Development of bioluminescent enzyme immunoassay for glycocholic acid based on singlechain variable fragment-nanoluciferase fusion

Abstract

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