Development of an Enzyme-Linked Immunosorbent Assay (ELISA) for Determining Neutrophil Elastase (NE) -a Potential Useful Marker of Multi-organ Damage Observed in COVID-19 and Post-Covid-19 (PCS)

Joanna Adamiec-Mroczek ¹ Joanna Kluz ² Sandra Chwałek ³ Maciej Rabczyński ² Kinga Gostomska-Pampuch ^4* Łukasz Lewandowski ⁴ Marta Misiuk-Hojło ¹ Beata Ponikowska ⁵ Goutam Chourasia ⁶ Ilias Dumas ⁷ Andrzej Gamian ⁸ Żanna Fiodorenko-Dumas ⁷ Bogusława Konopska ⁹ Agnieszka Gola ¹⁰ Klaudia Konikowska ¹¹ Daniel Strub ³ Agnieszka Bronowicka- Szydełko ⁴ Katarzyna Madziarska ²

• ¹ Clinical Department of Ophthalmology, Wroclaw Medical University, Wrocław, Silesian, Poland
• ² Clinical Department of Diabetology, Hypertension and Internal Disease, Wroclaw Medical University, Wroclaw, Poland
• ³ Faculty of Chemistry, Wrocław University of Science and Technology, Wroclaw, Poland
• ⁴ Department of Biochemistry and Immunochemistry, Wroclaw Medical University, Wrocław, Poland
• ⁵ Department of Physiology and Pathophysiology, Wroclaw Medical University, Wrocław, Poland
• ⁶ Department and Clinic of Emergency Medicine, Wroclaw Medical University, Wroclaw, Poland
• ⁷ Department of Clinical Physiotherapy and Rehabilitation, Wroclaw Medical University, Wroclaw, Poland
• ⁸ Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wrocław, Silesian, Poland
• ⁹ Department of Laboratory Diagnostics, Faculty of Pharmacy, Wroclaw Medical University, Wroclaw, Poland
• ¹⁰ Department of Physical Chemistry and Biophysics, Wroclaw Medical University, Wroclaw, Poland
• ¹¹ Department of Dietetics and Bromatology, Wroclaw Medical University, Wroclaw, Poland

The ongoing post-COVID-19 syndrome (PCS) epidemic, causing complications of diverse etiology (resulting from a prior COVID-19 infection), necessitates the search for new diagnostic markers and the development of widely accessible methods for their detection.. Confirming their presence in the bloodThis would enable the prognosis of PCS progression and, consequently, the faster implementation of targeted treatments. One potential marker appears to beis neutrophil elastase (NE), whose elevated levels in the blood during PCS may result from organ damage caused by increased secretion of severe inflammatory mediators or amyloidosis resulting from the interaction of NE with SARS-CoV-2.. The detection of this protein in the blood indicates organ damage. The aim of this publication is to present a step-by-step method for designing an enzymatic ELISA test, enabling the quantitative assessment of NE in the blood serum of patients. Methods: NE was measured using the designed ELISA test. Results: The study outlines all the steps necessary for designing and optimizing the ELISA test, including the selection of standards, primary and secondary antibodies, and their dilutions. Using the test, elevated NE levels were demonstrated in patients with advanced-stage diabetic nephropathy after symptomatic COVID-19, compared to a relative group of patients sampled before COVID-19. Conclusions: The undertaken efforts enabled the development of a test with high performance parameters (initially set sensitivity: ≥40 pg/µL; intra-assay precision: 7%; inter-assay precision <20%CV < 30%). No significant cross-reactivity with other tested proteins was observed. Serial dilution of plasma samples resulted in a proportional decrease in signal intensity.