Multiplexed Bead-based In Vitro Pediatric Allergy Test

描述(申请人提供)：这项建议的重点是开发一种高通量、高灵敏度、低血量、多重系统，用于儿科食物过敏的体外诊断。过敏包括一组免疫紊乱，构成了美国慢性疾病的第六大原因。在儿童过敏方面，有5000多万儿童受到影响，这一数字正在迅速增加，使其成为最常见的慢性儿童疾病。在许多情况下，及早识别过敏原触发因素可能是挽救生命的关键，也是管理可能限制儿童活动的衰弱症状的关键。的具体标识 个别过敏原也可能是开发治疗方法的关键，包括新形式的个性化脱敏治疗。 虽然体内皮肤点刺试验(SPT)和体外过敏原特异性IgE(SIgE)试验是过敏诊断的主要模式(连同体检和临床病史)，但体外sIgE试验的利用，特别是在儿科，具有许多优于SPT的优点，包括：i)通过简化操作和消除对受过训练的人员和专门设施/程序的需要来管理SPT，降低成本；ii)消除试验的可能的不良反应和患者对试验的耐受性，特别是在儿科受试者中；iii)在试验中使用高度标准化的分析程序和过敏原制剂，以及广泛可用的标准化(商业)FDA批准的测试，以及iv)sIgE水平的客观/定量读数以及自动化和高通量能力。 为了认识到儿科体外诊断过敏测试的巨大市场，有几个 公司一直在寻求开发多路系统，设计成只需几滴血就能检测多种过敏原和过敏原成分。例如，一种基于蛋白质微阵列技术的方法能够同时测量~100种过敏原成分(~40种过敏原类型)。然而，蛋白质微阵列的局限性，包括有限的灵敏度、重复性、可伸缩性和制造成本，阻碍了这种方法的实施。 取代儿科市场上更广泛使用的一对一变应原检测方法，如ELISA法和Phadia的免疫CAP法。 相比之下，AmberGen最近通过将Illumina开发的Veracode(TM)数字全息微珠技术应用于基因组应用，成功地开发了一种高灵敏度、高通量、低成本的多重免疫分析方法。这一新方法的核心依赖于AmberGen开发的专利方法，包括使用无细胞蛋白质表达来统一和可重复地生产高纯度过敏原成分，低免疫原性蛋白质附着在珠子表面，以及提供正常化和减少检测差异的2-Tag/2-激光过敏原检测。值得注意的是，这种方法的灵敏度与单独的ELISA免疫分析方法相似，但能够自动同时从10 L的血液中检测384种单独的过敏原蛋白。此外，配套的BeadXpress(Tm)平台每小时能够自动处理96个患者样本，从而使每分析一种过敏原的成本非常低。 在第一阶段，我们将评估这一新方法应用于低血量、多重、儿科食物过敏原检测。为此，我们将重点关注来自8种最常见的儿科食物过敏原的21种主要蛋白质，牛奶、大豆、小麦、鸡蛋、花生、坚果、鱼和贝类，它们占所有食物过敏的90%。无细胞表达的蛋白过敏原将 与多重平台上纯化的天然蛋白过敏原进行比较。结果将与在标准非多路传输的免疫CAP系统上进行的测量进行比较。为了进行检测验证并最终获得FDA的批准，AmberGen将与Illumina Inc.密切合作(请参阅支持函)，并与波士顿儿童医院过敏项目主任、哈佛医学院儿科副教授Lynda Schneider医学博士合作，后者将根据IRB批准提供咨询和验证的血液样本(请参阅支持函)。

DESCRIPTION (provided by applicant): This proposal focuses on the development of a high-throughput, high sensitivity, low blood-volume, multiplex system for in vitro diagnosis of pediatri food allergies. Allergies comprise a group of immune disorders constituting the 6th leading cause of chronic disease in the US. In the case of pediatric allergies, over 50 million children ar affected, a number which is rapidly increasing, making it the most common chronic childhood disease. Early identification of allergen triggers can be in many cases life-saving and a key to managing the debilitating symptoms which can limit a child's activities. Specific identification of individual allergens can also be critical for developing treatments including new forms personalized desensitizing therapies. While the in vivo skin prick test (SPT) and in vitro allergen-specific IgE (sIgE) tests are the majr modes of allergy diagnosis (along with physical examination and clinical history), the utilization of in vitro sIgE testing, especially in pediatrics, has many advantages over the SPT including: i) lower costs through ease of performance and elimination of the need for trained personnel and specialized facilities/procedures to administer SPT, ii) elimination of possible adverse reaction t the test and patient intolerance of the tests, particularly in pediatric subjects, iii) highly standardized assay procedures and allergen preparations used in the tests, as well as the wide-availability of standardized (commercial) FDA-approved tests, and iv) objective/quantitative readout of sIgE levels as well as automation and high throughput capability. In recognition of the significant market for pediatric in vitro diagnostic allergy testing, several companies have sought to develop multiplex systems designed to use only a few drops of blood to assay multiple allergens and allergen components. For example, one approach based on use of protein microarray technology is capable of measuring ~100 allergen components simultaneously (~40 allergen types). However, limitations of protein microarrays including limited sensitivity, reproducibility, scalability and expense of manufacture prevents this approach from replacing the more widely used one-by-one allergen assays such as ELISA and Phadia's ImmunoCAP in the pediatric market. In contrast, AmberGen has recently succeeded in developing a high sensitivity, high-throughput, low cost multiplex immunoassay approach by adapting the VeraCode(tm) digital holographic micro-bead technology developed by Illumina for genomic applications. The heart of this new approach relies on proprietary methods developed by AmberGen including the use of cell-free protein expression for uniform and reproducible production of highly pure allergen components, low immunogenic protein attachment to the bead surface and 2-tag/2-laser allergen detection providing normalization and reduced assay variance. Significantly, this approach results in sensitivities similar to individual ELISA immunoassays but is capable of automatically assaying 384 individual allergen proteins simultaneously from only 10 ?L of blood. Furthermore, the accompanying BeadXpress(tm) platform is capable of automatically processing 96 patient samples per hour resulting in very low costs per allergen assayed. During Phase I, we will evaluate this new approach applied to a low blood-volume, multiplex, pediatric food allergen test. For this purpose, we will focus on a panel of 21 major proteins from the 8 most common pediatric food allergens, milk, soy, wheat, egg, peanuts, tree nuts, fish and shellfish, which account for >90% of all food allergies. Cell-free expressed protein allergens will be compared to purified natural protein allergens on the multiplexed platform. Results will be compared with measurements performed on the standard non- multiplexed ImmunoCAP system. For the purpose of assay validation and eventual FDA approval, AmberGen will work closely with Illumina Inc. (see letter of support) and collaborate with Dr. Lynda Schneider, M.D., Director of the Allergy Program at Children's Hospital, Boston, Associate Professor of Pediatrics, Harvard Medical School, who will provide consulting and validated blood samples under IRB approval (see letter of support).