Journal of immunological methods · 2026
Engineered chimeric myeloperoxidase (MPO) antibodies as candidate reference materials for MPO-ANCA detection.
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Zhang Mingming, Chen Hong, Cheng Fangming, Zhang Youtao, Yu Pin, Li Li, Jiang Yuzhang, Dai Yaping, Qiu Fang, Qin Zhuye, Han Chongxu, Yang Yonghong, Wang Gang, Qian Chungen, Shi Xingjuan, Guo Xuancheng, Liu Xiangdong
Аннотация
BACKGROUND: The standardization of anti-neutrophil cytoplasmic antibodies (ANCA) targeting myeloperoxidase (MPO) represents a critical unmet need in diagnosing microscopic polyangiitis (MPA). MPO-ANCA testing remains challenging due to the diversity of methods and the absence of universally accepted quantitative reference standards.
OBJECTIVE: To develop and characterize human-mouse chimeric monoclonal antibodies specifically recognizing conformational MPO epitopes for use as candidate quantitative reference materials across MPO-ANCA detection immunoassays.
METHODS: Hybridoma-derived murine anti-MPO clones were screened for reactivity with native MPO antigens. The variable regions of two high-affinity clones (B61 and B62) were genetically engineered with human IgG1 constant domains and expressed using a mammalian expression system. Binding specificity and diagnostic utility were comprehensively assessed through four immunoassay modalities: indirect immunofluorescence (IIF) assay, line immunoassay (LIA), enzyme-linked immunosorbent assay (ELISA), and chemiluminescence immunoassay (CLIA).
RESULTS: The chimeric B61 and B62 antibodies demonstrated exclusive binding to native MPO, whereas they showed no reactivity with denatured MPO. In IIF, both antibodies produced characteristic perinuclear ANCA (p-ANCA) staining patterns on ethanol-fixed neutrophils, recapitulating the reactivity profiles of MPO-ANCA-positive clinical sera. LIA confirmed strict antigenic specificity and potency. Quantitative analyses using ELISA and CLIA produced well-characterized standard curves within defined quantifiable ranges (R2 > 0.99) based on polynomial regression. Competition assays indicated partial overlap of B61 and B62 antibodies with epitopes recognized by patient-derived MPO-ANCAs.
CONCLUSIONS: These engineered chimeric antibodies demonstrate consistent reactivity with native MPO antigens and exhibit full compatibility with multiple MPO-ANCA detection methods. They are proposed as candidate reference materials for relative assay harmonization and comparative performance evaluation. In this context, these antibodies may support titer-related assessments or serve as alternatives to MPO-positive serum controls in research or preliminary clinical settings.
OBJECTIVE: To develop and characterize human-mouse chimeric monoclonal antibodies specifically recognizing conformational MPO epitopes for use as candidate quantitative reference materials across MPO-ANCA detection immunoassays.
METHODS: Hybridoma-derived murine anti-MPO clones were screened for reactivity with native MPO antigens. The variable regions of two high-affinity clones (B61 and B62) were genetically engineered with human IgG1 constant domains and expressed using a mammalian expression system. Binding specificity and diagnostic utility were comprehensively assessed through four immunoassay modalities: indirect immunofluorescence (IIF) assay, line immunoassay (LIA), enzyme-linked immunosorbent assay (ELISA), and chemiluminescence immunoassay (CLIA).
RESULTS: The chimeric B61 and B62 antibodies demonstrated exclusive binding to native MPO, whereas they showed no reactivity with denatured MPO. In IIF, both antibodies produced characteristic perinuclear ANCA (p-ANCA) staining patterns on ethanol-fixed neutrophils, recapitulating the reactivity profiles of MPO-ANCA-positive clinical sera. LIA confirmed strict antigenic specificity and potency. Quantitative analyses using ELISA and CLIA produced well-characterized standard curves within defined quantifiable ranges (R2 > 0.99) based on polynomial regression. Competition assays indicated partial overlap of B61 and B62 antibodies with epitopes recognized by patient-derived MPO-ANCAs.
CONCLUSIONS: These engineered chimeric antibodies demonstrate consistent reactivity with native MPO antigens and exhibit full compatibility with multiple MPO-ANCA detection methods. They are proposed as candidate reference materials for relative assay harmonization and comparative performance evaluation. In this context, these antibodies may support titer-related assessments or serve as alternatives to MPO-positive serum controls in research or preliminary clinical settings.