Soluble endoglin (sENG) is an important biomarker of several cardiometabolic and vascular disorders. The accurate measurement of biomarkers that are simultaneously targeted by therapeutic monoclonal antibodies (mAbs) in preclinical models is a significant challenge. Traditional enzyme-linked immunosorbent assays (ELISAs) often fail due to epitope blocking, while advanced techniques like mass spectrometry are more expensive and require specialized expertise. To address these limitations, we developed a cost-effective, specific Western blot-based method for evaluating sENG in mouse plasma in a metabolic dysfunction-associated steatohepatitis (MASH) rescue model. Thus, from a methodological perspective, we significantly modified and extended the sENG detection method introduced in our previous work, focusing on developing a cost-effective approach for semi-quantitative analysis. For these purposes, we used a mouse MASH model treated with the anti-endoglin (ENG) mAb M1043. Plasma samples were concentrated, and proteins were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and probed with an anti-ENG antibody. We verified the method using human hepatic sinusoidal endothelial cells (HHSECs) culture media treated with the therapeutic human anti-ENG monoclonal antibody, TRC105. This Western blot-based approach avoids interference from therapeutic mAbs, delivering reliable and reproducible results. This method overcomes the limitations of ELISAs and mass spectrometer, providing a practical solution for semi-quantitative analysis of biomarkers in both preclinical and clinical research. Its versatility makes it applicable to various soluble protein biomarkers across diseases. As therapeutic mAbs become more widely used, this simple, cost-effective method facilitates mechanistic insights and accelerates targeted therapy development in research.

Department of Biological and Medical Sciences Faculty of Pharmacy in Hradec Kralove Charles University Heyrovskeho 1203 500 05 Hradec Kralove Czech Republic

Department of Clinical Immunology and Allergology Faculty of Medicine in Hradec Kralove Charles University Hradec Kralove Czech Republic

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