(T1330-01-07) A Novel LC-MS/MS-Based Method for Quantification of Human Serum Properdin: Development, Qualification, and Validation

Purpose: Properdin, a key regulator of the alternative complement pathway, is increasingly implicated in inflammatory and autoimmune diseases such as Myasthenia Gravis (MG). Traditional ELISA methods for properdin quantification suffer from limitations due to reliance on critical reagents and variability in assay performance. To address the lack of published reliable LC-MS/MS-based methods for human properdin quantification, this study aims to develop such a method, offering improved specificity, precision, accuracy, and robustness.

Methods: We developed and qualified a novel LC-MS/MS method for serum properdin quantification. Method performance was assessed for precision, accuracy, selectivity, interference, dilution integrity, and stability. Natural history MG serum samples and matched healthy serum samples were analyzed. Results were benchmarked against published data obtained with conventional ELISA assays.

Results: The LC-MS/MS method, with a dynamic range of 1–200 µg/mL, demonstrated enhanced selectivity, and precision (CV < 13%) and accuracy (91% of recovery) that were consistent across qualification studies. The method exhibited robust freeze-thaw and reinjection stability. Importantly, using this method, properdin levels in anti-acetylcholine receptor antibody-positive (AChR+) MG serum samples were significantly lower than matched healthy cohort. This finding aligned closely with published results from ELISA, validating this method's applicability for clinical measurements.

Conclusion: This novel LC-MS/MS method represents a significant advancement in the reliable quantification of serum properdin in disease samples. The elimination of critical reagent dependence associated with ELISA assays highlights its potential for clinical biomarker studies in MG and other complement-driven conditions, providing a powerful tool for both clinical research and therapeutic monitoring.

References: Ozawa Y, Uzawa A, Yasuda M, Kojima Y, Oda F, Himuro K, Kawaguchi N, Kuwabara S. Changes in serum complements and their regulators in generalized myasthenia gravis. Eur J Neurol. 2021 Jan;28(1):314-322. doi: 10.1111/ene.14500. Epub 2020 Sep 27. PMID: 32889770.


Table 1: Inter-assay precision and accuracy


Table 2: Assay selectivity


Figure 1: Validation of the LC-MS/MS method with Natural History MG serum samples and their matched healthy volunteer (HV) control samples