Multiplexed Bead-based In Vitro Pediatric Allergy Test

描述（由申请人提供）：该提案的重点是开发用于体外诊断小儿食物过敏的高通量、高灵敏度、低血容量、多重系统。过敏症包括一组免疫疾病，构成美国慢性病的第六大原因。在儿科过敏症的情况下，超过5000万儿童受到影响，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，早期识别过敏原触发因素可以挽救生命，也是控制可能限制儿童活动的衰弱症状的关键。特异性地鉴别 个体过敏原对于开发包括新形式的个性化脱敏疗法的治疗也是至关重要的。 而过敏原特异性IgE（sIgE）检测和体内皮肤点刺试验（SPT）是过敏诊断的主要方法（沿着体格检查和临床病史），体外sIgE检测的使用，特别是在儿科，具有许多优于SPT的优点，包括：㈠通过便于执行和消除对管理防范酷刑小组委员会的训练有素的人员和专门设施/程序的需要来降低成本，ii）消除了测试中可能的不良反应和患者对测试的不耐受性，特别是在儿科受试者中，iii）测试中使用的高度标准化的测定程序和过敏原制剂，以及标准化（商业）FDA批准的测试的广泛可用性，和iv）sIgE水平的客观/定量读出以及自动化和高通量能力。 认识到儿科体外诊断过敏试验的巨大市场， 公司已经寻求开发设计成仅使用几滴血来分析多种过敏原和过敏原成分的多重系统。例如，一种基于使用蛋白质微阵列技术的方法能够同时测量约100种过敏原组分（约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、再现性、可扩展性和制造成本，阻碍了这种方法 从取代更广泛使用的一个接一个的过敏原检测，如ELISA和Phadia的免疫CAP在儿科市场。 相比之下，AmberGen最近成功地开发了一种高灵敏度、高通量、低成本的多重免疫测定方法，该方法采用了由Illumina开发的用于基因组应用的VeraCodeTM数字全息微珠技术。这种新方法的核心依赖于AmberGen开发的专有方法，包括使用无细胞蛋白表达来均匀和可重复地生产高纯度的过敏原组分，低免疫原性蛋白附着在珠表面，以及2-标签/2-激光过敏原检测，提供标准化和降低的测定方差。值得注意的是，这种方法的结果类似于个别ELISA免疫测定的灵敏度，但能够自动测定384个单独的过敏原蛋白质，同时从只有10？我的血。此外，附带的BeadXpress（TM）平台能够每小时自动处理96个患者样本，从而导致每个过敏原测定的成本非常低。 在第一阶段，我们将评估这种新方法应用于低血容量，多重，儿科食物过敏原测试。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，鱼类和贝类，占所有食物过敏的90%以上。无细胞表达的蛋白过敏原将 与多路复用平台上纯化的天然蛋白过敏原进行比较。将结果与在标准非多重ImmunoCAP系统上进行的测量进行比较。为了进行检测验证和最终获得FDA批准，AmberGen将与Illumina Inc.密切合作。(see支持信），并与Lynda Schneider博士，医学博士，波士顿儿童医院过敏项目主任，哈佛医学院儿科副教授，将在IRB批准下提供咨询和经验证的血液样本（见支持函）。 公共卫生相关性：过敏是慢性病的第六大原因，每年至少涉及200亿美元的医疗保健费用。此外，过敏症的患病率正在以惊人的速度增长。小儿食物过敏的诊断尤其 由于可能发生严重的、危及生命的过敏反应，这一点很重要。AmberGen将评估构建一种低成本、高通量、基于微珠的系统的可行性，该系统用于同时体外测量多种过敏原特异性IgE，涵盖占所有食物过敏90%以上的前八种食物，并且仅需要与手指或医疗棒兼容的少量血液样本。

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). PUBLIC HEALTH RELEVANCE: Allergies constitute the 6th leading cause of chronic disease and involve at least $20 billion in annual health care costs. Furthermore, the prevalence of allergies is growing at an alarming rate. The diagnosis of pediatric food allergies is particularly important due to the possibility of severe, life-threatening anaphylactic reactions. During this proposed Phase I project, AmberGen will evaluate the feasibility of constructing a low-cost, high throughput, micro-bead based system for simultaneous in vitro measurements of multiple allergen-specific IgEs, covering the top eight foods which account for >90% of all food allergies, and requiring only a small blood sample volume compatible with a finger or heal-stick.