Nanobody-based enzyme immunoassay for naringenin in pummelos and herbal samples

Nanobodies have emerged as alternatives to conventional antibodies due to their unique advantages. In this study, two naringenin-specific nanobodies, Nb-C9 and Nb-C13, were successfully isolated. The obtained nanobodies demonstrated superior thermal stability compared to the previously reported naringenin monoclonal antibody (mAb). Based on Nb-C9, an indirect competitive enzyme-linked immunosorbent assay (Nb-ELISA) was developed for naringenin detection. The established assay exhibited a half-maximal inhibitory concentration (IC₅₀) of 15.31 ng mL^-1, with a linear range spanning from 4.55 to 60.60 ng mL^-1 (IC₂₀-IC₈₀). Furthermore, the Nb-ELISA was applied to quantify naringenin levels in peel and pulp samples from six pomelo varieties and four citrus-derived traditional Chinese herbal samples. The detection results showed strong consistency with those obtained by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). All results demonstrated that the developed Nb-ELISA is both accurate and reliable, while the identified naringenin-specific nanobodies provide a valuable foundation for developing novel immunoassays targeting naringenin.