我们感谢纽约州卫生部沃兹沃思中心对这个项目的支持。

以下方案已被纽约州卫生部沃兹沃思中心批准用于人体样本的道德使用，用于检测开发，方案批准号#03-019。
1. 安全
<ol><li>穿戴个人防护装备 （PPE），至少戴上护目镜（眼镜或面罩）、外科口罩和非乳胶手套。</li>
	<li>确保医务人员接种狂犬病疫苗，并且在过去 6 个月内证明滴度为 ≥0.5 IU/mL。</li>
</ol>2.抗原载玻片制备
注意：使用通用预防措施在生物安全柜 （BSC） 中执行所有病毒、脑脊液和血清操作。
<ol><li>在补充有 10% 胎牛血清 （EGM） 的 Eagle 最低必需培养基中制备 20 mL 小鼠神经母细胞瘤或 BHK-21 细胞，浓度为 3.0 x 105 个细胞/mL，并保持低温直至使用。</li>
	<li>通过在EGM中稀释至每毫升1.0 x10 6.5 50%组织培养感染剂量（TCID50）的工作稀释度来制备CVS-11病毒，并保持低温直至准备使用。 注：TCID50 由先前发表的 Reed 和 Muench 方法确定5.</li>
	<li>用70%乙醇清洁湿度载玻片室和聚四氟乙烯（PTFE）涂层的孔显微镜载玻片，并在BSC中风干。</li>
	<li>将蒸馏水（dH2O）添加到载玻片室中的吸水纸条中，以确保湿度在整个过程中保持恒定。</li>
	<li>用铅笔在每张载玻片上贴上批号、日期、细胞类型和储存所需的任何其他识别信息，然后放入载玻片室中。 注意：大多数记号笔、笔或标签都无法承受未来的丙酮固定步骤（步骤 2.9）。</li>
	<li>用重复移液管将 50 μL 病毒稀释液应用于显微镜载玻片上的每个孔。然后，将 50 μL 细胞稀释液应用于每个孔，注意不要让孔中已有的病毒污染移液器吸头。</li>
	<li>关闭湿度载玻片室，将其置于34-36°C的潮湿培养箱中。 24小时后评估细胞感染性。 注意：仅在一张幻灯片上执行步骤 2.8-2.12。</li>
	<li>从湿度室中取出载玻片，小心地吸出上清液。在充满磷酸盐缓冲盐水（PBS）的Coplin罐中洗涤载玻片2分钟，然后让载玻片风干。</li>
	<li>将载玻片放入Coplin罐中，并将载玻片在冷丙酮中固定至少1小时，在-20°C冰箱中批准用于易燃材料。在通风橱中执行所有丙酮浇注和风干程序。</li>
	<li>让丙酮闪光并滑动晾干。将根据制造商的说明制备的狂犬病直接荧光抗体（DFA）偶联物施加到载玻片的孔中，并在34-36°C潮湿的培养箱中孵育30分钟。</li>
	<li>在PBS的Coplin罐中洗涤载玻片两次，每次2分钟。风干载玻片。</li>
	<li>用0.05M Tris，0.15M NaCl（pH 9.0）和20%甘油封片剂安装盖玻片，并使用荧光显微镜以200倍放大倍率读取载玻片以评估感染性。如果细胞不是大约50%的感染，则在第二天重复步骤2.7-2.12，直到达到所需的感染性。 注：细胞感染性是根据目视评估载玻片孔中阴性细胞与狂犬病阳性细胞的比例来近似的。阴性细胞呈红色，而阳性细胞呈绿色荧光染色。</li>
	<li>从培养箱和湿度室中取出剩余的载玻片。小心地从每个孔中吸出上清液，然后将载玻片放入装有PBS的Coplin罐中1-2分钟。将载玻片风干约30分钟。将载玻片储存在-80°C直至准备使用。</li>
</ol>3. 样品制备
<ol><li>准备患者血清或脑脊液样本稀释液进行检测。制备必要的偶联物，以获得在PBS中用0.05%埃文斯蓝稀释的适当工作浓度。 注意：在应用前将偶联物保持在黑暗中以保持荧光基团的完整性</li>
</ol>4. IFA程序
<ol><li>取出每次测定所需的必要数量的制备抗原载玻片，并让载玻片解冻并完全干燥。</li>
	<li>将载玻片放入Coplin罐中，并将载玻片在冷丙酮中固定2小时至过夜，在-20°C冰箱中批准用于易燃材料。从丙酮中取出载玻片并风干。</li>
	<li>将载玻片放入 BSC 内的湿度箱中，并用 dH2O 浸泡的吸收条以保持湿度。将 50 μL 每种样品稀释液、对照样品或 PBS 施加到预定孔中。 注意：每张载玻片必须包含适当数量的患者样品孔、阳性对照、阴性对照和 PBS 细胞对照孔。</li>
	<li>将封闭的湿度载玻片室置于37°C，5%CO2 潮湿的培养箱中。将载玻片孵育 30 分钟，然后从培养箱中取出湿度载玻片室并将其放入 BSC 中。</li>
	<li>使用吸液吸头小心地从每个孔中吸出上清液，确保不会干扰细胞单层。使用无菌滴管移液器将一滴PBS滴入每个孔。</li>
	<li>重复仔细抽吸，然后将每个载玻片放入装有PBS的Coplin罐中，洗涤两次，总共15分钟。</li>
	<li>将载玻片放回湿度室盒中，并在每个孔中涂抹 50 μL 适当的抗人抗体偶联物。重复步骤 4.4 到 4.6。</li>
	<li>让载玻片风干，用封固剂安装盖玻片，并在荧光显微镜下读取载玻片。</li>
</ol>5. 幻灯片分析
<ol><li>根据染色模式和荧光强度对样品与已确认具有高抗狂犬病抗体滴度的阳性对照样品进行比较进行分级。</li>
	<li>以阴性、1+、2+、3+ 和 4+ 的等级对样品进行分级，阴性结果显示没有荧光染色，4+ 表示亮绿色苹果色荧光，染色模式类似于阳性对照样品1。 注意：青苹果色仅适用于FITC标记的抗体;颜色因荧光基团选择而异。</li>
	<li>为样品分配一个终点值，该终点值由稀释因子表示，在该浓度下，样品显示 1-2+ 等级。如果在初始测定中未达到终点，则在较高的稀释因子下测试样品。</li>
</ol>

快速、早期检测狂犬病抗体以评估患者的免疫反应

<ol>
	<li>Rupprecht, C. E., Fooks, A. R., Abela-Ridder, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laboratory+Techniques+in+Rabies.+Volume+1.">Laboratory Techniques in Rabies. Volume 1.</a> World Health Organization. , 232-245 (2018).</li><li>Moore, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Challenges+of+rabies+serology:+defining+context+of+interpretation.">Challenges of rabies serology: defining context of interpretation.</a> Viruses. 13 (8), 1516 (2021).</li><li>Zajac, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+evaluation+of+a+rabies+enzyme-linked+immunosorbent+assay+(ELISA)+targeting+IgM+and+IgG+in+human+sera.">Development and evaluation of a rabies enzyme-linked immunosorbent assay (ELISA) targeting IgM and IgG in human sera.</a> Viruses. , 40-49 (2019).</li><li>Mills, D. J., Lau, C. L., Mills, C., Furuya-Kanamori, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+persistence+of+antibodies+and+boostability+after+rabies+intradermal+pre-exposure+prophylaxis.">Long-term persistence of antibodies and boostability after rabies intradermal pre-exposure prophylaxis.</a> Journal of Travel Medicine. 29 (2), (2022).</li><li>Ramakrishnan, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+50%+endpoint+titer+using+a+simple+formula.">Determination of 50% endpoint titer using a simple formula.</a> World Journal of Virology. 5 (2), 85-86 (2016).</li><li>Rudd, R. J., Appler, K. A., Wong, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presence+of+cross-reactions+with+other+viral+encephalitides+in+the+indirect+fluorescent-antibody+test+for+diagnosis+of+rabies.">Presence of cross-reactions with other viral encephalitides in the indirect fluorescent-antibody test for diagnosis of rabies.</a> Journal of Clinical Microbiology. 51 (12), 4079-4082 (2013).</li><li>Fooks, A. R., Jackson, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Rabies: scientific basis of the disease and its management. , (2020).</li><li>Paldanius, M., Bloigu, A., Leinonen, M., Saikku, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+Chlamydia+pneumoniae-specific+immunoglobulin+A+(IgA)+antibodies+by+the+microimmunofluorescence+(MIF)+method:+comparison+of+seven+fluorescein-labeled+anti-human+IgA+conjugates+in+an+in-house+MIF+test+using+one+commercial+MIF+and+one+enzyme+immunoassay+kit.">Measurement of Chlamydia pneumoniae-specific immunoglobulin A (IgA) antibodies by the microimmunofluorescence (MIF) method: comparison of seven fluorescein-labeled anti-human IgA conjugates in an in-house MIF test using one commercial MIF and one enzyme immunoassay kit.</a> Clinical and Diagnostic Laboratory Immunology. 10 (1), 8-12 (2003).</li><li>Rodriguez, M. C., Fontana, D., Garay, E., Prieto, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+and+quantification+of+anti-rabies+glycoprotein+antibodies:+current+state+and+perspectives.">Detection and quantification of anti-rabies glycoprotein antibodies: current state and perspectives.</a> Applied Microbiology and Biotechnology. 105 (18), 6547-6557 (2021).</li><li>Katz, I. S. S., Guedes, F., Fernandes, E. R., Dos Ramos Silva, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunological+aspects+of+rabies:+a+literature+review.">Immunological aspects of rabies: a literature review.</a> Archives of Virology. 162 (1), 3251-3268 (2017).</li><li>Moore, S. M., Hanlon, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rabies-specific+antibodies:+measuring+surrogates+of+protection+against+a+fatal+disease.">Rabies-specific antibodies: measuring surrogates of protection against a fatal disease.</a> PLoS Neglected Tropical Diseases. 4 (3), 595 (2010).</li></ol>

作者没有什么可透露的。

IFA 检测利用抗原-抗体复合物，允许标记位点可视化狂犬病特异性抗体。将神经母细胞瘤或BHK细胞接种在多孔PTFE涂层的显微镜载玻片上，并接种狂犬病病毒实验室菌株CVS-11。一旦单层汇合并且细胞达到约50%的所需感染性，载玻片将被储存直至准备使用6。
将患者血清或脑脊液涂在感染的单层细胞上并孵育，以使任何狂犬病特异性抗体附着在病毒抗原7 上。在洗涤程序之后，应用荧光标记的抗人 IgG 或 IgM 抗体，并与来自样品应用的任何病毒结合抗体结合。然后可以在荧光显微镜下观察标记的抗体。
在准备进行该测定时，确定患者样品、对照品和偶联物的最佳制备方法非常重要。初始患者样本在低稀释因子或未稀释下进行筛选，以测试是否存在任何抗体。然后将配对样品或先前筛选的样品进一步稀释至终点。用于 IgG 检测的初始患者样本稀释液在市售 IFA 稀释剂中制备。IgM 检测样品最初是在市售的 IgG 阻断试剂中制备的。然后，在PBS中制备两种测试的后续连续稀释液。
用于检测的对照来自已知的狂犬病PrEP接受者或没有狂犬病疫苗接种史的个体。所有对照样品在使用前均进行了抗体检测。IgG 对照来自狂犬病疫苗接受者，其狂犬病中和抗体滴度已确定，经 FAVN 确认。IgM阳性对照样本是在完成疫苗接种后约2周从狂犬病疫苗接种者身上获得的。
使用适当的偶联物是进行IFA测试的另一个重要方面。用于每种检测的偶联物取决于该检测的抗体同种型靶标。在本例中，使用了市售的FITC标记的抗人IgG和抗人IgM抗体。在测试之前，根据制造商的建议确定工作抗体偶联物的稀释度。
该程序中有几个关键步骤将确保IFA测试的成功执行，也许最重要的是抗原载玻片制备。达到约 50% 的感染性可为可用抗体提供丰富的结合位点，同时在读取和分级样品时也能清晰地显示。狂犬病阴性细胞产生对比背景，以更好地可视化标记的抗体。当使用复杂的样品基质（如血清）进行荧光染色时，非特异性染色也会出现问题。在评估 IgM8 的存在时，使用 IgG 灭活试剂（例如 Gullsorb）有助于减少由于干扰 IgG 抗体而导致的非特异性染色。正确制备材料并将特殊试剂掺入程序有助于减少有问题的染色，同时保持高质量的结果。
已经开发了多种检测方法，专注于狂犬病抗体的检测、定量和鉴定。这些测试中的每一个都提供了可以根据所寻求的信息以各种方式使用的结果。狂犬病 IFA 检测是识别病毒特异性抗体同种型的有力工具，与其他检测方法（如 RFFIT 和 FAVN 检测）相比，可以在相对较短的时间内获得结果。
虽然IFA检测可以检测样本中的狂犬病抗体，但它不能提供抗体的标准化定量。IFA 检测提供血清稀释因子，抗体检测在该因子处结束。相比之下，RFFIT 和 FAVN 测试量化了样品中抗体的中和能力，从而得出国际单位 （IU） 的滴度，从而提供更详细和标准化的结果。FAVN 或 RFFIT 测试的结果表明存在中和抗体，这些抗体已被确定为 IgG 抗体9。IgM在中和活性中的作用被认为是有限的，因为它的结构10。因此，这些检查不能特异性检测IgM的存在。
当将它们应用于特定问题时，测试类型及其结果可能会有些问题。针对狂犬病病毒感染的抗体保护水平的概念不再用于评估对狂犬病疫苗接种的免疫反应。以前，保护性反应定义为 ≥0.5 IU。然而，目前的出版物通常将免疫后的抗体反应称为可接受（≥0.5 IU）或不可接受（<0.5 IU）。如前所述，IFA测试不能提供IU的抗体滴度，不应用于得出对狂犬病的保护水平。在评估患有狂犬病的患者时使用 IFA 检测可能并不总是导致抗体呈阳性，因为整个疾病期间免疫反应各不相同。由于狂犬病感染的致死率接近 100%，因此重要的是要考虑检测以及从这些结果中可以安全地获得多少信息11。由于这些原因，最好评估所有狂犬病抗体检测方法，并确定哪种方法在特定情况下最有效。

狂犬病间接荧光抗体（IFA）检测用于检测血清或脑脊液中的各种狂犬病特异性抗体同种型。它是可用于监测生前狂犬病患者的一系列测试之一。它对于早期检测抗体以评估患者对狂犬病感染的免疫反应特别有用。当与其他检测、病史和患者的疫苗接种状态结合使用时，IFA 检测可以帮助确定狂犬病病毒或疫苗的暴露情况1.当 IFA 检测测量 IgM 和/或 IgG 时，特异性抗体的值可以指示从暴露于抗原1 开始的大致时间范围。该测试在列出的应用程序或其他尚未探索的应用程序中可能有用。
有几种狂犬病血清学检测方法可用。快速荧光聚焦抑制试验 （RFFIT）、荧光抗体病毒中和 （FAVN） 试验或这些试验的改良是测量狂犬病病毒中和抗体 （RVNA） 的主要方法1。然而，这些测试不能区分 IgM 和 IgG 抗体。当鉴别抗体同种型对监测狂犬病免疫应答很重要时，可使用狂犬病 IFA 和狂犬病酶联免疫吸附测定 （ELISA） 检测，但不检测 RVNA。尽管 IFA 和 ELISA 检测可用于确定样本中是否存在狂犬病特异性抗体，但它们的执行方式存在一些差异。IFA 检测使用细胞培养的活病毒作为其抗原底物，而用于狂犬病检测的典型 ELISA 使用一种或多种病毒蛋白。在可以培养狂犬病病毒的实验室环境中，IFA 检测可能更容易进行，而不是购买或培养用于 ELISA 的单个病毒蛋白。在决定选择哪种方法时，应考虑检测目的和从任何狂犬病血清学检测结果中获得的信息2.
IgM 最先产生反应，直到在第 28 天左右观察到类别转换，此时 IgG 成为主要的循环抗体3。因此，IgM只会在暴露于狂犬病病毒或接种疫苗后的有限时间内出现。检测血清和脑脊液 （CSF） 可以表明暴露是通过疫苗接种（其中抗体仅在血清中可见）还是来自病毒感染（可能在 CSF1 中显示抗体）。
已经确定，狂犬病抗体在暴露前预防 （PrEP） 后会持续数年4。IFA测试可以成为在接种疫苗或暴露后的不同时间点证明这一点的有用工具。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>25x55mm glass cover slips</td>
			<td>Any</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>Any</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Human IgG Labeled Conjugate</td>
			<td>Sigma-Aldrich</td>
			<td>F9512</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Human IgM Labeled Conjugate</td>
			<td>SeraCare</td>
			<td>5230-0286</td>
			<td></td>
		</tr>
		<tr>
			<td>Aspirating pipette tip</td>
			<td>Any</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BHK-21 Cells</td>
			<td>ATCC</td>
			<td>CCL-10</td>
			<td></td>
		</tr>
		<tr>
			<td>BION IFA Diluent</td>
			<td>MBL BION</td>
			<td>DIL-9993</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Culture water</td>
			<td>Sigma-Aldrich</td>
			<td>W3500</td>
			<td>EGM</td>
		</tr>
		<tr>
			<td>Coplin Jars</td>
			<td>Any</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>F2442</td>
			<td>EGM</td>
		</tr>
		<tr>
			<td>Fluorescent microscope with FITC filter</td>
			<td>Any</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma-Aldrich</td>
			<td>G7893</td>
			<td>Mountant</td>
		</tr>
		<tr>
			<td>Gullsorb IgM inactivation reagent</td>
			<td>Fisher Scientific</td>
			<td>23-043-158</td>
			<td>IgG Inactivation Reagent</td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Sigma-Aldrich</td>
			<td>G-7513</td>
			<td>EGM</td>
		</tr>
		<tr>
			<td>Minimum Essential Media Eagle &#8211; w/Earle&#8217;s salts, L-glutamine, and non-essential amino acids, w/o sodium bicarbonate</td>
			<td>Sigma-Aldrich</td>
			<td>M0643</td>
			<td>EGM</td>
		</tr>
		<tr>
			<td>Mouse Neuroblastoma Cells</td>
			<td>ATCC</td>
			<td>CCL-131</td>
			<td></td>
		</tr>
		<tr>
			<td>Multi-well Teflon coating glass slides</td>
			<td>Any</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Any</td>
			<td></td>
			<td>pH 7.6&#160;</td>
		</tr>
		<tr>
			<td>Penicillin</td>
			<td>Sigma</td>
			<td>P-3032</td>
			<td>EGM</td>
		</tr>
		<tr>
			<td>Rabies Direct Fluorescent Antibody Conjugate</td>
			<td>Millipore Sigma</td>
			<td>5100, 5500 or 6500</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>Sigma-Aldrich</td>
			<td>S-5761</td>
			<td>EGM</td>
		</tr>
		<tr>
			<td>Sodium Chloride crystals</td>
			<td>Sigma-Aldrich</td>
			<td>S5886</td>
			<td>Mountant</td>
		</tr>
		<tr>
			<td>Sterile dropper</td>
			<td>Any</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Streptomycin sulfate salt</td>
			<td>Sigma</td>
			<td>S9137</td>
			<td>EGM</td>
		</tr>
		<tr>
			<td>Trizma pre-set crystals pH 9.0</td>
			<td>Sigma-Aldrich</td>
			<td>S9693</td>
			<td>Mountant</td>
		</tr>
		<tr>
			<td>Tryptose Phosphate Broth</td>
			<td>BD</td>
			<td>260300</td>
			<td>EGM</td>
		</tr>
		<tr>
			<td>Vitamin mix</td>
			<td>Sigma-Aldrich</td>
			<td>M6895</td>
			<td>EGM</td>
		</tr>
	</tbody>
</table>

detection of rabies igg and igm antibodies using the rabies indirect fluorescent antibody test

狂犬病间接荧光抗体 （IFA） 检测旨在检测血清或脑脊液中的各种狂犬病特异性抗体同种型。该测试可快速获得结果，可用于在几种不同情况下检测狂犬病抗体。狂犬病IFA检测对于快速和早期检测抗体以评估狂犬病患者的免疫反应特别有用。尽管其他生前狂犬病诊断方法优先，但该检测可用于通过抗体检测证明近期狂犬病病毒暴露情况。IFA 检测不提供病毒中和抗体 （VNA） 滴度，但可以通过抗体阳性或阴性来评估暴露前预防 （PrEP） 反应。该测试可用于各种情况，并且可以为许多不同的目标提供结果。在这项研究中，我们使用了来自接受PrEP的个体的几个配对血清样本，并使用IFA测试证明了他们狂犬病抗体随时间推移的存在。

所有血清样本均在PrEP后大致相同的时间范围内从患者身上收集。样本在以下时间点对五名不同的患者进行了测试：最后一次狂犬病疫苗接种后 2 周、狂犬病疫苗系列接种后 6 个月和狂犬病疫苗系列接种后 18 个月。将每个血清样品串联稀释，并根据 IgM 和 IgG 的存在进行分级，如方案步骤 5.2 和 5.3 中所述。分配的抗体值表示样品达到终点等级 1-2+ 的稀释因子。
PrEP后每个时间点的检测结果证明了该检测能够检测不同水平的抗体存在。初次接种疫苗后不久，患者样本中都存在高水平的 IgM 和 IgG，如图 1 所示。亮绿色荧光细胞染色区域表明抗体存在阳性;红细胞是狂犬病阴性细胞，没有抗体可以结合。接种疫苗后约 6 个月，患者样本中 IgM 和 IgG 水平均显著降低，但 IgM 几乎完全消失。在接种疫苗后 18 个月的最后一个时间点，在任何患者样本中均未检测到 IgM 抗体，如图 2 所示，未观察到绿色荧光细胞染色。然而，IgG 水平持续存在，并且与从 2 周时间点开始下降后的 6 个月时间点检测到的水平相似。接种后2周、接种后6个月和接种后18个月样本的IgG和IgM检测结果分别列于表1、表2和表3中。图 3 显示了执行阶段的流程图。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65459/65459fig01.jpg"> 图 1：狂犬病 IgM IFA 阳性染色。 在完成 PrEP 疫苗系列后不久，以 1：8 稀释度的患者血清显示出阳性染色模式。图像上的比例尺表示 100 μm。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65459/65459fig01large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65459/65459fig02.jpg"> 图 2：狂犬病 IgM IFA 阴性染色。 1：2稀释度的患者血清在完成PrEP疫苗系列后约18个月未显示阳性染色。图像上的比例尺表示 100 μm。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65459/65459fig02large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65459/65459fig03.jpg"> 图 3：狂犬病 IFA 流程图。流程图显示了IFA程序中主要步骤的执行阶段，以帮助实现过程可视化。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65459/65459fig03large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td>患者样本</td>
			<td>免疫球蛋白（IgM）</td>
			<td>IgG蛋白</td>
		</tr><tr><td>PS1-1系列</td>
			<td>1:32</td>
			<td>1:512</td>
		</tr><tr><td>PS2-1</td>
			<td>1:8</td>
			<td>1:128</td>
		</tr><tr><td>PS3-1的</td>
			<td>1:16</td>
			<td>1:256</td>
		</tr><tr><td>PS4-1的</td>
			<td>1:64</td>
			<td>1:512</td>
		</tr><tr><td>PS5-1版</td>
			<td>1:16</td>
			<td>1:128</td>
		</tr></tbody></table>表 1：接种疫苗后 2 周的结果。 在完成 PrEP 疫苗系列后约 2 周收集的血清样本的患者样本结果。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td>患者样本</td>
			<td>免疫球蛋白（IgM）</td>
			<td>IgG蛋白</td>
		</tr><tr><td>PS1-2系列</td>
			<td>1:2</td>
			<td>1:128</td>
		</tr><tr><td>PS2-2系列</td>
			<td>1:1</td>
			<td>1:128</td>
		</tr><tr><td>PS3-2系列</td>
			<td>1:1</td>
			<td>1:64</td>
		</tr><tr><td>PS4-2系列</td>
			<td>1:1</td>
			<td>1:256</td>
		</tr><tr><td>PS5-2系列</td>
			<td>1:1</td>
			<td>1:32</td>
		</tr></tbody></table>表 2：接种疫苗后 6 个月的结果。 完成 PrEP 疫苗系列后约 6 个月收集的血清样本的患者样本结果。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td>患者样本</td>
			<td>免疫球蛋白（IgM）</td>
			<td>IgG蛋白</td>
		</tr><tr><td>PS1-3系列</td>
			<td>未检测到</td>
			<td>1:128</td>
		</tr><tr><td>PS2-3系列</td>
			<td>未检测到</td>
			<td>1:128</td>
		</tr><tr><td>PS3-3系列</td>
			<td>未检测到</td>
			<td>1:64</td>
		</tr><tr><td>PS4-3系列</td>
			<td>未检测到</td>
			<td>1:64</td>
		</tr><tr><td>PS5-3系列</td>
			<td>未检测到</td>
			<td>1:32</td>
		</tr></tbody></table>表 3：接种疫苗后 18 个月的结果。 在完成 PrEP 疫苗系列后约 18 个月收集的血清样本的患者样本结果。

Watch this Scientific Journal Video about Rabies-Specific Antibody Isotypes Detection in Sera or Cerebral Spinal Fluid Using an IFA Test at JoVE.com

作者聚焦： 使用 IFA 检测血清或脑脊髓液中的狂犬病特异性抗体同种型 

本手稿的目的是研究使用狂犬病间接荧光抗体检测来检测狂犬病特异性 IgG 和 IgM 抗体。

使用狂犬病间接荧光抗体检测狂犬病 IgG 和 IgM 抗体

The rabies indirect fluorescent antibody (IFA) test was developed to detect various rabies-specific antibody isotypes in sera or cerebral spinal fluid. ...

IFA 测试评估狂犬病感染期间的抗体反应或评估疫苗的免疫反应。它可用于确定样品中是否存在抗体，并区分存在哪些抗体同种型。中和抗体检测是狂犬病抗体检测的金标准，用于评估接受过暴露前或暴露后预防的患者的抗体滴度。尽管IFA检测可能产生与中和抗体检测不同的结果，但作为替代方法时，它仍然可以提供有价值的信息。通常，测量狂犬病中和抗体的检测方法会评估免疫反应。然而，这些测试需要数天时间，需要病毒和活细胞培养。IFA检测允许对抗原载玻片进行高级制备，与几天相比，在几个小时内即可获得结果。在人类狂犬病病例中使用IFA测试可能会带来更多关于个人免疫系统如何应对狂犬病感染的答案。病例之间存在很大差异，该测试可以在疾病期间实时提供结果。

detection-rabies-igg-igm-antibodies-using-rabies-indirect-fluorescent

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

Detection of Rabies IgG and IgM Antibodies Using the Rabies Indirect Fluorescent Antibody Test

1.8K 次观看.  New York State Department of Health. IFA 测试评估狂犬病感染期间的抗体反应或评估疫苗的免疫反应。它可用于确定样品中是否存在抗体，并区分存在哪些抗体同种型。中和抗体检测是狂犬病抗体检测的金标准，用于评估接受过暴露前或暴露后预防的患者的抗体滴度。尽管IFA检测可能产生与中和抗体检测不同的结果，但作为替代方法时，它仍然可以提供有价值的信息。通常，测量狂犬病中和抗体的检测方法...

视频: 作者聚焦： 使用 IFA 检测血清或脑脊髓液中的狂犬病特异性抗体同种型 

Generation of Recombinant Human IgG Monoclonal Antibodies from Immortalized Sorted B Cells

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, including the development of immunotherapeutics. However, the techniques to identify and produce such antibodies tend to be arduous and sometimes the heavy and light chain pair of the antibodies are dissociated. Here, we describe a relatively simple, straightforward protocol to produce human recombinant monoclonal antibodies from human peripheral blood mononuclear cells using immortalization with Epstein-Barr Virus (EBV) and Toll-like receptor 9 activation. With an adequate staining, B cells producing antibodies can be isolated for subsequent immortalization and clonal expansion. The antibody transcripts produced by the immortalized B cell clones can be amplified by PCR, sequenced as corresponding heavy and light chain pairs and cloned into immunoglobulin expression vectors. The antibodies obtained with this technique can be powerful tools to study relevant human immune responses, including autoimmunity, and create the basis for new therapeutics.

Synthesis of human monoclonal antibodies is the first step in studies aimed at unraveling the pathophysiological mechanisms of auto-antibody-mediated immune responses. We have developed a protocol to generate recombinant human immunoglobulin G (IgG) monoclonal antibodies from blood sorted B cells, including B-cell isolation, antibody cloning and in vitro synthesis.

从排序的永生B细胞产生的重组人IgG单克隆抗体

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, ...

科研

免疫与感染

Antibody Binding Specificity for Kappa (V&#954;) Light Chain-containing Human (IgM) Antibodies: Polysialic Acid (PSA) Attached to NCAM as a Case Study

Antibodies of the IgM isotype are often neglected as potential therapeutics in human trials, animal models of human diseases as well as detecting agents in standard laboratory techniques. In contrast, several human IgMs demonstrated proof of efficacy in cancer models and models of CNS disorders including multiple sclerosis (MS) and amyotrophic lateral sclerosis (ALS). Reasons for their lack of consideration include difficulties to express, purify and stabilize IgM antibodies, challenge to identify (non-protein) antigens, low affinity binding and fundamental knowledge gaps in carbohydrate and lipid research. 
This manuscript uses HIgM12 as an example to provide a detailed protocol to detect antigens by Western blotting, immunoprecipitations and immunocytochemistry. HIgM12 targets polysialic acid (PSA) attached to the neural cell adhesion molecule (NCAM). Early postnatal mouse brain tissue from wild type (WT) and NCAM knockout (KO) mice lacking the three major central nervous system (CNS) splice variants NCAM180, 140 and 120 was used to evaluate the importance of NCAM for binding to HIgM12. Further enzymatic digestion of CNS tissue and cultured CNS cells using endoneuraminidases led us to identify PSA as the specific binding epitope for HIgM12.

We present a protocol to identify protein moieties containing antigens for human and mouse IgM antibodies with specific kappa (V&#954;) light chains. This protocol is not limited to IgM class antibodies but applies to all immunoglobulin isotypes that target their antigen with sufficiently high affinity during immunoprecipitations.

附NCAM作为一个案例研究聚唾液酸（PSA）:为河童（Vκ）抗体结合特异性含轻链人类（IgM抗体）抗体

Antibodies of the IgM isotype are often neglected as potential therapeutics in human trials, animal models of human diseases as well as detecting agents ...

Generation of Two-color Antigen Microarrays for the Simultaneous Detection of IgG and IgM Autoantibodies

Autoantibodies, which are antibodies against self-antigens, are present in many disease states and can serve as markers for disease activity. The levels of autoantibodies to specific antigens are typically detected with the enzyme-linked immunosorbent assay (ELISA) technique. However, screening for multiple autoantibodies with ELISA can be time-consuming and requires a large quantity of patient sample. The antigen microarray technique is an alternative method that can be used to screen for autoantibodies in a multiplex fashion. In this technique, antigens are arrayed onto specially coated microscope slides with a robotic microarrayer. The slides are probed with patient serum samples and subsequently fluorescent-labeled secondary antibodies are added to detect binding of serum autoantibodies to the antigens. The autoantibody reactivities are revealed and quantified by scanning the slides with a scanner that can detect fluorescent signals. Here we describe methods to generate custom antigen microarrays. Our current arrays are printed with 9 solid pins and can include up to 162 antigens spotted in duplicate. The arrays can be easily customized by changing the antigens in the source plate that is used by the microarrayer. We have developed a two-color secondary antibody detection scheme that can distinguish IgG and IgM reactivities on the same slide surface. The detection system has been optimized to study binding of human and murine autoantibodies.

We describe here a method to generate customizable antigen microarrays that can be used for the simultaneous detection of serum IgG and IgM autoantibodies from humans and mice. These arrays allow for high-throughput and quantitative detection of antibodies against any antigens or epitopes of interest.

双色抗原微阵列产生的IgG和IgM自身抗体的同时检测

Autoantibodies, which are antibodies against self-antigens, are present in many disease states and can serve as markers for disease activity. The levels ...

Antibody Labeling with Fluorescent Dyes Using Magnetic Protein A and Protein G Beads

Antibodies labeled with small molecules like fluorescent dyes, cytotoxic drugs, and radioactive tracers are essential tools in biomedical research, immunodiagnostics and more recently as therapeutic agents. Traditional methods for labeling antibodies with small molecules require purified antibodies at relatively high concentration, involve multiple dialysis steps and have limited throughput. However, several applications, including the field of Antibody Drug Conjugates (ADCs), will benefit from new methods that will allow labeling of antibodies directly from cell media. Such methods may allow antibodies to be screened in biologically relevant assays, for example, the receptor-mediated antibody internalization assay in the case of ADCs. Here, we describe a method (on-bead method) that enables labeling of small amounts of antibodies directly from cell media. This approach utilizes high capacity magnetic Protein A and Protein G affinity beads to capture antibodies from the cell media followed by labeling with small molecules using either amine or thiol chemistry and subsequent elution of the labeled antibodies. Taking fluorescent dyes as surrogates for small molecules, we demonstrate the on-bead labeling of three different mouse antibodies directly from cell media using both amine and thiol labeling chemistry. The high binding affinity of antibodies to Protein A and Protein G ensures high recoveries as well as high purity of the labeled antibodies. In addition, use of magnetic beads allows multiple samples to be handled manually, thereby significantly improving labeling throughput.

The on-bead method for labeling antibodies with small molecules enables labeling of a small amount of antibodies directly from cell media. This method is compatible with amine and thiol chemistry, and can handle multiple samples in parallel, manually or using automated platforms.

抗体标记用荧光染料使用磁蛋白A和G蛋白珠

Antibodies labeled with small molecules like fluorescent dyes, cytotoxic drugs, and radioactive tracers are essential tools in biomedical research, ...

Enhanced Rabies Surveillance Using a Direct Rapid Immunohistochemical Test

Laboratory-based surveillance is integral for rabies prevention, control and management efforts. While the DFA is the gold standard for rabies diagnosis, there is a need to validate additional diagnostic techniques to improve rabies surveillance, particularly in developing countries. Here, we present a standard protocol for the DRIT as an alternative, laboratory or field-based testing option that uses light microscopy as compared to the DFA. Touch impressions of brain tissue collected from suspect animals are fixed in 10% buffered formalin. The DRIT uses rabies virus-specific monoclonal or polyclonal antibodies (conjugated to biotin), a streptavidin-peroxidase enzyme, and a chromogen reporter (such as acetyl 3-amino-9-ethylcarbazole) to detect viral inclusions within infected tissue. In approximately 1 h, a brain tissue sample can be tested and interpreted by the DRIT. Evaluation of suspect animal brains tested from a variety of species in North America, Asia, Africa, and Europe have illustrated high sensitivity and specificity by the DRIT approaching 100% with results compared to DFA. Since 2005, the United States Department of Agriculture&#8217;s Wildlife Services (USDA WS) program has conducted large-scale enhanced rabies surveillance efforts using the DRIT to test &#62;94,000 samples collected from wildlife in strategic rabies management areas. The DRIT provides a powerful, economical tool for rabies diagnosis that can be used by laboratorians and field biologists to improve current rabies surveillance, prevention and control programs globally.

The direct rapid immunohistochemical test (DRIT) offers a World Organization for Animal Health and World Health Organization (OIE/WHO) recognized alternative to the direct fluorescent antibody (DFA) test for rabies diagnosis. This test allows for field-based applications that can be performed in approximately 1 h upon brain impressions using light microscopy.

使用直接快速免疫性化学测试增强狂犬病监测

Laboratory-based surveillance is integral for rabies prevention, control and management efforts. While the DFA is the gold standard for rabies diagnosis, ...

In Vitro ELISA Test to Evaluate Rabies Vaccine Potency

The growing global concern for the animal welfare is encouraging manufacturers and the National Control Laboratories (OMCLs) to follow the 3Rs strategy for the Replacement, Reduction, and Refinement of the laboratory animal testing. The development of in vitro approaches is recommended at the WHO and European levels as alternatives to the NIH test for evaluating the rabies vaccine potency. At the surface of the rabies virus (RABV) particle, trimers of glycoprotein constitute the major immunogen to induce Viral Neutralizing Antibodies (VNAbs). An ELISA test, where Neutralizing Monoclonal Antibodies (mAb-D1) recognize the trimeric form of the glycoprotein, has been developed to determine the contents of the native folded trimeric glycoprotein along with the production of the vaccine batches. This in vitro potency test demonstrated a good concordance with the NIH test and has been found suitable in collaborative trials by RABV vaccine manufacturers and OMCLs. Avoidance of animal use is an achievable objective in the near future.
The method presented is based on an indirect ELISA sandwich immunocapture using the mAb-D1 which recognizes the antigenic sites III (aa 330 to 338) of the trimeric RABV glycoprotein, i.e., the immunogenic RABV antigen. mAb-D1 is used for both coating and detection of glycoprotein trimers present in the vaccine batch. Since the epitope is recognized because of its conformational properties, the potentially denatured glycoprotein (less immunogenic) cannot be captured and detected by the mAb-D1. The vaccine to be tested is incubated in a plate sensitized with the mAb-D1. Bound trimeric RABV glycoproteins are identified by adding the mAb-D1 again, labeled with peroxidase and then revealed in the presence of substrate and chromogen. Comparison of the absorbance measured for the tested vaccine and the reference vaccine allows for the determination of the immunogenic glycoprotein content.

Here we describe an indirect ELISA sandwich immunocapture to determine the immunogenic glycoprotein contents in rabies vaccines. This test uses a neutralizing Monoclonal Antibody (mAb-D1) recognizing glycoprotein trimers. It is an alternative to the in vivo NIH test to follow the consistency of vaccine potency during production.

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 ...

Pan-lyssavirus Real Time RT-PCR for Rabies Diagnosis

Molecular assays are rapid, sensitive and specific, and have become central to diagnosing rabies. PCR based assays have been utilized for decades to confirm rabies diagnosis but have only recently been accepted by the OIE (World Organisation for Animal Health) as a primary method to detect rabies infection. Real-time RT-PCR assays provide real-time data, and are closed-tube systems, minimizing the risk of contamination during setup. DNA intercalating fluorochrome real-time RT-PCR assays do not require expensive probes, minimizing the cost per sample, and when the primers are designed in conserved regions, assays that are specific across virus genera rather than specific to just one virus species are possible. Here we describe a pan-lyssavirus SYBR real-time RT-PCR assay that detects lyssaviruses across the Lyssavirus genus, including the most divergent viruses IKOV, WCBV and LLEBV. In conjunction with dissociation curve analysis, this assay is sensitive and specific, with the advantage of detecting all lyssavirus species. The assay has been adopted in many diagnostic laboratories with quality assured environments, enabling robust, rapid, sensitive diagnosis of animal and human rabies cases.

This real-time RT-PCR using dsDNA intercalating dye is suitable to diagnose lyssavirus infections. The method begins with RNA extracted from rabies suspected ante-mortem or post-mortem samples, detailing master mix preparation, RNA addition, setup of the real-time machine and correct interpretation of results.

用于狂犬病诊断的泛源病毒实时RT-PCR

Molecular assays are rapid, sensitive and specific, and have become central to diagnosing rabies. PCR based assays have been utilized for decades to ...

Field Postmortem Rabies Rapid Immunochromatographic Diagnostic Test for Resource-Limited Settings with Further Molecular Applications

Functional rabies surveillance systems are crucial to provide reliable data and increase the political commitment necessary for disease control. To date, animals suspected as rabies-positive must be submitted to a postmortem confirmation using classical or molecular laboratory methods. However, most endemic areas are in low- and middle-income countries where animal rabies diagnosis is restricted to central veterinary laboratories. Poor availability of surveillance infrastructure leads to serious disease underreporting from remote areas. Several diagnostic protocols requiring low technical expertise have been recently developed, providing opportunity to establish rabies diagnosis in decentralized laboratories. We present here a complete protocol for field postmortem diagnosis of animal rabies using a rapid immunochromatographic diagnostic test (RIDT), from brain biopsy sampling to the final interpretation. We complete the protocol by describing a further use of the device for molecular analysis and viral genotyping. RIDT easily detects rabies virus and other lyssaviruses in brain samples. The principle of such tests is simple: brain material is applied on a test strip where gold conjugated antibodies bind specifically to rabies antigens. The antigen-antibody complexes bind further to fixed antibodies on the test line, resulting in a clearly visible purple line. The virus is inactivated in the test strip, but viral RNA can be subsequently extracted. This allows the test strip, rather than the infectious brain sample, to be safely and easily sent to an equipped laboratory for confirmation and molecular typing. Based on a modification of the manufacturer&#39;s protocol, we found increased test sensitivity, reaching 98% compared to the gold standard reference method, the direct immunofluorescence antibody test. The advantages of the test are numerous: rapid, easy-to-use, low cost and no requirement for laboratory infrastructure, such as microscopy or cold-chain compliance. RIDTs represent a useful alternative for areas where reference diagnostic methods are not available.

We present a complete protocol for postmortem diagnosis of animal rabies under field conditions using a rapid immunochromatographic diagnostic test (RIDT), from brain biopsy sampling to final interpretation. We also describe further applications using the device for molecular analysis and viral genotyping.

现场验尸狂犬病快速免疫性诊断测试资源有限设置与进一步分子应用

Functional rabies surveillance systems are crucial to provide reliable data and increase the political commitment necessary for disease control. To date, ...

Immunohistochemistry Test for the Lyssavirus Antigen Detection from Formalin-Fixed Tissues

One of the primary diagnostic modalities for rabies is the detection of viral ribonucleoprotein (RNP) complex (antigen) in the infected tissue samples. While the direct fluorescent antibody (DFA) test or the direct rapid immunohistochemical test (DRIT) are most commonly utilized for the antigen detection, both tests require fresh and/or frozen tissues for impressions on slides prior to the antigen detection using antibodies. If samples are collected and fixed in formalin, neither test is optimal for the antigen detection, however, testing can be performed by conventional immunohistochemistry (IHC) after embedding in paraffin blocks and sectioning. With this IHC method, tissues are stained with anti-rabies antibodies, sections are deparaffinized, antigen retrieved by partial proteolysis or other methods, and incubated with primary and secondary antibodies. Antigens are stained using horseradish peroxidase / amino ethyl carbazole and counterstained with hematoxylin for the visualization using a light microscope. In addition to the specific antigen detection, formalin fixation offers other advantages like the determination of histological changes, relaxed conditions for specimen storage and transport (under ambient temperatures), ability to test retrospective cases and improved biological safety through the inactivation of infectious agents.

Here, we present an immunohistochemistry test protocol for the detection of rabies virus antigen as an alternative diagnostic test for formalin-fixed tissues.

从正式固定组织检测利沙病毒抗原的免疫化学测试

One of the primary diagnostic modalities for rabies is the detection of viral ribonucleoprotein (RNP) complex (antigen) in the infected tissue samples. ...

Visualization of Bacterial Resistance using Fluorescent Antibiotic Probes

Fluorescent antibiotics are multipurpose research tools that are readily used for the study of antimicrobial resistance, due to their significant advantage over other methods. To prepare these probes, azide derivatives of antibiotics are synthesized, then coupled with alkyne-fluorophores using azide-alkyne dipolar cycloaddition by click chemistry. Following purification, the antibiotic activity of the fluorescent antibiotic is tested by minimum inhibitory concentration assessment. In order to study bacterial accumulation, either spectrophotometry or flow cytometry may be used, allowing for much simpler analysis than methods relying on radioactive antibiotic derivatives. Furthermore, confocal microscopy can be used to examine localization within the bacteria, affording valuable information about mode of action and changes that occur in resistant species. The use of fluorescent antibiotic probes in the study of antimicrobial resistance is a powerful method with much potential for future expansion.

Fluorescently tagged antibiotics are powerful tools that can be used to study multiple aspects of antimicrobial resistance. This article describes the preparation of fluorescently tagged antibiotics and their application to studying antibiotic resistance in bacteria. Probes can be used to study mechanisms of bacterial resistance (e.g., efflux) by spectrophotometry, flow cytometry, and microscopy.