Enhanced detection of N-acetylneuraminic acid using a chemiluminescence enzyme-linked immunosorbent assay

N-Acetylneuraminic acid (Neu5Ac) is a key sialic acid in food and food products that influences nutritional quality, glycosylation, and microbial interactions. Neu5Ac is integral to cellular recognition, immune modulation, and neural development, and it is a promising diagnostic biomarker, with applications spanning clinical diagnostics, food safety, pharmaceuticals, and targeted therapeutics. By optimizing assay parameters, we developed an improved chemiluminescence enzyme-linked immunosorbent assay (CLEIA) that significantly outperforms our previously established indirect competitive ELISA (ic-ELISA), which had limitations in precision and was prone to interference. The optimized CLEIA achieved an IC₅₀ of 1.864 ng mL^-1, a limit of detection (LOD) of 0.272 ng mL^-1, and a limit of quantification (LOQ) of 1.321 ng mL^-1, demonstrating superior sensitivity over the ic-ELISA's LOD of 0.57 ng mL^-1 and LOQ of 1.14 ng mL^-1. Spiked recovery experiments using edible bird's nest, milk, saliva, and urine confirmed the CLEIA's high stability, sensitivity, minimal matrix effects, and suitability for food quality control, safety, clinical, and biomedical applications. This advancement sets a new benchmark in analytical detection and application of Neu5Ac over traditional methods, promising to revolutionize food safety monitoring and clinical diagnostics.