Single-Eye NfL Measurement Using NULISA Technology Enables Reduction in Animal Use.

Neurofilament light chain (NfL) is a pivotal biomarker for neuroaxonal damage. However, its quantification in volume-limited matrices, such as mouse aqueous humor (AH) (2 to 6 µL per eye), has historically required pooling samples from multiple animals due to the low sensitivity and high sample volume requirements of standard immunoassays. Such pooling increases animal requirements and precludes individual-level data analysis. To overcome these limitations, we developed and evaluated a NfL HomeBrew (HB) assay using the NUcleic acid-Linked Immuno-Sandwich Assay (NULISA) technology. NULISA offers superior analytical sensitivity and requires less than 10 µL volume. Our results demonstrate that the NfL HB NULISA performs comparably to the Simoa reference assay and provides superior sensitivity to the Ella platform. Crucially, the assay reliably quantified NfL in as little as 2 µL of individual mouse AH, with 90% of samples yielding quantifiable results. This approach eliminates the need for sample pooling, effectively reducing animal use by up to eightfold while preserving data integrity at the individual animal level. These findings establish the NULISA platform as a highly sensitive, ethically advantageous solution for neurobiological research in volume-restricted models.