Multiplexed Bead-based In Vitro Pediatric Allergy Test

描述(申请人提供)：本提案致力于发展一种高通量、高灵敏度、低血量、多重分析的过敏症体外诊断方法。 名为AllerBead(Tm)。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。及早识别过敏原诱因可以挽救生命，是管理可能限制儿童活动的衰弱症状的关键，也是开发包括新形式的个性化脱敏疗法在内的治疗方法的关键。 与更常见的体内皮肤点刺试验(SPT)相比，使用体外过敏原特异性IgE(SIgE)检测具有许多优势，包括：i)成本较低，ii)消除了可能的不良反应和患者对测试的不耐受，尤其是在儿科患者中，iii)测试中使用的高度标准化的分析程序和过敏原制剂，以及iv)客观/定量读出sIgE水平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 各公司一直在寻求开发只需几滴血就能检测多种过敏原和过敏原成分蛋白的多重系统。例如，一种基于蛋白质微阵列技术[2，3]的方法能够同时测量~100种过敏原成分。然而，蛋白质微阵列在灵敏度、重复性、可伸缩性、非线性性和制造成本等方面的局限性阻碍了这种方法取代儿科市场上更广泛使用的逐个过敏原分析方法，如ELISA法和Phadia的免疫CAP(R)。 相比之下，AmberGen在第一阶段开发和评估了一种高灵敏度、高通量、低成本的多重免疫分析方法，采用了Illumina为基因组应用开发的Veracode(R)数字全息微珠技术。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均匀和可重复地生产高纯度过敏原成分，低免疫原性一步蛋白质附着在珠子表面，以及使用可光切割抗体消除干扰IGG。值得注意的是，基于来自8个主要食品组的27种过敏原的多重分析的第一阶段结果实现了所有三个主要目标。这项工作表明，AllerBeadTM的灵敏度和变异系数类似于FDA批准的针对个人过敏原的金标准免疫CAP(R)分析，但能够自动快速地从每项专利仅10？L的血液中检测多达384种个人过敏原的96个样本。 在第二阶段，将开发最全面和最低成本的儿童食物过敏血液检测，包括60种过敏原蛋白和整个食物提取物，包括占所有儿童食物过敏90%的所有“八大”食物类别。使用AllerBead(Tm)技术的结果将与从非多路传输的免疫CAP(R)测试以及多路传输的Phadia ISAC(R)微阵列上获得的商业测量结果进行比较。为了进行检测验证并最终获得FDA的批准，AmberGen将与INOVA诊断公司密切合作，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，后者将提供900份IRB批准的有效血液样本。Life Technologies首席医疗官Paul Billings博士以及勃林格-英格尔海姆公司和拜耳医疗保健公司全球战略业务发展前负责人Kevin Xie博士也将担任顾问。

DESCRIPTION (provided by applicant): This proposal focuses on the development of a high-throughput, high sensitivity, low blood-volume, multiplex assay for in vitro diagnosis of allergies termed AllerBead(tm). 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 life-saving and a key to managing the debilitating symptoms which can limit a child's activities, as well as critical for developing treatments including new forms personalized desensitizing therapies. Utilization of in vitro allergen-specific IgE (sIgE) testing, especially in pediatrics, has many advantages over the more common in vivo skin prick test (SPT) including: i) lower costs, ii) elimination of possible adverse reactions and patient intolerance of the tests, particularly in pediatric subjects, iii) highly standardized assay procedures and allergen preparations used in the 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 component proteins. For example, one approach based on use of protein microarray technology [2, 3] is capable of measuring ~100 allergen components simultaneously. However, limitations of protein microarrays including in sensitivity, reproducibility, scalability, non-linearity 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(r) in the pediatric market. In contrast, AmberGen has developed and evaluated during Phase I a high sensitivity, high-throughput, low cost multiplex immunoassay approach by adapting the VeraCode(r) 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 1-step protein attachment to the bead surface and elimination of interfering IgGs using photocleavable antibodies. Significantly, Phase I results based on a multiplex assay of 27-allergens from the 8 major food groups achieved all 3 major aims. This work demonstrated that AllerBeadTM results in sensitivities and CVs similar to the gold-standard FDA-approved ImmunoCAP(R) assays for individual allergens, but is capable of automatically and rapidly assaying 96- samples for up to 384 individual allergens from only 10 ?L of blood per patent. During Phase II, the most comprehensive and low cost blood test for childhood food allergies comprising 60- allergen proteins and whole food extracts, including all of the "Big 8" food groups which account for >90% of all childhood food allergies, will be developed. Results using AllerBead(tm) technology will be compared with measurements obtained commercially from the non-multiplexed ImmunoCAP(r) assay and also on the multiplexed Phadia ISAC(r) Microarray. For the purpose of assay validation and eventual FDA approval, AmberGen will work closely with INOVA Diagnostics 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 900 validated blood samples under IRB approval. Dr. Paul Billings, the Chief Medical Officer at Life Technologies and Dr. Kevin Xie, former Head, Global Strategic Business Development at Boehringer-Ingelheim and at Bayer Healthcare will also serve as consultants.