A digital <scp>ELISA</scp> for multiplexed detection of allergen‐specific <scp>IgE</scp> against Der p 1, Der p 2, and Der p 23

The house dust mite <i>Dermatophagoides pteronyssinus</i> produces major allergens (Der p 1, Der p 2, and Der p 23) that require precise IgE detection for clinical diagnosis. We developed a multiplex digital ELISA using fluorescence-encoded micromagnetic beads (532 nm/638 nm dual-wavelength system) coupled with microfluidics to simultaneously quantify serum IgE against these components, with comprehensive evaluation against the clinical standard UniCAP system. The 532 nm channel measured allergen-specific signals via average brightness increase (ABMB) of enzymatically amplified fluorescence, while 638 nm enabled spectral bead differentiation. Comparative evaluation with UniCAP showed the improved digital ELISA achieved uniform 75.0% sensitivity but variable specificity (42.9%-54.5%) across allergens at the 15.8% ABMB threshold. Sample classification results (Der p 1: 9 positive/6 negative; Der p 2: 7/8; Der p 23: 7/8) demonstrated suboptimal positive predictive values (33.3%-60.0%) versus more favorable negative predictive values (60.0%-85.7%), with likelihood ratios (LR+: 1.31-1.65) and Cohen's <i>κ</i> (0.12-0.25) suggesting limited diagnostic reliability. The automated platform offered 60% reduced sample volume (20 μL vs. 50 μL), multiplex capability, and maintained sensitivity for low-titer samples, representing an efficient screening solution pending specificity enhancement.