Mechanisms of Antibody-Mediated Toxin Neutralization

描述（由申请人提供）：一个多世纪以来，抗体一直被用于防止生物毒素的影响。人们普遍认为，抗体的保护机制是通过阻止毒素进入细胞。然而，这忽略了一个重要的证据，证明A-B毒素a链抗体与抗b链抗体具有相同或更大的中和活性。我们将用蓖麻毒素的数据来证明这一点。基于这些结果，我们已经形成了一个假设，即抗体可以通过改变毒素的细胞内加工和路径来保护，抗体结合的亲和力可能决定了它这样做的能力。为了验证这一假设，我们提出以下具体目标：

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.