SBIR-STTR Award

This proposal focuses on the development of a sample-sparing and multiplexable diagnostic for detecting and monitoring the immune marker IgE against food allergen components using Isotype- Specific Agglutination-PCR (ISAP) technology. Early identification of marking IgE can prevent severe allergic reactions by enabling allergen avoidance and linking patients to targeted immunotherapies to induce allergen de-sensitization. Current food allergy IgE diagnostics mostly use whole food extract as antigens, which leads to low assay specificity due to contamination from cross-reactive proteins. Up to 77% patients positive for peanut extract IgE are not at risk for severe allergic reactions. In contrast, diagnostics that use individually purified peanut components can identify allergy risks with significantly improved accuracy. However, these component- resolved IgE diagnostics require large sample volumes and have high assay costs that adversely restrict their adoption. To address these limitations, the proposed ISAP-based allergy diagnostic uses a homogenous ligation- based DNA barcoding assay to sensitively quantify multiple allergen IgE types in a 1?L volume sample. The sample-sparing nature of the ISAP assay permits pediatric-friendly near-painless blood collection and also preserves precious samples for additional immunological testing such as genomic and cellular analysis. Notably, since this assay uses inexpensive reagents and widely-available qPCR instruments for readout, it answers the call for a low cost, yet easily-adoptable assay for both clinical and research environments. Specific Aim I seeks to synthesize and characterize ISAP reagents for a peanut allergy panel and validate them with Co-Investigators at the Stanford University Sean Parker Center for Allergy & Asthma Research using samples collected from their peanut allergy immunotherapy trial (POISED). First, we will synthesize ISAP reagents and optimize them for cross-reactivity in detecting sIgE using positive control samples. Next, we will assess potential technical pitfalls including reproducibility of the conjugation protocol, stability of key reagents and potential interference from hemolytic and lipemic samples. Finally, we will perform a head-to-head comparison of ISAP-based assays with the current standard - ImmunoCAP. The results from this aim will provide the basis for an ISAP-based allergy test that can significantly improve pediatric and adult patient compliance and disease management. Specific Aim 2 expands upon the peanut allergy panel developed in the previous aim to create a 13-plex food allergy test that covers over 80% of major food allergens. As before, we will synthesize ISAP reagents for each component and validate them using pre-characterized serum samples. A second head-to-head comparison will be performed between ISAP and ImmunoCAP using samples derived from the multi-food allergy trial (POISED and MAP-X). In an era of rising prevalence of food allergy, the study can establish ISAP as a sample-sparing assay to better serve millions of pediatric and adult patients, while providing proof-of-principle for ISAP use for the detection of a wide array of other allergens, including environmental, drug and those implicated in asthma and dermatitis.

Project Terms:
Address; Adopted; Adoption; Adult; Agglutination; Allergens; Allergic Reaction; Allergy to peanuts; American; anti-IgE; Antibodies; Antigens; Asthma; Attention; base; Benchmarking; Biological Assay; Biological Sciences; Blood; Child; Childhood; Clinical; Clinical Research; cohort; Collection; Compliance behavior; Consumption; cost; cross reactivity; Data; Dermatitis; Detection; Development; Diagnostic; Disease; Disease Management; DNA; Drops; Early identification; Environment; Fat emulsion; Food; food allergen; Food Hypersensitivity; food preparation; Freezing; Gel; Genomics; head-to-head comparison; Hypersensitivity; IgE; Immune System Diseases; Immunologic Markers; Immunologic Monitoring; Immunologic Tests; Immunotherapy; immunotherapy trials; improved; Individual; instrument; Legal patent; Life; Ligation; Link; Methods; Molecular; Monitor; Nature; novel; Painless; Patients; pediatric patients; Pharmaceutical Preparations; Plasma; Positioning Attribute; Preparation; Prevalence; prevent; Proteins; Protocols documentation; Quality of life; Reagent; Recovery; Reproducibility; Research; Research Personnel; response; Risk; Sampling; Sensitivity and Specificity; Serum; Specificity; success; Technology; Testing; Time; tool; Universities; Validation; Variant; Vendor;

Food allergy is a serious and growing health concern currently affecting 8% of children in the US. Blood tests based on the immunology marker IgE facilitate assessment of food allergy risks, and are required to recommend food avoidance or to implement new and innovative immunotherapies. However, current methods for allergy blood testing are expensive, require large volume phlebotomy and often result in false positives and negatives. In our Phase I study, Enable Biosciences created a highly novel allergy immune test based on Isotype-Specific Agglutination-PCR (ISAP) technology. This ISAP allergy panel detects over 80% of common food allergens, including peanut, egg, milk, hazelnut, cashew, and shrimp. Unlike existing methods, ISAP requires only a single microliter of blood to perform, facilitating easier testing in children who may not be able to endure large blood draws. ISAP displayed 100% specificity and 75-100% sensitivity for all food allergens in comparison to existing methods that exhibit 38-60% specificity and 71-96% sensitivity, giving ISAP the potential to greatly reduce false positives and negatives in allergy testing. ISAP also showed very low inter- and intra- assay variability (<15%). Finally, we demonstrated that ISAP can be adapted for use with dried blood spot with near perfect correlation with plasma (R = 0.90-0.99). Building upon these significant technical achievements, Enable has secured pivotal business partnerships with Hamilton Robotics to provide marketing and automation support for ISAP assays. We are in the final stage of CLIA certification for our South San Francisco laboratory, creating a new clinical testing channel for commercialization of ISAP tests. In this Phase II proposal, we seek support for further product development and quality control efforts to optimize ISAP testing for the clinical marketplace. First, we will expand our current food allergy panels to cover 95% of all common food allergens. Second, we will automate the ISAP chemistry to reduce labor costs and further improve reproducibility. Third, we will generate and validate quality control and manufacturing protocols to ensure seamless scalability. Finally, we will validate the suitability of ISAP for use with dried blood spot samples to permit even less invasive testing. This Phase II proposal can revolutionize allergy testing and enable therapeutic success by increasing accessibility, accuracy and reproducibility while reducing costs for patients and payors alike.

Public Health Relevance Statement:
Food allergy is a growing and potentially fatal condition affecting over 8% of children in the United States. Tests that diagnose or establish risk for allergy guide treatment decisions and support clinical trials for new and innovative prevention therapies. However, current allergy tests are expensive, inaccurate and require large volumes of blood which makes it challenging to test small children. Enable Biosciences is developing a low-cost, next-generation allergy test with much better accuracy that requires only a minute blood sample that can be collected by fingerstick or dried blood spot in the convenience of the home.

Project Terms:
Achievement; Affect; Agglutination; Agreement; Allergens; Allergic; assay development; Automation; base; Biological Assay; Biological Sciences; Blood; Blood specimen; Blood Tests; Blood Volume; Businesses; Cashew nut; Certification; Chemistry; Child; Childhood; Clinical; Clinical Trials; cohort; commercialization; cost; cross reactivity; design; Development; Devices; Diagnosis; Diagnostic; DNA; egg; Enrollment; Ensure; Evaluation; Exhibits; Food; food allergen; food avoidance; Food Hypersensitivity; Generations; Hazelnuts; Health; Home environment; Hypersensitivity; IgE; Immunologic Markers; Immunologic Tests; Immunology; Immunotherapy; improved; Infant; innovation; Juglans; Laboratories; Liquid substance; Manuals; manufacturing process; Marketing; Measures; Methods; Milk; Monitoring Clinical Trials; next generation; novel; Output; Patients; Performance; Phase; phase 1 study; Plasma; Prevention; Prevention therapy; Process; product development; Protocols documentation; quality assurance; Quality Control; Reagent; Reproducibility; research clinical testing; Risk; Robotics; ROC Curve; Sampling; San Francisco; Secure; Sensitivity and Specificity; Shrimp; Signal Transduction; software development; soy; Specificity; Spottings; success; Technology; Test Result; Testing; testing services; Therapeutic; Transportation; United States; Validation; Variant; Venous; Venous blood sampling; Wheat