Screening of a novel VHH pair targeting different epitopes of D-dimer and development of a sandwich luminex immunoassay for plasma D-dimer detection

Background: Early screening for venous thromboembolism and the precise use of anticoagulant drugs are crucial, and further basic innovations are urgently needed to advance related diagnostic and therapeutic practices. This study aimed to develop an innovative antibody for detecting D-dimer and to verify its performance. The core aim was to screen a pair of variable domains of the heavy chain of heavy-chain antibodies (VHH) that can specifically recognize unique epitopes on the D-dimer molecule. This study is expected to promote technological advancements in the diagnosis, risk assessment, and efficacy monitoring of thrombotic diseases. Results: D-dimer-positive clones underwent positive and negative screening across seven rounds of phage biopanning, yielding a pair of VHHs. The superior thermal stability of the sandwich VHH pair was demonstrated after continuous heating at 80 °C for up to 1 h by comparison with a commercial monoclonal antibody. Subsequently, by integrating the reliable and convenient Luminex platform with these novel VHHs, a new detection method for D-dimer was established under optimal conditions, with a wide quantitative range of 0.5 to 1000 ng/mL and detection limit as low as 0.25 ng/mL. The recovery rate of spiked samples ranged from 99.3% to 106.0%, with a relative standard deviation of 1.1% to 3.2%. Furthermore, this newly developed VHH-based Luminex assay was utilized to examine clinical D-dimer samples, and its performance was evaluated by correlation analysis with the clinical INNOVANCE® D-Dimer Assay, resulting in a Pearson correlation coefficient of 0.99086 (P < 0.0001). Significance: This study identified a novel VHH pair targeting D-dimer, demonstrating good binding activity and exceptional thermal stability, making it highly effective for bioanalysis experiments. Utilizing the reliable and efficient Luminex platform, a detection method for D-dimer was developed, exhibiting substantial potential for applications in venous thrombus screening, anticoagulant efficacy assessment, and related fundamental research.