:380px;">Propyl-iophenoxic acid, 99 % (w/w)</td>
			<td style="width:193px;">PR EuroChem Ltd.</td>
			<td style="width:304px;">N/A</td>
			<td style="width:159px;">Lot PR100612108RR</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:380px;">Repeat pipettor</td>
			<td style="width:193px;">Eppendorf</td>
			<td style="width:304px;">M4</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:380px;">Screw-top autosampler vial caps, PTFE-lined</td>
			<td style="width:193px;">Agilent</td>
			<td style="width:304px;">5190-7024</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:25px;width:380px;">Sodium chloride, Certified ACS grade</td>
			<td style="width:193px;">Fisher</td>
			<td style="width:304px;">S271</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:380px;">Statistical Software Package</td>
			<td style="width:193px;">SAS Institute, Cary, North Carolina, USA</td>
			<td style="width:304px;">N/A</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:380px;">Trifluoroacetic acid, 99 %</td>
			<td style="width:193px;">Alfa Aesar</td>
			<td style="width:304px;">L06374</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:18px;width:380px;">UPLC</td>
			<td style="width:193px;">Agilent</td>
			<td style="width:304px;">1290 Series</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:380px;">Vortex Mixer</td>
			<td style="width:193px;">Glas-Col</td>
			<td style="width:304px;">099A PV6</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:380px;">0.2-mL pipettor tips</td>
			<td style="width:193px;">Eppendorf</td>
			<td style="width:304px;">30089.413</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:380px;">0.5-mL pipettor tips</td>
			<td style="width:193px;">Eppendorf</td>
			<td style="width:304px;">30089.421</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:380px;">1.5-mL microcentrifuge tubes</td>
			<td style="width:193px;">Fisher</td>
			<td style="width:304px;">14-666-325</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:380px;">1250-&#181;L capacity pipette tips</td>
			<td style="width:193px;">GeneMate</td>
			<td style="width:304px;">P-1233-1250</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:380px;">1-mL pipettor tips</td>
			<td style="width:193px;">Eppendorf</td>
			<td style="width:304px;">30089.43</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:380px;">2-mL amber screw-top autosampler vials</td>
			<td style="width:193px;">Agilent</td>
			<td style="width:304px;">5182-0716</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:380px;">5-mL pipettor tips</td>
			<td style="width:193px;">Eppendorf</td>
			<td style="width:304px;">30089.456</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:380px;">80-position microcentrifuge tube rack</td>
			<td style="width:193px;">Fisher</td>
			<td style="width:304px;">05-541-2</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:380px;">8-mL amber vials with PTFE-lined caps</td>
			<td style="width:193px;">Wheaton</td>
			<td style="width:304px;">224754</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:380px;">70 % (v/v) isopropanol</td>
			<td style="width:193px;">Fisher</td>
			<td style="width:304px;">A459</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:380px;">100-1000 &#181;L air displacement pipette</td>
			<td style="width:193px;">Eppendorf</td>
			<td style="width:304px;">ES-100</td>
			<td style="width:159px;"></td>
		</tr>
	</tbody>
</table>

analysis of iophenoxic acid analogues in small indian mongoose (herpestes auropunctatus) sera for use as an oral rabies vaccination biological marker

小型印度驼鹿(赫皮斯特龙),是波多黎各狂犬病病毒(RABV)的宿主,每年报告的动物狂犬病病例占70%以上。控制野生生物水库的RABV循环通常是通过口服狂犬病疫苗接种(ORV)策略实现的。目前波多黎各不存在野生动物ORV项目。对口服狂犬病疫苗和各种诱饵类型的研究已经展开,取得了可喜的成果。监测ORV的成功依赖于估计目标物种的诱饵吸收,这通常涉及评估接种后RABV中和抗体(RVNA)的变化。在有活跃的野生动物或病毒计划的地区,或在RABV是动物的和在水库物种中存在RVNA背景水平的地区,这种策略可能难以解释。在这种情况下,与疫苗或诱饵基质结合的生物标志物可能很有用。我们提供16个圈养的驼鹿安慰剂ORV诱饵含有乙基-硫磷酸(et-IPA)的浓度在诱饵内和0.14%在诱饵内和0.14%的诱饵内。我们还提供了12个含有甲基-硫磷酸(me-IPA)的圈养驼鹿ORV诱饵,其浓度为0.035%、0.07%和0.14%。我们在提供诱饵之前收集了血清样本,然后每周在提供后八周内收集。我们从血清中提取硫磷酸到醋酸,并使用液相色谱/质谱法进行定量。我们通过液相色谱-质谱法分析了血清的et-IPA或me-IPA。我们发现,对于et和me-IPA,分别至少8周和4周的标记能力。两种IPA衍生物都适用于对驼鹿的ORV诱饵摄入量进行实地评估。由于在蒙古塞的标记的寿命,必须小心,不要混淆结果,使用相同的IPA衍生物在连续评估。

图1中给出了来自me-IPA分析的代表性的环位图。对照蒙古斯血清(图1A)说明了et-IPA(代理Anlya)的保留时间和在指定保留时间没有me-IPA。质量控制样本(图1B)说明了me-IPA与et-IPA的基线分离,以及me-IPA的量化和限定符转换。图1C显示了该研究的代表性样本,观察到血清浓度为33.5微克/mL me-IPA。
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Watch this Scientific Journal Video about Analysis of Iophenoxic Acid Analogues in Small Indian Mongoose Herpestes Auropunctatus Sera for Use as an Oral Rabies Vaccination Biological Marker at JoVE.co

Analysis of Iophenoxic Acid Analogues in Small Indian Mongoose Herpestes Auropunctatus Sera for Use as an Oral Rabies Vaccination Biological Marker

我们提供以乙基或甲基硫磷酸为生物标志物的圈养驼鹿安慰剂口服狂犬病疫苗诱饵,并使用新型液相色谱法与串联质谱法(LC-MS/MS)方法验证诱饵的捕杀。

小印度蒙古狼的异苯抗酸模拟分析(赫皮斯特奥罗普塔图斯)塞拉,用作口服狂犬病疫苗接种生物标记

The small Indian mongoose (Herpestes auropunctatus) is a reservoir of rabies virus (RABV) in Puerto Rico and comprises over 70% of animal rabies cases ...

我们概述了检测和量化驼鹿血清中的碘氧酸的程序。结果表明，碘氧酸可用作生物标记物，以验证该物种的β吸收。这种方法的主要优点是，它允许使用简单的蛋白质沉淀技术，将碘氧酸类比在十亿级部分进行定量。对于移动阶段 A，将一毫升甲酸与一升超纯水混合。对于移动阶段 B，将一毫升甲酸与一升乙酰三叶酸混合。对于稀释剂，将一毫升三氟乙酸与200毫升乙酰酸合并。接下来，在微平衡上重约10毫克甲基-IPA，并记录质量为正负0001毫克。定量地将甲基-IPA转移到10毫升A类体积烧瓶中，使用4至5毫升的乙酰三酯。声波一分钟溶解所有固体，然后用醋酸酯体积。将每股约8毫升的琥珀色玻璃瓶与PTFE衬里瓶，并在室温下储存样品。然后，将剩余库存转移到危险废物中。对于25X库存7甲基IPA库存准备的甲基IPA和乙酰三叶酸库存，每毫升约200微克。将一毫升的库存转移到五毫升的烧瓶中，然后用乙酰三叶酸稀释成体积。然后，将库存转移到一个八毫升琥珀色玻璃瓶与PTFE衬里盖和储存在室温下。使用重复移液器，在文本协议中描述的六个附加的 25X 甲基 IPA 库存中，用 PTFE 衬里盖准备 8 毫升琥珀玻璃小瓶，并在室温下储存。对于 25X 代理库存，使用 100 微升玻璃注射器将 0.1 毫升浓缩乙基-IPA 库存转移到 10 毫升 A 级卷瓶中，然后用乙酰三酯稀释至体积。将代理库存的大约 8 毫升转移到带 PTFE 衬里盖的 8 毫升琥珀色玻璃瓶中，并在室温下储存。将剩余库存转移到危险废物中。接下来，在10毫升螺丝顶玻璃自动取样器小瓶中制备含有甲基-IPA和乙基-IPA的4X库存，如文本协议中所述。例如，要准备库存 4X7，使用具有 0.5 毫升容量尖端的重复移液器将 25X 库存 7 甲基 IPA 库存的 0.2 毫升添加到两毫升小瓶中。使用具有 0.5 毫升容量尖端的重复移液器，加入 25X 代理乙基 IPA 库存的 0.2 毫升。使用具有 1 毫升容量尖端的重复移液器添加 0.85 毫升的乙酰三叶酸。安全盖住小瓶，倒置五次，以混合。之后，按照文本协议中所述，在两毫升螺丝顶自动采样器小瓶中准备标准曲线。例如，要准备标准七，将 4X7 库存的 0.2 毫升添加到两毫升小瓶中，使用具有 0.5 毫升容量尖端的重复移液器。使用具有 1 毫升容量尖端的重复移液器加入 0.6 毫升的超纯度电离水。安全盖住小瓶，倒置五次以混合。对于每个样品，准备一个1.5毫升的微型离心管，内含200至300毫克氯化钠，并安排在80位置的塑料架上。对于每个样品，将一个 1.5 毫升微型离心管标记为 A，将第二个微型离心管标记为 B。将血清提取所需的以下材料和设备放在二类生物安全柜中：微型离心管、 涡流混合器、具有 0.5 毫升和 5 毫升容量的重复移液器、带 1000 微升尖端的 100 至 1000 微升空气位移液器、每个具有约 100 毫升稀释剂和超纯度电离水的容器以及生物危害废物容器。接下来，从冷冻储存中去除血清样本，并在生物安全柜中加热至室温。漩涡在取样前混合每个血清样本。使用具有 0.5 毫升容量尖端的重复移液器，将 05 毫升的驼鹿血清分配到管 A 中，并加入 25 倍代理库存的 05 毫升。然后使用具有 5 毫升容量尖端的重复移液器向管 A 添加 0.95 毫升稀释剂。牢固地盖住管子，将涡流混合10至15秒。接下来，将控制驼鹿血清添加到管A.使用具有0.5毫升容量尖端的重复移液器，用我-IPA强化每个4QC样品。将 25 倍代理库存添加到 QC 样本中。然后，将稀释剂添加到 QC 样本中。盖住管子和涡流混合。将预称氯化钠分配到管 A 中，在 8 到 12 秒内将涡流混合三次。然后，使用 70%异丙醇擦拭装有管 A 的小瓶架外表面。从生物安全柜中去除样品后，在1200次G下离心管S1分钟分离水位和乙酰三叶酸相。移液器 0.8 毫升的上乙酰三角到管 B，使用 100 至 1000 微升空气位移液器。将管 A 中剩余的溶液转移到危险废物中，将空管丢弃在生物危害废物容器中。现在，在45摄氏度的水浴中，用温和的氮气从B管中去除乙酮和三氟乙酸酯。使用重复的移液器和涡流混合，将0.25毫升乙酰酸杆菌加入B管，进行4至5秒钟的涡流混合。然后，在1200倍G下离心2至4秒，收集管底的液体。离心后，使用具有5毫升容量的重复移液器将0.75毫升的超纯去离子水加入B管，并加入4至5秒的涡流混合。在 1200 倍 G 下离心一分钟，以澄清样品。然后，使用1000微升空气位移液器将0.75毫升的上流液转移到自动取样器小瓶中，并丢弃生物危害废物容器中的移液器尖端。安全盖住每个自动取样器小瓶，进行LC-MS/MS分析。将B管中剩余的溶液转移到危险废物中，将空管丢弃在生物危害废物容器中。使用文本协议中描述的所有参数配置 LC-MS/MS。打开 LC-MS/MS 电源，使柱达到 70 摄氏度，然后将流量设置为每分钟 0.8 毫升。在数据采集软件中设置一个序列，在每批由质量控制样品和未知样品组成的批次之前和之后注入标准曲线。使用文本协议中列出的参数注入所有标准和样本并获取 MR 离子色谱图。序列完成后，关闭 LC-MS/MS 并处置危险废物中的所有自动取样器小瓶。对照驼鹿血清的离子色谱说明了乙基-IPA的保留时间以及甲基-IPA在指示的保留时间没有。质量控制样品的离子色谱图说明了甲基-IPA与乙基-IPA的基线分离，以及甲基-IPA的限定符和限定符过渡。研究中代表性样品的离子色谱图显示，观察到的血清浓度为每微升甲基 IPA 33.5 微克。此处显示了用甲基 IPA 强化的控制驼鹿血清的准确性和精度结果。回收百分比从96.9%到109%不等，三个防御工事水平的相对偏差率分别是3.4%和2.3%。此处显示了使用乙基-IPA 强化的控制驼鹿血清的精度和精度结果。回收率从89.5%到115%不等，五个防御工事水平的相对标准偏差率分别是4.3%1.5%2.3%5.6%和1.1%。所有提供乙基-IPA和甲基-IPA诱饵的驼鹿在24小时时间限制内消耗了至少25%的诱饵，并分别在其血清中含有乙基-IPA和甲基-IPA的可量化水平。通过配置LC-MS来收集其他异丙酸类比的数据，如丙烯和丁基氧氧酸，可以增加该方法的潜在效用。我们鼓励人员咨询免疫做法咨询委员会或机构生物安全委员会的最新建议，以指导是否需要接种接触前狂犬病疫苗。本协议涉及未稀释血清的第 2.3 至 2.6 节应在第二类生物安全柜中执行。

Research

JoVE Journal

Immunology and Infection

Analysis of Iophenoxic Acid Analogues in Small Indian Mongoose (Herpestes Auropunctatus) Sera for Use as an Oral Rabies Vaccination Biological Marker

An Analytical Tool-box for Comprehensive Biochemical, Structural and Transcriptome Evaluation of Oral Biofilms Mediated by Mutans Streptococci

口腔生物膜的生物化学，结构和转录综合评价分析工具盒介导变形链球菌

Biofilms are highly dynamic, organized and structured communities of microbial cells enmeshed in an extracellular matrix of variable density and ...

科研

免疫与感染

Introducing Shear Stress in the Study of Bacterial Adhesion

在细菌粘附的研究剪应力

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

寄生蜂简介<em&gt;果蝇</em&gt;和Antiparasite的免疫反应的

Most known parasitoid wasp species attack the larval or pupal stages of Drosophila. While Trichopria drosophilae infect the pupal stages of the host (Fig. ...

Locked Nucleic Acid Flow Cytometry-fluorescence in situ Hybridization (LNA flow-FISH): a Method for Bacterial Small RNA Detection

Locked Nucleic Acid Flow Cytometry-fluorescence in situ Hybridization LNA flow-FISH: a Method for Bacterial Small RNA Detection

锁核酸流式细胞仪，荧光原位杂交（LNA流FISH）：细菌小RNA检测方法

Fluorescence in situ hybridization (FISH) is a powerful technique that is used to detect and localize specific nucleic acid sequences in the cellular ...

Use of Galleria mellonella as a Model Organism to Study Legionella pneumophila Infection

Use of Galleria mellonella as a Model Organism to Study Legionella pneumophila Infection

使用<em&gt;大蜡螟</em&gt;作为模式生物来研究<em&gt;嗜肺军团菌</em&gt;感染

Legionella pneumophila, the causative agent of a severe pneumonia named Legionnaires&#39; disease, is an important human pathogen that infects and ...

Intralymphatic Immunotherapy and Vaccination in Mice

淋巴免疫治疗和疫苗接种小鼠

Vaccines are typically injected subcutaneously or intramuscularly for stimulation of immune responses. The success of this requires efficient drainage of ...

Porphyromonas gingivalis as a Model Organism for Assessing Interaction of Anaerobic Bacteria with Host Cells

Porphyromonas gingivalis as a Model Organism for Assessing Interaction of Anaerobic Bacteria with Host Cells

牙龈卟啉单作为模式生物的评价厌氧细菌的相互作用与宿主细胞

Anaerobic bacteria far outnumber aerobes in many human niches such as the gut, mouth, and vagina. Furthermore, anaerobic infections are common and ...

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

高通量设置的建立筛选小分子能调节C-二GMP信令<em&gt;绿脓杆菌</em

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. ...

In Vitro Methods for Comparing Target Binding and CDC Induction Between Therapeutic Antibodies: Applications in Biosimilarity Analysis

In Vitro Methods for Comparing Target Binding and CDC Induction Between Therapeutic Antibodies: Applications in Biosimilarity Analysis

体外方法比较标的结合和CDC诱导治疗性抗体之间：应用在Biosimilarity分析

Therapeutic monoclonal antibodies (mAbs) are relevant to the treatment of different pathologies, including cancers. The development of biosimilar mAbs by ...

In Vitro ELISA Test to Evaluate Rabies Vaccine Potency

体外ELISA测试评估狂犬病疫苗效力

The growing global concern for the animal welfare is encouraging manufacturers and the National Control Laboratories (OMCLs) to follow the 3Rs strategy ...