ENHANCED LOCAL LYMPH NODE ASSAY USING FLOW CYTOMETRY

动物福利法(AWA)和政府机构要求，当可用时，使用替代方法来解决动物研究的减少、改进和替换(3R)。局部淋巴结节试验(LLNA)是豚鼠致敏试验的替代方法，后者被制药、化工、消费品和化妆品行业用来识别和表征具有免疫毒性的物质。这项建议的目的是确定应用流式细胞术增强LLNA的可行性。目前形式的LLNA的一个主要限制是它不能区分致敏剂的亚类(即接触性过敏原和呼吸道过敏原)。此外，该分析使用了放射性和大量的动物。基于流式细胞术的终点将增强LLNA的灵敏度和辨别能力，同时(1)通过直接寻址3R来遵守AWA；(2)与现有方法(豚鼠试验)相比，提高产生的数据的质量和数量；以及(3)通过避免使用放射性来大幅降低分析成本。这种增强型检测的开发将满足行业的需求，并测试我们将流式细胞术应用于现有检测和开发新的毒性测试的长期计划的可行性。拟议的商业应用：MB Research将向制药、生物技术、化妆品、化工和消费品行业提供使用流式细胞术的LLNA。该提案包括几家大公司(默克公司、强生高级护理产品公司、互联网服务提供商和普渡制药公司)的支持信，表明有兴趣增强LLNA。对现有客户的调查表明，对LLNA和其他基于流式细胞仪的分析有很大的需求(参见与未来研究和开发的关系)。流式细胞术是一项在毒理学和药理学测试中未得到充分利用的强大技术，除了提高LLNA的市场化程度外，还可以用于开发其他商业上可行的测试。

The Animal Welfare Act (AWA) and government agencies require, when available, the use of alternative methods which address the Reduction, Refinement and Replacement (3R's) of animal studies. The Local Lymph Node Assay (LLNA) is an alternative to the guinea pig sensitization test which is employed by the pharmaceutical, chemical, consumer product and cosmetic industries to identify and characterize substances with immunotoxic properties. The purpose of this proposal is to determine the feasibility of applying flow cytometry to enhance the LLNA. A major limitation of the LLNA in its current form is that it cannot differentiate between subclasses of sensitizing agents (i.e., contact vs. respiratory allergens). Furthermore, the assay uses radioactivity and large numbers of animals. Flow cytometry-based endpoints will enhance the sensitivity and discriminating power of the LLNA, while (1) complying with the AWA by directly addressing the 3R's; (2) increasing the quality and quantity of data generated when compared to existing methods (guinea pig test); and (3) substantially reducing the cost of analysis by avoiding the use of radioactivity. The development of this enhanced assay will satisfy a demand in the industry and test the feasibility of our long term plan to apply flow cytometry to existing assays and to develop new toxicity tests. PROPOSED COMMERCIAL APPLICATIONS: MB Research will offer the LLNA using flow cytometry to the pharmaceutical, biotech, cosmetic, chemical and consumer products industries. Included with this proposal are letters of support from several major companies (Merck, Johnson & Johnson Advanced Care Products, ISP, and Purdue Pharma) demonstrating interest in an enhanced LLNA. A survey of existing clients indicates a great demand for the LLNA and other flow cytometric-based assays (see Relationship to Future Research and Development). Flow cytometry is a powerful technology that is underutilized in toxicology and pharmacology testing which, in addition to improving the marketability of the LLNA, can be applied to develop other commercially viable tests.