Applicability of the Ella system for automatic Elisa to detect biomarkers in endometriosis

Aim. Endometriosis is a chronic gynecological disorder characterized by ectopic endometrial tissue, inflammation, and angiogenesis. Non-invasive biomarkers in serum and urine could improve diagnosis and disease monitoring. This study evaluates the applicability of the Ella automated ELISA system for quantifying key endometriosis- associated biomarkers. Methods. The Ella platform (ProteinSimple/Bio-Techne) allows automated, multiplexed quantification of cytokines, chemokines, and growth factors from minimal sample volumes. Plasma and urine were considered as matrices for biomarker detection. System performance, including sensitivity, reproducibility, and multiplexing capability, was assessed based on previously reported biomarker levels in women with and without endometriosis. Results. Previous studies reported elevated levels of IGFBP-1 ( UniProt Consortium 2025b, National Center for Biotechnology Information 2025c ), CD5L Hong 2025 ,ANXA1 (Fang 2024), MCP-1 ( National Center for Biotechnology Information 2025b ), VEGF-A (Didžiokaitė 2025 ), TNF-α ( National Center for Biotechnology Information 2025a ), and ‡ ‡ § | | © Mihaylova E et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. IL-6 in serum ( UniProt Consortium 2025a ), reflecting systemic inflammation and angiogenic activity. Urine samples showed increased IL-6 ( UniProt Consortium 2025a ), IL-8 (UniProt Consortium. 2025a ), VEGF (Didžiokaitė 2025 ), IL-6-ST (National Center for Biotechnology Information. 2025 ), Alpha-Enolase ( UniProt Consortium. 2025b ), VDBP (Fernando 2020 ), and CA125 ( Felder 2014 ), indicating both local and systemic disease- related changes. For better visual representation, the proteins described above and their functions are listed in Table 1. These findings support the feasibility of using automated ELISA platforms like Ella to detect disease-relevant biomarkers in both biofluids. Conclusions. The Ella automated ELISA system provides a reliable and efficient approach for measuring serum and urinary biomarkers associated with endometriosis. Its multiplexing capacity and low sample requirements enable non-invasive biomarker studies, supporting early diagnosis, monitoring of disease progression, and evaluation of therapeutic interventions. Gene Full name Protein Function IGFBP1 Insulin like growth factor binding protein 1 IGFBP-1 Regulates the bioavailability and acitvity of insulin-like growth factors; important in cell migration and metabolism CD5L CD5 molecule like CD5L Key regulator of lipid synthesis; regulates mechanisms in inflammatory responses ANXA1 Annexin A1 ANXA1 Ubiquitous protein;Inhibits phospholipase A2 and has anti- inflammatory activity CCL-2 C-C motif chemokine ligand 2 MCP-1 Regulates leukocyte recruitment; Chemotactic activity for monocytes and basophils TNF Tumor necrosis factor TNF-αInvolved in the regulation of a wide spectrum of biological processes including cell proliferation, differentiation, apoptosis, lipid metabolism, and coagulation VEGF- A Vascular endothelial growth factor A VEGF-A Key mediator of angiogenesis and inflammation; Implicated in reproductive and immune processes IL-6 Interleukin 6 IL-6 Potent inducer of the acute phase response; Induces the production of VEGF CXCL8 C-X-C motif chemokine ligand 8 IL-8 Mediates inflammatory response by attracting neutrophils, basophils, and T-cells to clear pathogens and protect the host from infection IL6ST Interleukin 6 cytokine family signal transducer IL-6ST Part of the cytokine-receptor complex; Suggested role in regulating myocyte apoptosis Table 1. Applicability of the Ella system for automatic Elisa to detect biomarkers in endometriosis. 2 Mihaylova E et al Gene Full name Protein Function ENO1 Enolase 1 Alpha- enolase Glycolytic enzyme; Involved in various processes such as growth control, hypoxia tolerance and allergic responses GC GC vitamin D binding protein Vitamin D- binding protein Immunoregulation, glucose metabolism, and regulation of blood pressure; involved in maintaining an environment of tolerance to paternal and fetal tissue and the augmentation of pro-inflammatory states MUC16 Mucin 16, cell surface associated CA 125 Immune attack resistance; Plays role in metastasis; Contributes to ovarian tumor growth

automated Elisa (Ella), endometriosis, biomarkers Presenting author Dragomira Nikolova Presented at International Conference on Natural Sciences and Biotechnology – Kliment’s Days 2025

This study is a part of the project D-156/4.6.25 financed by the Council of Medical Science, Medical University - Sofia, Bulgaria. Funding program Project D-156/4.6.25 Conflicts of interest The authors have declared that no competing interests exist. Applicability of the Ella system for automatic Elisa to detect biomarkers ... 3

• Didžiokaitė G, et al. (2025) Serum VEGF Potential as a Biomarker for Diagnosis of Female Unexplained Infertility: Does Atopy Matter? International Journal of Molecular Sciences https://doi.org/10.3390/ijms26199499 • Fang L, et al. (2024) Annexin A1 binds PDZ and LIM domain 7 to inhibit adipogenesis and prevent obesity. Signal Transduction and T argeted Therapy https://doi.org/10.1038/ s41392-024-01930-0 • Felder M, et al. (2014) MUC16 (CA125): tumor biomarker to cancer therapy, a work in progress. Molecular Cancer https://doi.org/10.1186/1476-4598-13-129 • Fernando M, et al. (2020) Vitamin D-Binding Protein in Pregnancy and Reproductive Health. Nutrients https://doi.org/10.3390/nu12051489 • Hong H, et al. (2025) Characterizing Kupffer Cell Production of CD5 Antigen-Like and Its Function on Regulating Migration of NaturalKiller T Cells. The American Journal Of Pathology https://doi.org/10.1016/j.ajpath.2025.06.003 • National Center for Biotechnology Information (2025a) TNF tumor necrosis factor [Homo sapiens (human)] – Gene ID: 7124. https://www.ncbi.nlm.nih.gov/gene/7124 • National Center for Biotechnology Information (2025b) CCL2 C-C motif chemokine ligand 2 [Homo sapiens (human)] – Gene ID: 6347. https://www.ncbi.nlm.nih.gov/gene/6347 • National Center for Biotechnology Information (2025c) IGFBP1 insulin like growth factor binding protein 1 [Homo sapiens (human)] – Gene ID: 3484. https://www.ncbi.nlm.nih.gov/ gene/3484. Accessed on: 2025-10-21. • National Center for Biotechnology Information. (2025) IL6ST interleukin 6 cytokine family signal transducer [Homo sapiens (human)]. https://www.ncbi.nlm.nih.gov/gene/3572 • UniProt Consortium (2025a) P05231 – IL6 – Interleukin-6 – Homo sapiens (Human). https://www.uniprot.org/uniprotkb/P05231/entry • UniProt Consortium (2025b) P08833 – IGFBP1 – Insulin-like growth factor-binding protein 1 – Homo sapiens (Human). https://www.uniprot.org/uniprotkb/P08833/entry. Accessed on: 2025-10-21. • UniProt Consortium. (2025a) P10145 – CXCL8 – Interleukin-8 – Homo sapiens (Human). https://www.uniprot.org/uniprotkb/P10145/entry • UniProt Consortium. (2025b) P06733 – ENO1 – Alpha-enolase – Homo sapiens (Human). https://www.uniprot.org/uniprotkb/P06733/entry 4 Mihaylova E et al