ENHANCED LOCAL LYMPH NODE ASSAY USING FLOW CYTOMETRY

《动物福利法》（AWA）和政府机构要求在可用时使用替代方法，以解决动物研究的减少、改进和替代（3R）问题。局部淋巴结试验（LLNA）是豚鼠致敏试验的替代方法，用于制药、化工、消费品和化妆品行业，以识别和表征具有免疫毒性的物质。本提案的目的是确定应用流式细胞术增强LLNA的可行性。LLNA目前形式的主要限制是它不能区分敏化剂的亚类（即，接触与呼吸道过敏原）。此外，该测定使用放射性和大量动物。基于流式细胞术的终点将提高LLNA的灵敏度和鉴别能力，同时（1）通过直接解决3R来遵守AWA;（2）与现有方法（豚鼠试验）相比，提高了生成的数据的质量和数量;（3）通过避免使用放射性大大降低了分析成本。这种增强的检测方法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方法和开发新的毒性检测的长期计划的可行性。拟议的商业应用：MB Research将为制药、生物技术、化妆品、化学和消费品行业提供使用流式细胞术的LLNA。本提案包括几家主要公司（默克、约翰逊和约翰逊高级护理产品、ISP和普渡制药）的支持信，表明对增强LLNA感兴趣。对现有客户的调查表明，对LLNA和其他基于流式细胞术的检测的需求很大（见与未来研究和开发的关系）。流式细胞术是一种强大的技术，在毒理学和药理学测试中未得到充分利用，除了提高LLNA的可销售性外，还可以应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.