Development and optimization of a new aptamer-based method for P-selectin (CD62p) measurement
Ömer Erdoğan1, Mohammed SattarAbduljabbar Al-saadi2, Evrim Cevik3, Ozge Cevik11Department of Biochemistry, Aydin Adnan Menderes University, Faculty of Medicine, Aydin, Turkey2Department of Molecular Biotechnology, Graduate School of Health Sciences, Aydin Adnan Menderes University, Aydin, Turkey
3Department of Machinery and Metal Technologies, Kocarli Vocational High School, Aydin Adnan Menderes University, Aydin, Turkey
INTRODUCTION: Disease-specific biomarkers are an essential tool for the efficient management of pathological conditions, including susceptibility determination, diagnosis, and preventive monitoring. P-selectin (CD62p) is selectively expressed after platelet activation, and is involved in thrombus formation and immune response. The serum and plasma level of CD62p increases in conditions such as heart attack, stroke, some immune diseases, and cancer. The aim of this study was to develop a new, aptamer-based method for CD62p measurement.
METHODS: Aptamers can be used to target specific biomarkers based on their molecular shape. The systematic evolution of ligands by exponential enrichment (SELEX) process is a method to identify aptamers with a high affinity for a specific macromolecular target. This study explored using aptamers to measure CD62p. First, aptamers that specifically bind to CD62p were isolated using the SELEX method. The aptamers that demonstrated the highest binding affinity to the CD62p protein were used to coat 96-well plates. Next, the level of CD62p in human serum was measured using this aptamer and the test performance parameters of sensitivity, specificity, and precision were evaluated.
RESULTS: Among the aptamers used, Apt-1, Apt-2, and Apt-3, bound to CD62p protein with high affinity. Apt-2 had the greatest binding affinity to CD62, demonstrating a binding constant of -9.6 kcal/mol, and a dissociation constant (Kd) of 18.15±2.36 nM. Bovine serum albumin was used in the specificity test as a negative control. No binding between the selected aptamers and this protein was observed. The performance showed that of intra-assay coefficient of variation (CV) was <6.52% and inter-assay CV was <3.96%. The recovery values were between 95.06% and 107.95%, and the linearity values were between 99.49% and 113.17%. Sensitivity was calculated at 0.30 ng/mL of CD62p.
DISCUSSION AND CONCLUSION: The aptamer method to measure CD62p proved to be a sensitive, specific, time-saving, and low-cost option.
Manuscript Language: English