Anti-neutrophil cytoplasmic antibodies (ANCA) are critical serological markers for ANCA-associated vasculitis (AAV), primarily targeting neutrophil autoantigens such as proteinase 3 (PR3) and myeloperoxidase (MPO) (Jennette et al., 2013). PR3 is a 29-kDa neutral serine protease localized in the azurophilic granules of neutrophils and monocytes and serves as the primary antigen for cytoplasmic ANCA (c-ANCA) in granulomatosis with polyangiitis (GPA). PR3-ANCA exhibits over 90 % specificity for GPA when detected via immunoassays, with the majority of antibodies targeting conformational epitopes on PR3 (Sun et al., 1998).

The revised international consensus recommends high-quality antigen-specific immunoassays as the primary screening method for PR3-ANCA detections (Bossuyt et al., 2017). Efforts to standardize PR3-ANCA measurement have led to the development of international reference materials, such as the PR3-ANCA human reference serum #16 (Center for Disease Control, Atlanta, GA, USA), assigned a quantitative content of 100 IU/mL. Additionally, the Institute for Reference Materials and Measurements (IRMM) of the European Union has released certified reference materials (ERM-DA483/IFCC) based on plasmapheresis samples (Monogioudi et al., 2019).

Despite these advancements, a stable, non-serum-derived PR3-ANCA reference material suitable for various diagnostic platforms remains unavailable. Given the limited supply of the International Union of Immunological Societies-Centers for Disease Control and Prevention (IUIS-CDC) reference serum, which is primarily used to calibrate secondary standards, we aimed to develop an alternative PR3-ANCA reference with an unlimited supply. We identified a high-affinity mouse anti-PR3 antibody (A07), sequenced its variable regions, and generated a chimeric antibody incorporating human IgG1 constant regions. Utilizing a CHO-based expression system, we produced the A07 antibody in large quantities and evaluated its performance across multiple in vitro PR3-ANCA assays.