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Difference in antigenic determinant profiles between human and rat myeloperoxidase
Y C Patry1, P H Nachman, M A P Audrain
1INSERM U419, Nantes, France.
Abstract:
We tested whether rat and human MPO have similar antigenic determinants using 36 human MPO-ANCA positive sera, one mouse anti-rat MPO and four mouse anti-human MPO monoclonal reagents. Purified rat and human MPO were used in ELISA, with or without crossinhibition by preincubation with human MPO or irrelevant antigen in the liquid phase. Only one human MPO ANCA positive serum exhibited significant binding in rat MPO ELISA. This binding was poorly inhibited by preincubation with human MPO in the liquid phase, but was conserved after adsorption of non specific anti-rat activity in a chromatography column. Three mouse anti-human MPO IgG monoclonal antibodies did not recognize rat MPO. Only one mouse anti-human MPO IgA monoclonal antibody bound to rat MPO. This binding was poorly inhibited by preincubation with human MPO (35% at 2 micro g/ml). Conversely, the mouse anti-rat MPO monoclonal did not bind human MPO. We have concluded that: (1) Most human MPO-ANCA recognize antigenic determinants on human MPO which are absent on rat MPO. Therefore, human auto-antibodies bind to epitopes which recently appeared after species evolution; (2) Inversely, the mouse anti-rat MPO monoclonal do not bind human MPO. Therefore, rat MPO epitopes have been altered during species evolution; (3) Mice injected with human MPO preferentially develop antibodies against xeno-epitopes which are not present in rodents. Therefore, human MPO may not be the best antigen to raise ANCA in animal models and (4) A comparison of the amino acid sequences of rat and human MPO may help elucidate the major antigenic epitopes.
Insights
Human myeloperoxidase (MPO) autoantibodies primarily target unique epitopes absent in rat MPO, suggesting recent evolutionary changes. Rat MPO also possesses distinct epitopes, indicating evolutionary alterations in this species.
Area of Science:
- Immunology
- Biochemistry
- Evolutionary Biology
Background:
- Myeloperoxidase (MPO) is a key enzyme implicated in autoimmune diseases, particularly those associated with anti-neutrophil cytoplasmic antibodies (ANCA).
- Understanding the antigenic determinants of MPO is crucial for developing accurate diagnostic tools and effective animal models for research.
Purpose of the Study:
- To investigate the cross-reactivity of human MPO-ANCA with rat MPO.
- To compare the antigenic epitopes of human and rat MPO.
- To evaluate the suitability of human MPO as an antigen in animal models for generating ANCA.
Main Methods:
- Enzyme-linked immunosorbent assay (ELISA) was employed using purified rat and human MPO.
- Human MPO-ANCA positive sera and monoclonal antibodies (mouse anti-rat MPO, mouse anti-human MPO) were utilized.
- Cross-inhibition studies were performed by preincubation with human MPO or irrelevant antigen.
Main Results:
- Only one out of 36 human MPO-ANCA positive sera showed significant binding to rat MPO, with poor inhibition by human MPO.
- Three out of four mouse anti-human MPO monoclonal antibodies did not recognize rat MPO; one IgA antibody showed weak binding.
- The mouse anti-rat MPO monoclonal antibody did not bind human MPO, indicating distinct epitopes between species.
Conclusions:
- Human MPO-ANCA predominantly recognize epitopes absent in rat MPO, suggesting recent evolutionary divergence.
- Rat MPO contains unique epitopes altered during evolution, and human MPO may not be optimal for raising ANCA in rodent models.
- Comparative amino acid sequence analysis of rat and human MPO is recommended to identify key antigenic epitopes.