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).
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.