Institute for Biomolecular Research, Hochschule Fresenius, University of Applied Sciences, Limburger Straße 2, 65510 Idstein, Germany.
Clinical Lipidomics Unit, Institute of Physiological Chemistry, University Medical Center, 55131 Mainz, Germany.
J Proteome Res. 2023 Oct 6;22(10):3135-3148. doi: 10.1021/acs.jproteome.3c00082. Epub 2023 Sep 6.
Procalcitonin (PCT) is a biomarker for bacterial sepsis, and accurate quantification of PCT is critical for sepsis diagnosis and treatment. Immunological PCT quantification methods are routinely used in clinical laboratories, yet there is a need for harmonization of PCT quantification protocols. An orthogonal method to clinical immunological assays, such as LC-MS/MS, is required. In this study, a highly sensitive and robust immunoaffinity LC-MRM quantitative method for detecting procalcitonin in human serum has been developed. An initial comparison of immunocapture of PCT with a polyclonal anti-PCT antibody immobilized on polystyrene nanoparticles (Latex) and magnetic beads demonstrated superior performance with magnetic beads. Three tryptic PCT peptides from the N- and C-terminal regions of PCT were selected for LC-MS/MS quantification. For PCT quantification, an LLOQ of 0.25 ng/mL of PCT in human serum was achieved using a sample volume of 1 mL. The method's trueness and precision consistently lie within the 15% margin. The parallel measurement of three PCT peptides may allow future differentiation of intact PCT vs other PCT forms originating from potential degradation, processing, or polymorphisms. An established and validated LC-MRM-based quantification of PCT will be relevant as an orthogonal method for harmonization and standardization of clinical assays for PCT.
降钙素原 (PCT) 是细菌性败血症的生物标志物，准确测量 PCT 对败血症的诊断和治疗至关重要。免疫测定法常用于临床实验室中 PCT 的定量，但需要对 PCT 定量方案进行协调。需要一种与临床免疫测定法（如 LC-MS/MS）正交的方法。本研究开发了一种用于检测人血清中降钙素原的高灵敏度和稳健的免疫亲和 LC-MRM 定量方法。初步比较了与固定在聚苯乙烯纳米颗粒（乳胶）和磁性珠上的多克隆抗 PCT 抗体的 PCT 免疫捕获，结果表明磁性珠具有更好的性能。选择 PCT 的 N-和 C-末端区域的三个胰蛋白酶 PCT 肽用于 LC-MS/MS 定量。对于 PCT 的定量，使用 1 mL 样本体积，人血清中 PCT 的LLOQ 为 0.25ng/mL。该方法的准确性和精密度始终在 15%的范围内。三个 PCT 肽的平行测量可能允许未来区分完整的 PCT 与其他可能源自潜在降解、加工或多态性的 PCT 形式。已建立和验证的基于 LC-MRM 的 PCT 定量方法将作为 PCT 临床测定的协调和标准化的正交方法具有重要意义。
