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Mechanisms of Antibody-Mediated Toxin Neutralization

抗体介导的毒素中和机制

基本信息

批准号：
7346980
负责人：
Seth H. Pincus
金额：
$ 29.05万
依托单位：
CHILDREN'S HOSPITAL (NEW ORLEANS)
依托单位国家：
美国
项目类别：
财政年份：
2004
资助国家：
美国
起止时间：
2004-02-01 至 2010-01-31
项目状态：
已结题

项目摘要

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.

描述（由申请人提供）：一个多世纪以来，抗体一直被用来防止生物毒素的影响。人们普遍认为抗体的保护机制是阻止毒素进入细胞。然而，这忽略了一个重要的证据，证明 A-B 毒素的 A 链抗体具有与抗 B 链抗体相同或更强的中和活性。我们将提供用蓖麻毒素证明这一点的数据。基于这些结果，我们形成了这样的假设：抗体可以通过改变毒素的细胞内加工和路径来提供保护，并且抗体结合的亲和力可能决定其这样做的能力。为了检验这一假设，我们提出以下具体目标：具体目标 1：生产并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。具体目标 2：研究在中和和非中和抗体存在下荧光标记的蓖麻毒素的细胞内路径。共聚焦、反卷积、电子显微镜和亚细胞分离将用于研究抗体对蓖麻毒素亚细胞定位的影响。具体目标 3：研究毒素和抗体之间的细胞内关联。双标记研究和荧光能量转移（FRET）将用于确定抗体在细胞内处理过程中与毒素结合的时间。蓖麻毒素是 A-B 毒素的原型，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对于开发疫苗和中毒治疗也具有实用性。