Development of a bioluminescent enzyme immunoassay for glycocholic acid based on a single-chain variable fragment-nanoluciferase fusion

Abstract

As glycocholic acid (GCA) has been recognized as a biomarker for hepatocellular carcinoma (HCC), its detection is crucial for the prevention, diagnosis, and treatment of HCC. With the advantages of being simple, rapid and sensitive, immunoassay is one of the optimal choices for the timely detection of GCA. The genetic antibody is an effective approach to generate a bi-functional antibody, which can further improve the performance of the immunoassay. In this work, the bi-functional antibody was created by the fusion of a GCA-specific single-chain variable fragment (scFv) with nanoluciferase (Nluc), named scFv-Nluc. Using scFv-Nluc, the developed bioluminescent enzyme immunoassay (BLEIA) was carried out without the addition of a secondary antibody, which could reduce assay time and enhance assay performance. After optimizing the assay conditions, the half maximal inhibitory concentration (IC₅₀) of BLEIA was calculated to be 0.959 µg mL⁻¹, with a limit of detection (LOD) of 0.122 µg mL⁻¹. The BLEIA provided not only high selectivity for GCA analogues but also the satisfactory recoveries ranging from 99.26% to 117.18%. Thus, these results indicated that scFv-Nluc was an effective immunodetection tool for developing BLEIA, which has significant potential for GCA analysis.