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Updated: Mar 13, 2026
Quantifying Myeloperoxidase-DNA and Neutrophil Elastase-DNA Complexes from Neutrophil Extracellular Traps by Using a Modified Sandwich ELISA
Published on: May 12, 2023
Engineered chimeric myeloperoxidase (MPO) antibodies as candidate reference materials for MPO-ANCA detection
Mingming Zhang1, Hong Chen1, Fangming Cheng2
1Key Laboratory of Developmental Genes and Human Diseases, School of Life Science and Technology, Southeast University, Nanjing, Jiangsu 210088, China.
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.
Insights
Researchers developed chimeric antibodies to standardize anti-neutrophil cytoplasmic antibodies (ANCA) testing for myeloperoxidase (MPO). These antibodies show consistent reactivity and can serve as reference materials for improved MPO-ANCA assay harmonization and evaluation.
Area of Science:
- Immunology
- Biotechnology
- Clinical Diagnostics
Background:
- Standardization of anti-neutrophil cytoplasmic antibodies (ANCA) targeting myeloperoxidase (MPO) is crucial for diagnosing microscopic polyangiitis (MPA).
- Current MPO-ANCA testing faces challenges due to method diversity and lack of quantitative reference standards.
Purpose of the Study:
- To develop and characterize human-mouse chimeric monoclonal antibodies targeting conformational MPO epitopes.
- To establish candidate quantitative reference materials for MPO-ANCA detection immunoassays.
Main Methods:
- Screened hybridoma-derived murine anti-MPO clones for native MPO reactivity.
- Engineered high-affinity clones (B61, B62) with human IgG1 constant domains.
- Assessed binding specificity and diagnostic utility via indirect immunofluorescence (IIF), line immunoassay (LIA), ELISA, and chemiluminescence immunoassay (CLIA).
Main Results:
- Chimeric B61 and B62 antibodies exclusively bound native MPO, showing no reactivity with denatured MPO.
- Antibodies produced characteristic p-ANCA staining in IIF and confirmed specificity in LIA.
- Quantitative ELISA and CLIA yielded standard curves (R² > 0.99); competition assays showed partial epitope overlap with patient-derived MPO-ANCAs.
Conclusions:
- Engineered chimeric antibodies exhibit consistent native MPO reactivity and compatibility with multiple MPO-ANCA assays.
- Proposed as candidate reference materials for assay harmonization and performance evaluation.
- May support titer assessments or serve as alternatives to MPO-positive serum controls in research/preliminary clinical settings.
