Multiplexed Bead-based In Vitro Pediatric Allergy Test

摘要/摘要 本方案的重点是开发一种高通量、高灵敏度、低输血量、多路传输的 过敏症的体外诊断试验称为AllerBeadTM。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。在儿童过敏的情况下，超过5000万 儿童受到影响，这个数字正在迅速增加，使其成为最常见的慢性儿童 疾病。及早识别过敏原触发因素可以挽救生命，也是管理衰弱的关键 可限制儿童活动的症状，以及开发包括新形式在内的治疗方法的关键 个性化脱敏疗法。 利用体外过敏原特异性IgE(SIgE)检测，特别是在儿科，有许多优点超过 更常见的活体皮肤点刺试验(SPT)包括：i)较低的成本，ii)消除可能的不良反应 反应和患者对检测的不耐受性，特别是在儿科受试者中，三)高度标准化的检测 测试中使用的程序和过敏原制剂以及IV)sIgE水平的客观/定量读数 以及自动化和高吞吐量能力。 为了认识到儿科体外诊断过敏测试的巨大市场，几家公司已经 寻求开发设计为仅使用几滴血来检测多种过敏原的多重系统，并 过敏原成分蛋白。例如，一种基于蛋白质微阵列技术的方法[2，3] 能够同时检测~100种过敏原成分。然而，蛋白质的局限性 包括灵敏度、重复性、可扩展性、非线性度和制造成本 防止这种方法取代更广泛使用的逐个过敏原检测方法，如酶联免疫吸附试验和 Phadia公司的免疫CAP(R)进入儿科市场。 相比之下，AmberGen在第一阶段开发和评估了高灵敏度、高通量、低 采用Veracode(R)数字全息微珠技术的成本多重免疫分析方法 由Illumina开发用于基因组应用。这种新方法的核心依赖于专有方法 由AmberGen开发，包括使用无细胞蛋白表达，以实现统一和可重复性 生产高纯度的过敏原成分，低免疫原性的一步蛋白附着在珠子上 利用可光切抗体对干扰性免疫球蛋白进行表面和消除。值得注意的是，第一阶段的结果 基于对来自8个主要食品组的27种过敏原的多重分析，实现了所有3个主要目标。这部作品 证明了AllerBeadTM导致的敏感性和简历类似于FDA批准的黄金标准 免疫CAP(R)检测个别过敏原，但能够自动和快速检测96- 每项专利仅从L的10份血液中提取多达384种个体过敏原样本。 在第二阶段，针对儿童食物过敏的最全面和最低成本的血液测试包括60- 过敏原蛋白质和全食物提取物，包括占90%的“八大”食物组 所有儿童时期的食物过敏，都会发生。使用AllerBeadTM技术的结果将与 从非多路复用的免疫CAP(R)测试中获得的商业测量结果 多路复用Phadia ISAC(R)微阵列。为了测试验证和最终FDA批准的目的， AmberGen将与INOVA诊断公司密切合作，并与医学博士琳达·施奈德合作。 波士顿儿童医院过敏项目主任，哈佛大学儿科学副教授 医学院将提供900份经IRB批准的有效血液样本。保罗·比林斯博士，酋长 生命科技医疗官和前全球战略业务发展主管谢凯文博士 勃林格-英格尔海姆和拜耳医疗集团也将担任顾问。

SUMMARY/ABSTRACT This proposal focuses on the development of a high-throughput, high sensitivity, low blood-volume, multiplex assay for in vitro diagnosis of allergies termed AllerBeadTM. 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 are 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 AllerBeadTM 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.