Screening and characterization of aptamers for recombinant human programmed death-1 and recombinant extracellular domain of human programmed death ligand-1.

OBJECTIVE: Programmed death ligand-1 (PD-L1) is expressed on tumor cells and macrophages. The detection of PD-L1 expression in cancer and the treatment by targeting the PD-L1/programmed death-1 (PD-1) are of great clinical significance. This work aims to screen the aptamers with high affinity and specificity for recombinant human PD-1 (rhPD-1)/recombinant human PD-L1 extracellular domain (rhPD-L1). MATERIALS AND METHODS: In this study, we have expressed, purified, prepared, and identified rhPD-1 and rhPD-L1. The rhPD-L1/rhPD-1 aptamers with high affinity and specificity were obtained by systematic evolution of ligands by exponential enrichment technique. Ten aptamers sequences to rhPD-L1 and 10 aptamers sequences to rhPD-1 were obtained by cloning and sequencing. The affinity and specificity of candidate aptamers were analyzed by gold nanoparticles-based colorimetric assay, dot blot assay, and electrophoretic mobility shift assay. RESULTS: The aptamers named A6 were picked out as the optimal aptamers that recognize PD-1, specifically with the Kd value of 47.84 ± 24.78 nM. The aptamers named B10 were picked out as the optimal aptamers that recognize PD-L1, specifically with the Kd value of 59.72 ± 15.87 nM. CONCLUSIONS: The study lays a foundation for the development of detection methods and therapeutic drugs targeting PD-L1/PD-1.

Matrix interference in serum total thyroxin (T4) time-resolved fluorescence immunoassay (TRFIA) and its elimination with the use of streptavidin-biotin separation technique.

In our development of total serum thyroxin TRFIA using an immobilized second-antibody (S-Ab) as the separation agent, we observed a significant measurement bias caused by a matrix interference when the immobilized S-Ab had a relatively low binding capacity for the primary anti-T4 monoclonal antibody (McAb). Therefore, we employed a new separation system based on the highly active surface streptavidin and biotinylated anti-T4 McAb. Our results indicate that the matrix interference was removed and the assay performance was improved with the use of streptavidin-biotin separation technique. In our method, microwells were first coated with biotinylated BSA and then a streptavidin solution in the presence of 1% BSA was added to allow streptavidin to be immobilized via the pre-coated BSA-biotin. Surface streptavidin prepared in this protocol expressed a significantly increased binding capacity for the biotinylated anti-T4 McAb, compared to the passively adsorbed S-Ab for binding the original anti-T4 McAb. The immunoreactions between the biotinylated anti-T4 McAb and the T4 in the standard or sample or the europium-labeled T4-BSA conjugate mainly occurred in liquid solution, and then the immune complex was specifically trapped by the surface streptavidin and isolated from the free trace by washing. Serum TT4 TRFIA based on surface streptavidin was accurate, precise and economic, maintained all the merits of the directly immobilized surface antibodies.