This proposal focuses on the development of a sample-sparing and multiplexable diagnostic fordetecting 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 allergicreactions by enabling allergen avoidance and linking patients to targeted immunotherapies to induce allergende-sensitization. Current food allergy IgE diagnostics mostly use whole food extract as antigens, which leadsto low assay specificity due to contamination from cross-reactive proteins. Up to 77% patients positive for peanutextract IgE are not at risk for severe allergic reactions. In contrast, diagnostics that use individually purifiedpeanut 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 theiradoption. 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. Thesample-sparing nature of the ISAP assay permits pediatric-friendly near-painless blood collection and alsopreserves 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 thecall for a low cost, yet easily-adoptable assay for both clinical and research environments. Specific Aim I seeksto synthesize and characterize ISAP reagents for a peanut allergy panel and validate them with Co-Investigatorsat the Stanford University Sean Parker Center for Allergy & Asthma Research using samples collected from theirpeanut allergy immunotherapy trial (POISED). First, we will synthesize ISAP reagents and optimize them forcross-reactivity in detecting sIgE using positive control samples. Next, we will assess potential technical pitfallsincluding reproducibility of the conjugation protocol, stability of key reagents and potential interference fromhemolytic and lipemic samples. Finally, we will perform a head-to-head comparison of ISAP-based assays withthe current standard - ImmunoCAP. The results from this aim will provide the basis for an ISAP-based allergytest that can significantly improve pediatric and adult patient compliance and disease management. Specific Aim2 expands upon the peanut allergy panel developed in the previous aim to create a 13-plex food allergy test thatcovers over 80% of major food allergens. As before, we will synthesize ISAP reagents for each component andvalidate them using pre-characterized serum samples. A second head-to-head comparison will be performedbetween 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 betterserve millions of pediatric and adult patients, while providing proof-of-principle for ISAP use for the detection ofa wide array of other allergens, including environmental, drug and those implicated in asthma and dermatitis.