Production and characterization of a single-chain variable fragment-alkaline phosphatase fusion protein for glycocholic acid detection in a one-step enzyme-linked immunosorbent assay

Abstract

A single-chain variable fragment (scFv)-alkaline phosphatase (AP) fusion protein for glycocholic acid (GCA) was produced and characterized. The scFv gene with a 218 linker was generated by splicing by overlap extension (SOE)-polymerase chain reaction (PCR) and sequentially inserted into the expression vector pecan45 containing AP gene to express the scFv-AP fusion protein in Escherichia coli (E. coli). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses revealed that the fusion protein showed the expected molecular weight of about 80 kDa. Both the antibody binding capacity and AP enzyme activity of the scFv-AP fusion protein were validated by colorimetric analysis. One-step competitive direct enzyme-linked immunosorbent assay (ELISA) based on the scFv-AP fusion protein indicated that the average concentration required for 50% inhibition of binding (IC₅₀) and limit of detection (LOD) for GCA were 216 ng mL⁻¹ and 37.0 ng mL⁻¹, respectively, and the linear response range extended from 71.0 to 657 ng mL⁻¹. The cross-reactivity (CR) of the scFv-AP fusion protein was similar to those of its parental scFv antibody. The scFv-AP fusion protein was bifunctional, retaining both antibody binding specificity and AP enzyme activity. This work indicates that the production of the scFv-AP fusion protein in E. coli strain BL21(DE3)pLysS is feasible and suggests that it could be further used as convenient one-step detection probes for GCA.