Mechanisms of Antibody-Mediated Toxin Neutralization

描述（由申请方提供）：抗体已被用于防止生物毒素的影响超过世纪。一般认为，抗体的保护机制是通过阻断毒素进入细胞。然而，这忽略了一个重要的证据主体，该证据表明针对A-B毒素的A链的抗体具有与抗B链抗体相等或更大的中和活性。我们将提供蓖麻毒素的数据来证明这一点。基于这些结果，我们已经形成了这样的假设，即抗体可以通过改变毒素的细胞内加工和路由来保护，并且抗体结合的亲和力可能决定其这样做的能力。为了验证这一假设，我们提出以下具体目标： 具体目标1：制备和表征一组高亲和力抗蓖麻毒素A链抗体。将研究抗体亲和力、体外中和和体内保护之间的关系。 具体目的2：研究荧光标记蓖麻毒素在中和和非中和抗体存在下的细胞内路径。共聚焦，去卷积，电子显微镜，和亚细胞分离将被用来研究抗体对蓖麻毒素的亚细胞定位的影响。 具体目的3：研究毒素与抗体的细胞内结合。将使用双标记研究和荧光能量转移（FRET）来确定抗体在细胞内处理期间保持与毒素结合多长时间。 蓖麻毒素是一种原型A-B毒素，包括许多细菌和植物毒素。除了定义基本过程之外，本申请中提出的研究还可用于开发中毒的疫苗和治疗。

DESCRIPTION (provided by applicant): Antibodies have been used to protect against the effects of biological toxins for over a century. It is generally accepted that the mechanism whereby antibodies protect is by blocking the entry of toxins into cells. However, this ignores an important body of evidence demonstrating that antibodies to the A-chain of A-B toxins have equal or greater neutralizing activity than anti-B chain antibodies. We will present data demonstrating this with ricin toxin. Based upon these results we have formed the hypothesis that antibodies can protect by altering the intracellular processing and routing of the toxin and that the affinity of antibody binding may determine its ability to do so. To test this hypothesis we propose the following Specific Aims: Specific Aim 1: To produce and characterize a panel of high affinity anti-ricin A chain antibodies. The relationship between antibody affinity, in vitro neutralization and in vivo protection will be studied. Specific Aim 2: To study the intracellular routing of fluorescent-labeled ricin toxin in the presence of neutralizing and non-neutralizing antibodies. Confocal, deconvolution, and electron microscopy, and subcellular isolations will be used to study effects of antibody on subcellular localization of ricin. Specific Aim 3: To study the intracellular association between toxin and antibody. Double-label studies and fluorescence-energy transfer (FRET) will be used to determine how long antibody remains bound to the toxin during intracellular processing. Ricin is a prototype A-B toxin, a group that includes many bacterial and plant toxins. In addition to defining basic processes, the studies proposed in this application have utility for the development of vaccines and treatments for intoxications.