A novel sandwich immunoassay for Staphylococcus aureus using horseradish peroxidase-conjugated trivalent nanobody

Nanobodies (Nbs), owing to their lack of the fragment crystallizable region, are a promising alternative to conventional antibodies for the specific and efficient detection of pathogenic bacteria by minimizing nonspecific interactions. This study reports a trivalent nanobody (3Nb156) targeting Staphylococcus aureus (S. aureus) engineered via multivalent design and directly conjugated with horseradish peroxidase (HRP). A sandwich enzyme-linked immunosorbent assay (TNb-sELISA) was developed using Nb55 as the capture antibody and HRP-3Nb156 as the detection probe. The TNb-sELISA achieved a detection limit of 2.84 × 10⁴ CFU/mL, representing a 3.2-fold improvement in sensitivity over the monovalent nanobody-based ELISA. The assay demonstrated high specificity and excellent reproducibility. In spiked milk and chicken samples diluted 10-fold, recoveries ranged from 89.49% to 110.37%. After 6 h of enrichment, 10 CFU of S. aureus was detectable in real samples. This study establishes a rapid, sensitive, and reliable platform for detecting S. aureus in complex food matrices.