Development of immunoaffinity enrichment techniques for measurement of gfap protein in serum by id-lc-ms/ms method

Abstract

The high rates of mortality and disability caused by neurological diseases represent a significant global public health concern, with a further increase in the coming decades. In conditions like Alzheimer's disease (AD), Parkinson's disease (PD), and traumatic brain injury (TBI), early and accurate diagnosis is essential for the timely interventions that can help slow down disease progression. However, the definitive diagnosis of many neurological disorders often occurs only after symptoms have significantly advanced, which severely restricts the window for effective treatment. Overcoming this challenge requires developing reliable, accessible biomarkers that directly reflect disease pathology. In this context, Glial Fibrillary Acidic Protein (GFAP), a major structural component of astrocytes in the central nervous system (CNS), emerges as a particularly promising candidate. As a type III intermediate filament protein, GFAP is essential for preserving cellular structure, offering mechanical support, and managing the blood-brain barrier (BBB). When the CNS experiences trauma, ischemia, or degeneration, astrocytes undergo reactive changes known as "astrogliosis," characterised by increased GFAP production. Furthermore, when the integrity of the blood-brain barrier is disrupted, GFAP can leak into the peripheral bloodstream, where it can be detected, serving as a specific marker for the severity of brain injury and prognosis. Currently, GFAP measurement in serum primarily relies on immunoassays like Enzyme-Linked Immunosorbent Assay (ELISA) and Single-Molecule Array (SIMOA). However, ensuring SI (International System of Units) traceability in accordance with ISO 17511 standards is a clinical necessity to achieve harmonization and comparability between these methods. In this thesis, to establish a foundation for the metrological traceability chain, recombinant human GFAP protein was successfully expressed and obtained at high purity using chromatographic techniques. The purification of the polyhistidine-tagged protein was carried out using Immobilized Metal Affinity Chromatography (IMAC), and the quality of the purification was confirmed through Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE). Using this purified protein, the study aimed to develop a candidate Reference Measurement Procedure (RMP) via Isotope Dilution Liquid Chromatography-Tandem Mass Spectrometry (ID-LC-MS/MS). This approach intends to fill the existing gap in the traceability chain by directly linking patient results to SI units. To overcome low protein concentrations and matrix complexity in serum, an immunoaffinity (IA) enrichment strategy using antibody-magnetic-bead complexes was employed. Optimization experiments determined that binding 100 μg of antibody to 1 mg of magnetic nanoparticles was optimal, and utilizing 10 μl of this synthesized complex was sufficient for GFAP enrichment from 1 ml of serum. Following these optimizations, the developed ID-LC-MS/MS method produced Limits of Quantification (LOQ) of 0.3, 0.93, 0.77, and 0.78 μg/L, and Limits of Detection (LOD) of 0.09, 0.28, 0.23, and 0.23 μg/L for the four selected peptides (LEAENNLAAYR, ALAAELNQLR, FADLTDAAAR, LADVYQAELR), respectively. Although the current sensitivity levels—limited by existing mass spectrometry capabilities and the complexity of the serum matrix—are insufficient to measure picogram levels found in healthy individuals or in early-stage neurodegeneration, the established SI traceability indicates that this method serves as a strong candidate reference measurement procedure, crucial for developing certified reference materials. Future research should focus on improving analytical sensitivity and selectivity through advancements in sample preparation, enrichment efficiency, and instrumental performance. In particular, the use of more efficient immunoenrichment strategies, highly selective affinity binders such as next-generation monoclonal antibodies or aptamers, and optimized chromatographic workflows may further reduce matrix interference and enhance low-level GFAP detection in serum. Furthermore, it is recommended to apply this candidate reference method to large-scale clinical samples to compare it with existing immunoassays and to fully demonstrate the clinical applicability of GFAP for early diagnosis and monitoring of disease using standardized data. In conclusion, this thesis standardizes the entire process from GFAP production to reference measurement, establishing a strategic academic foundation for a reference measurement system compatible with international standards for the diagnosis of neurological diseases.