Development of a chimeric monoclonal antibody as a potential diagnostic reference in PR3-ANCA detection

The quantification of antineutrophil cytoplasmic antibodies (ANCA) against proteinase 3 (PR3) is crucial for diagnosing and monitoring granulomatosis with polyangiitis (GPA). However, a standardized and widely available quantitative reference material for PR3-ANCA detection remains lacking.

This study aims to develop and evaluate the properties of a novel chimeric human-mouse PR3 monoclonal antibody as a potential quantitative reference for PR3-ANCA detection using various in vitro diagnostic methods.

Mouse monoclonal antibodies were screened from hybridoma cells derived from mice immunized with recombinant human PR3 (rPR3). A high-affinity monoclonal antibody (A07) was sequenced, and a chimeric antibody incorporating human IgG1 constant regions was produced using a mammalian cell expression system. The antibody's binding to PR3 and its applicability in indirect immunofluorescence (IIF) assay, line-blotting, enzyme-linked immunosorbent assay (ELISA), and chemiluminescence immunoassay (CLIA) were analyzed.

The chimeric A07 antibody recognized the conformational epitopes with high affinity for only non-denatured PR3 and demonstrated the correct cytoplasmic staining pattern in IIF. Line-blotting assays confirmed its specificity and potency. ELISA and CLIA experiments showed well-defined standard curves, with the chimeric antibody displaying consistent reactivity comparable to native PR3-ANCA in serum samples across different antigen conditions.

These findings indicate that the chimeric A07 antibody effectively recognizes both recombinant and native PR3 antigens and can be applied across multiple in vitro PR3-ANCA assays. Furthermore, this antibody may serve as a standard reference for determining serum PR3-ANCA titers or as a substitute for PR3-positive serum in diagnostic applications.