Bioluminescent enzyme immunoassay for cortisol by exploitation of single-chain variable fragment and single-chain variable fragment-nanoluciferase fusion protein.

BACKGROUND

Measurement of cortisol levels is still a complex multi-step process in immunoassay. The isolation of single-chain variable fragment (scFv) from phage-display library is an effective approach to generate antibody-enzyme fusion protein, which can be used to improve the performance of immunoassay.

RESULTS

In this work, the specific scFv against cortisol was isolated from native chicken-derived phage display library by the stringent bio-panning strategy. The obtained scFv was further fused with nanoluciferase (Nluc), named scFv-Nluc. Compared with scFv-based enzyme-linked immunosorbent assay (ELISA), the bioluminescent enzyme immunoassay (BLEIA) based on scFv-Nluc was conducted in one-step strategy, which could reduce the assay time. The half maximal inhibitory concentration (IC50) of the one-step BLEIA was calculated to be 203 ng/mL, which was 5-fold lower than the scFv-based ELISA (IC50 of 1139 ng/mL). When the BLEIA and ELISA are applied to serum samples subjected to 200-fold dilution, the corresponding original-serum equivalent IC50 values are 41 μg/mL for BLEIA and 228 μg/mL for ELISA, respectively. The established BLEIA exhibited excellent selectivity for cortisol among the analogues, while the satisfactory recoveries between 85.1 % and 120.1 % were obtained.

SIGNIFICANCE: The results above demonstrated that both scFv and scFv-Nluc could be utilized as useful reagents for immunoassay, but the scFv-Nluc-based BLEIA could further enhance the performance of immunoassay, which may represent the promising method for cortisol analysis.