Luminex Technology for the Quantification of Cytokines in Non-Human Primates

描述(申请人提供)：非人灵长类动物(NHP)是临床前研究的首选动物模型，因为它们在生理和遗传学上比其他任何动物都更接近人类。通过使用NHP模型，免疫学研究取得了重大进展，最显著的是在艾滋病的发病机制、治疗和疫苗开发方面。细胞因子和趋化因子是免疫系统的可溶性介质，在细胞间信号传递和细胞向炎症部位募集方面发挥关键作用。识别NHP中的这些分子对于理解这些物种中发生的复杂的生理和病理机制以及证明这些机制在人类中是否具有类似的功能至关重要。Luumex100系统是一种台式流式细胞仪，允许用户在一根试管中同时定量多达100个分子。最近，我们鉴定了几种针对人类细胞因子的抗体，它们具有识别同源趋化因子和NHP来源的细胞因子的能力。目前，我们拥有一组试剂，可以同时鉴定黑猩猩和东半球猕猴、恒河猴、狒狒、食蟹猴、猪尾猕猴和非洲绿猴的23种细胞因子和趋化因子。在这项R-24应用中，我们建议通过完成以下具体目标来促进和扩大Luminex技术在NHP中的应用：1)通过增加旧大陆猴子物种中可检测的分子的数量，提高我们23-plex NHP Luminex小组的广度和分析能力；2)通过提高一些目前用于定量旧世界猴子物种细胞因子的次优试剂的灵敏度，改善我们23-plex NHP Luminex小组的质量和深度；以及3)开发重组抗体，用于同时定量绒猴的细胞因子，这是一种越来越多地用于疫苗开发、衰老和肥胖症研究的新世界猴物种。我们将对现有的针对旧大陆猴细胞因子的单抗进行广泛的测试，并采用最先进的重组抗体技术来创建针对绒猴细胞因子的抗体。这些研究产生的信息和方案将在SNPRC的流式细胞术网站上列出；新的抗体将分发给在成本回收基础上证明其必要性的科学家。

DESCRIPTION (provided by applicant): Nonhuman primates (NHP) are the preferred animal models for pre-clinical research because they approximate humans in physiology and genetics more closely than any other animal. Critical advances in immunologic research have been made through the use of the NHP model, most notably in AIDS pathogenesis, treatment, and vaccine development. Cytokines and chemokines are soluble mediators of the immune system that play a crucial role in intercellular signaling, and in the recruitment of cells to inflammation sites. Identification of these molecules in NHP is crucial for the understanding of complex physiological and pathological mechanisms that occur in these species, and to demonstrate whether these mechanisms function similarly in humans. The Luminex100 system is a bench-top flow cytometer that allows the user to quantify up to 100 molecules simultaneously in a single tube. Recently, we identified several antibodies specific for human cytokines that have the capacity to recognize homologous chemokines and cytokines of NHP origin. Currently, we have a panel of reagents that allow for the simultaneous identification of 23 cytokines and chemokines from chimpanzees and the Old World monkeys rhesus macaques, baboons, cynomolgus macaques, pig-tailed macaques, and African green monkeys. In this R-24 application we propose to facilitate and expand the use of the Luminex technology in NHP by completing the following Specific Aims: 1) to improve the breadth and analytical power of our 23-plex NHP Luminex panel by increasing the number of detectable molecules in Old World monkey species; 2) to improve the quality and depth of our 23-plex NHP Luminex panel by increasing the sensitivity of some suboptimal reagents currently used for quantification of cytokines from Old World monkey species; and 3) to develop recombinant antibodies for the simultaneous quantification of cytokines in the marmoset, a New World monkey specie increasingly used in vaccine development, aging, and obesity research. We will perform extensive testing of available monoclonal antibodies against Old World monkey cytokines, and state-of-the-art recombinant antibody technology for the creation of antibodies against marmoset cytokines. The information and protocols generated from these studies will be listed in the Flow Cytometry website of the SNPRC; novel antibodies will be distributed to scientists who justify their need on cost-recovery bases.