Construction of a biotinylated cameloid-like antibody for lable-free detection of apolipoprotein B-100

Nanobodies (Nbs), also known as the variable domain of the heavy-chain-only antibody (VHH), are single-domain antigen-binding fragments derived from heavy-chain antibodies that occur naturally in sera of camelids. Due to their unique properties of small size (15 kD), intrinsic stability, high affinity and specificity, Nbs are suitable for detecting clinical relevant antigens. Apolipoprotein B-100 (ApoB-100) is a highly predictive marker for coronary artery disease (CAD), which is frequently detected in clinical diagnosis. Herein, we successfully obtained anti-ApoB-100 Nbs for the first time and further fabricated a label-free and sensitive immunosensor for ApoB-100 based on isolated anti-ApoB-100 nanobody (Nb) using the electrochemical impedance spectroscopy (EIS) technique. We have generated an immunized phage display library against ApoB-100 and isolated four anti-ApoB-100 Nbs with high affinity and stability. The Nb with the highest affinity was biotinylated based on in vivo BirA system. Further, we developed a label-free electrochemical impedance immunosensor for ApoB-100 using this anti-ApoB-100 Nb. The attachment of ApoB-100 onto the anti-ApoB-100 Nb-immobilized sensing layer led to the increased electron-transfer resistance, which was proportional to ApoB-100 concentration in the range from 0.05 to 5 ng mL−1 with a detection limit of 0.03 ng mL−1. This proposed immunosensor revealed high specificity to detect ApoB-100, acceptable intra-assay precision and good stability, functioning as a feasible technique for CAD diagnosis.