B-120 Enhanced Detection of Brazil Nut-Specific IgE Using an Optimized Protein Extraction Protocol Integrated with the AutoLumo® 2000 Plus Analyzer

Abstract Background Brazil nut allergy is an IgE mediated hypersensitivity that can cause anaphylaxis. Its prevalence among tree nut allergies ranges from 12 percent to 22 percent. The major allergens in Brazil nut are the 9 kDa Ber e 1 and the 29 kDa Ber e 2 proteins, which can form dimers and hexamers, respectively. Current diagnostic tests use extracts that show low sensitivity compared with clinical history. Thus, a novel allergen extract is needed to improve detection accuracy. In response, we developed a procedure that chemically modifies Brazil nut extracts to enhance serum specific IgE detectability. This method is integrated with the automated AutoLumo® 2000 Plus Analyzer, a platform known for rapid chemiluminescence detection and high throughput performance that improves assay reliability. Methods Raw Brazil nut materials for multiple vendors were used to extract the allergen material using a newly developed extraction method followed by a chemical modification. Allergenic protein profiles were characterized using HPLC, with subsequent analysis by SDS PAGE to assess differences in protein composition and structural integrity. The profiles were then purified by FPLC, and functional allergenicity was evaluated in vitro using human serum samples. The optimized Brazil nut allergen extract was applied to develop a chemiluminescence immunoassay for sIgE detection on the AutoLumo® 2000 Plus Analyzer. Key performance metrics including clinical sensitivity, clinical specificity, intra run and inter run precision, linearity, and accelerated stability were determined and benchmarked against the established Phadia ImmunoCAP™ assay. Results The extracted material displayed distinct bands on HPLC and SDS-PAGE corresponding to Ber e 1, Ber e 2, their precursor forms, and polymers. Purification and testing confirmed strong allergenicity when the components were exposed to sIgE positive serum samples. Results from the AutoLumo® 2000 Plus Analyzer showed that the chemically modified extract significantly enhanced assay performance. The novel extract achieved a clinical sensitivity of 89 percent based on 91 samples and a clinical specificity of 99 percent based on 75 samples, compared with a sensitivity of 49 percent and a specificity of 100 percent for commercial extracts. Precision studies showed intra run and inter run coefficients of variation of 1.6 percent and 3.4 percent, respectively. The assay demonstrated excellent linearity, with an R squared value of 1.000 and a slope of 1.002 across IgE concentrations from 0.1 kU per L to 100 kU per L. Conclusion A novel protein extraction method has thus been successfully developed and validated, which significantly improves the sensitivity and specificity of Brazil nut specific sIgE detection. Coupled with the AutoLumo® 2000 Plus Analyzer, this approach enables a fully automated, high throughput chemiluminescence immunoassay that meets the clinical demand for accurate and timely diagnosis of Brazil nut allergy.