Immunology and Infection
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使用捕获测定法检测基于病毒样颗粒（VLP）的疫苗上显示的抗原中和敏感表位
Chapters
Summary February 10th, 2022
Please note that all translations are automatically generated.
Click here for the English version.
在这里，我们提出了一种检测抗原显示病毒样颗粒（VLP）的中和表位的方案。使用包膜糖蛋白特异性单克隆抗体偶联至蛋白G偶联磁珠进行人免疫缺陷病毒（HIV）衍生VLP的免疫沉淀。随后使用病毒核心蛋白Gag特异性抗体对捕获的VLP进行SDS-PAGE和蛋白质印迹分析。
Transcript
在我的实验室，我们为病毒载体和病毒样颗粒或基于VLP的疫苗开发生产系统。VLP捕获测定允许对VLP上显示的靶抗原进行非常灵敏的检测。在该测定中，我们使用针对中和表位的广泛中和抗体来证明这些表位在VLP表面上的暴露。
对于候选疫苗来说，这是一项重要的质量评估，因为只有显示中和敏感表位的VLP才能在疫苗使用者中引发中和抗体反应。首先通过上下移液或在旋转器上以50 RPM混合至少五分钟来悬浮磁珠。同时，制备含有bNB的抗体溶液。
每次反应在200微升抗体结合和洗涤缓冲液中使用10微克每个bNAB。对于每次反应，将50微升磁珠溶液转移到1.5毫升的反应管中，然后将管放在磁分离架上。等到珠子聚集在管球处，以确保收集所有珠子，然后取出上清液。
取出磁铁并将磁珠悬浮在先前制备的bNAB溶液的200微升中。在室温下以50 RPM在旋转器上混合时孵育30分钟至3小时。孵育后，将反应管放入磁分离架中，等待并除去上清液。
从磁铁中取出试管，通过重新悬浮在200微升抗体结合和洗涤缓冲液中洗涤微珠。用抗体结合和洗涤缓冲液重复洗涤，完成后尽可能多地去除洗涤缓冲液。将样品添加到磁珠结合的bNB中。
如果添加的样品体积低于1毫升，则加入PBS以将样品体积调节至1毫升，然后通过轻轻移液重新悬浮微球。将样品和珠子在室温下在旋转器上孵育2.5小时，确保珠子保持悬浮状态，并且在孵育过程中将溶液充分混合。将管子放在磁铁上并除去上清液，然后将磁珠悬浮在200微升的洗涤缓冲液中洗涤。
将磁珠悬浮在100微升的洗涤缓冲液中，并将悬浮液转移到清洁，耐热的反应管中。将管子放在磁分离架上，并完全除去上清液。为了制备变性的STS-PAGE样品，将珠子悬浮在20至80微升的Laemmli缓冲液中，并在95摄氏度下孵育5分钟。
直接使用SDS-PAGE，将管子放在磁性架上，将磁珠与溶液分离。或者，将样品储存在零下20摄氏度。这里显示了首先使用bNABs从无细胞培养物上清液和VLP沉淀中捕获的VLP的代表性结果，随后进行蛋白质印迹分析以检测病毒核心蛋白。
用于捕获测定的珠子涂有三种不同的bNABs，直接针对包膜糖蛋白的中和表位和作为阴性对照的同种型抗体。使用同位素抗体包被的微球，在含有秃头VLP的VLP样品中未检测到Gag蛋白，即Env阴性VLP或Env显示VLP，表明VLP与包被人抗体的珠子的非特异性结合不介导VLP捕获。秃头VLP也不被bNABs包被的珠子结合，因此，在蛋白质印迹分析中检测不到Gag蛋白。
相比之下，所有三种bNABs都捕获了显示Env蛋白的VLP，因此，Gag蛋白很容易被检测到，这表明VLP上显示的Env-glycrot蛋白中存在中和表位。这最好使用旋转下大于500微升的体积来实现。
或者，捕获的VLP可以在非还原条件下洗脱。这使得能够使用免疫电子显微镜分析VLP，或使用天然PAGE研究显示的抗原。
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