Use of Mass Spectrometry to Assess Anti-Neutrophil Cytoplasmic Antibodies (ANCA) in ANCA-Associated Vasculitis (AAV)

Anti-Neutrophil Cytoplasmic Antibodies (ANCA)-Associated Vasculitis (AAV) is a group of rare disorders characterised by the necrotising inflammation of small blood vessels. Patients may present with severe organ-threatening or life-threatening disease. The detection of Myeloperoxidase (MPO)-ANCA and Proteinase (PR3)-ANCA aids in the rapid diagnosis of AAV. Since the discovery of ANCA, inconsistencies in their measurement have presented problems in terms of monitoring disease severity, activity, and progression. Therefore, prediction of disease onset and flares remains suboptimal.

Mass Spectrometry (MS) techniques are used routinely to quantify small molecules such as drugs of abuse in clinical biochemistry laboratories and the results of these are widely accepted by regulatory and official bodies. Whilst this technology is beginning to flourish as an exciting and powerful tool for biomarker discovery, very few studies to date have used it in autoantibody investigations. In collaboration with the specialist protein company ‘Binding Site,’ this study aims to establish an MS technique that is novel within the setting of AAV.

By successfully conjugating MPO and PR3 antigens to the surface of magnetic beads, these were utilised to extract MPO-ANCA and PR3-ANCA from serum samples. A process known as immunoprecipitation. These purified MPO-ANCA and PR3-ANCA eluates were then subjected to MALDI-ToF acquisition, and their presence and quantity analysed.

Although only on a small scale, this feasibility study provided evidence that MS technology has the potential to reliably detect, quantify and standardise the measurement of MPO-ANCA and PR3-ANCA. Longitudinal assessment of patient samples by MALDI-ToF may provide an improved understanding of the dynamic clonal evolution of ANCA in each patient that will better inform AAV therapeutic
interventions, playing a role to prevent relapse and minimise the burden of therapy induced adverse effects.

Although not a high throughput solution, confirmation that the method can be established in larger cohort studies of AAV patients will demonstrate its wider applicability in other autoantibody diseases.

Outputs

• Poster Presentation at the American Association of Immunologists (AAI) Annual Conference in Honolulu, Hawaii, May 2025
• Poster Presentation at the British society of Immunology – Clinical Immunology Professional Network (BSI-CIPN) Annual conference in Liverpool, Dec 2025