Screening for novel variable domains of the heavy chains of heavy-chain antibodies targeting different epitopes of thrombomodulin and Luminex fluorescence immunoassay for soluble thrombomodulin.

Venous thromboembolism (VTE), a potentially fatal disease, poses considerable health risks that should not be overlooked. Early disease screening and timely intervention with anticoagulant drugs are crucial for its management. However, effective screening methods, real-time anticoagulant efficacy monitoring approaches, and high-performance diagnostic antibodies are currently lacking for VTE. In this work, VHHs spanning multiple regions of the extracellular domain of thrombomodulin were confirmed using a progressive peptide truncation strategy. For the first time, this study identified novel soluble thrombomodulin-targeting variable domain of the heavy chain of heavy-chain antibody (VHH) pairs with excellent binding activity. Furthermore, by integrating the convenient and reliable Luminex platform with the novel VHH pairs, this study established a new detection method for soluble thrombomodulin with a wide quantitative range of 0.25-1000 ng/mL and detection limit as low as 0.18 ng/mL. In the spiked actual samples, recovery rate was 95.8-99.1 %, with a relative standard deviation of 1.9-4.6 %. Overall, the identified antibodies and our new detection method exhibit great potential for basic and applied research on thrombomodulin and soluble thrombomodulin.