An antigen-detection assay to diagnose Babesia microti infection

项目摘要 人类巴贝斯虫病是一种类似疟疾的多系统疾病，主要由小巴贝斯虫引起， 在人体红细胞内感染和发育的顶复体寄生虫。寄生虫会传播到 人类通过蜱媒介肩胛硬蜱传播，也可以通过输血传播。感染 可引起流感样症状，严重感染可致命，特别是在免疫抑制和 老人目前诊断巴贝虫病的方法包括显微镜检查法、PCR法、IFA法和ELISA法 检测病人或捐赠者血清中的抗体这些方法都有很大的局限性，例如 灵敏度不足、复杂性高和吞吐量低。此外，许多这些测定的结果 在活动性感染消退后数月或数年内保持阳性。 在该项目的第一阶段，我们开发了一种捕获ELISA测定法，可以检测寄生虫的蛋白质， BmGPI12/BmSAI。我们已经鉴定了可用于捕获BmGPI 12的单克隆抗体对， 小鼠和人类样品。我们的初步结果表明，这种方法具有良好的灵敏度。 使用从受感染小鼠（一种已建立的人类疾病模型）收集的样本，我们证明， 成功治疗后，我们的检测结果为阴性，而PCR和血清学检测结果仍为阳性。 因此，我们的检测方法可能有助于区分活动性感染和既往感染。 在该项目的第二阶段，我们将进一步开发该检测试剂盒，目标是申请FDA批准。我们 将分析数百个动物和人类样本，以确定检测的重要特征 包括灵敏度和特异性。这些第二阶段的实验旨在让我们充分了解 潜在的生物学适应症和商业实用性的测定。

PROJECT SUMMARY Human babesiosis is a malaria-like multisystem disease caused primarily by Babesia microti, an emerging apicomplexan parasite that infects and develops within human erythrocytes. The parasite is transmitted to humans by the tick vector Ixodes scapularis and can also be introduced through blood transfusion. Infection can cause flu-like symptoms, and severe infection can be fatal, in particular in the immunosuppressed and the elderly. Current methods for babesiosis diagnosis include microscopy, PCR, IFA and ELISA-based methods that detect antibodies in serum from patients or donors. Each of these methods has major limitations, such as insufficient sensitivity, high complexity, and low throughput. In addition, results from many of these assays remain positive for months or years after resolution of active infection. In Phase I of this project, we have developed a capture ELISA assay that can detect a protein of the parasite, BmGPI12/BmSAI. We have identified pairs of monoclonal antibodies that can be used to capture BmGPI12 in mouse and human samples. Our preliminary results demonstrate that this assay has an excellent sensitivity. Using samples collected from infected mice, a well-established model of human disease, we demonstrated that following successful treatment, our assay gives negative results while PCR and serology remain positive. Thus, our assay may be useful in distinguishing active infection from past exposure. In Phase II of this project, we will further develop this assay with the goal of applying for FDA clearance. We will analyze several hundred animal and human samples to establish important characteristics of the assay including sensitivity and specificity. These Phase II experiments are designed to give us a full understanding of the potential biological indications and commercial usefulness of the assay.