NEW APPROACHES TO THE DIAGNOSIS OF AMEBIASIS

由溶组织内阿米巴引起的阿米巴病是发展中国家的一个主要问题。在发达国家，这种情况在从第三世界返回的移民和旅行者中最常见，在男男性行为者和免疫抑制者中不太常见。在以前的SBIR研究中，我们开发了一种粪便抗原检测E。溶组织粘附素是一种有效的体外诊断标记物。我们的测试，微孔ELISA，比显微镜和其他商业ELISA更准确，比“金标准”培养/酶模式更快速，需要2周才能完成。我们现在已经开发出抗原亲和纯化抗体，使我们能够将测试的灵敏度提高十倍。我们建议在试纸中使用改进的抗体，该试纸可在现场用作快速粪便抗原检测。为了使仅检测固定标本的实验室能够使用免疫测定法（在美国，阿米巴病的误诊最多），我们将开发检测福尔马林稳定的内阿米巴抗原的免疫测定法。我们还建议开发一种基于抗体的内阿米巴包囊检测方法。将开发针对包囊的多克隆和单克隆抗体，并将其用于对受污染的水和食品进行免疫检测（最近分别在弗吉尼亚州雷斯顿和格鲁吉亚共和国第比利斯发生的食源性和水源性疫情使其成为一种局部和紧迫的需要）。囊特异性检测的发展将遵循贾第虫和隐孢子虫的囊和卵囊特异性抗原的发展模式。收集的数据将用于编制510（k）文件，以提交给FDA并由FDA审查。该项目将导致一系列测试，在阿米巴病的诊断中具有广泛的应用。这些测试将是评估我们单独的热带疾病研究股开发的阿米巴病候选疫苗的关键工具。拟议的商业应用：将开发新的阿米巴病诊断测试，以解决美国社区和参考实验室的误诊问题，能够对供水进行寄生虫筛查，并允许对疫苗试验进行实地评估。我们的研究结果将导致商业测试，这将对世界各地的健康产业具有重大价值。

Amebiasis, caused by Entamoeba histolytica, is a major problem in developing countries. In developed countries, it is most common in immigrants and travelers returning from the third world, and less common but present in men who have sex with men and immunosuppressed individuals. In previously sponsored SBIR research, we developed a stool antigen test for the detection of the E. histolytica adhesin, a protein that is an effective in vitro diagnostic marker. Our test, a microwell ELISA, is more accurate than microscopy and other commercial ELISAs, and more rapid than the "gold standard" culture/zymodeme, which takes 2 weeks to complete. We now have developed antigen affinity-purified antibody that has enabled us to increase the sensitivity' of the test ten-fold. We propose to use the improved antibody in dipsticks that can be used in the field as a rapid stool antigen test. In an effort to make immunoassays available to laboratories that test only fixed specimens (where most misdiagnosis of amebiasis occurs in the U.S.), we will develop immunoassays that detect formalin-stable Entamoeba antigens. We also propose to develop an antibody-based test for Entamoeba cysts. Polyclonal and monoclonal antibodies against cysts will be developed and used in screening immunoassays for contaminated water and food (recent food and water- borne outbreaks in Reston Virginia and Tblissi, Republic of Georgia respectively, make this a topical and immediate need). The development of cyst-specific tests will be patterned after those for the cyst- and oocyst- specific antigens of Giardia and Cryptosporidium. The data collected will be used to develop 510(k) documents for submission to and review by the FDA. This project will lead to a battery of tests that have broad application in the diagnosis of amebiasis. The tests will be crucial tools for evaluation of amebiasis vaccine candidates developed under our separate Tropical Disease Research Unit. PROPOSED COMMERCIAL APPLICATIONS: New diagnostic tests will be developed for amebiasis to solve the problems of misdiagnosis at community and reference labs in the U.S., enable screening of water supplies for the parasite, and to permit field evaluation of vaccine trials. Our results should lead to commercial tests that will be of significant value to the health industry around the world.