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Interaction between Myeloperoxidase (MPO), Ceruloplasmin and Anti-MPO Antibodies from Patients with Microscopic Polyangiitis

Author: ChenJie
Tutor: LiHong；ZhaoMingHui
School: Zhengzhou University
Course: Internal Medicine
Keywords: Myeloperoxidase Anti- neutrophil cytoplasmic antibody Under the microscope polyangiitis Ceruloplasmin Inhibition
CLC: R593.2
Type: Master's thesis
Year: 2007
Downloads: 66
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Abstract

[Background and Purpose] anti-neutrophil cytoplasmic antibodies (antineutrophil cytoplasmic antibodies, ANCA) associated vasculitis is a group of autoimmune diseases. ANCA serological diagnostic tools, alcohol-fixed neutrophils as substrate, indirect immunofluorescence method can be divided into the cytoplasm (cytoplasmic, c-ANCA) and ring karyotype (perinuclear p-ANCA) its main target antigens proteinase 3 (proteinase 3, PR3) and myeloperoxidase (myeloperoxidase in MPO). Which c-ANCA/PR3-ANCA mainly seen in Wegener's granulomatosis (Wegener's granulomatosis, WG) patients, the while p-ANCA/MPO-ANCA mainly seen under the microscope polyangiitis (microscopic polyangiitis, MPA) patients. MPA is the most common ANCA-associated systemic vasculitis (ANCA associated systemic vasculitis, AASV). Anti-MPO antibody is considered to be important serological markers of MPA diagnosis, but their pathogenesis is not entirely clear. MPO is a neutrophil cytoplasmic oxidase, ceruloplasmin is a physiological inhibitor of MPO. The interaction between the anti-MPO antibodies, plasma copper, ceruloplasmin and MPO may play an important role in the pathogenesis of MPA, but the domestic yet to see the reports. Purpose of this experiment is to study using affinity chromatography and extraction in patients with primary MPA anti-MPO antibody activity of MPO oxidation inhibition, and further study of the MPO, the relationship between ceruloplasmin and anti-MPO antibodies to the pathogenesis of MPA provide a theoretical basis. [Experimental Method] 11 patients with plasma from anti-MPO antibodies in patients with primary MPA Peking University First Hospital, Department of Nephrology, October 1998, admitted in April 2006, the sera of 12 healthy blood donors served as normal controls. Purified by Protein G affinity chromatography method in serum immunoglobulin G (IgG), anti-MPO antibody application MPO affinity chromatography purified IgG, the application of highly purified human neutrophil MPO 0.127mg / the ML sCP amine 0.001% over the hydrogen peroxide under conditions to establish a stable enzyme reaction system. Were measured in different concentrations in patients with anti-MPO antibodies, of ceruloplasmin and normal IgG exist the MPO enzyme oxidation activity under the conditions, and the MPA patients with anti-MPO antibodies and ceruloplasmin inhibit the the MPO enzymatic oxidation activity differences. The application of competitive enzyme-linked immunosorbent assay (ELISA) for further determination of the interaction between the anti-MPO antibody, ceruloplasmin and MPO. Under the same conditions, the decline of the MPO enzymatic oxidation activity = 30% as a disincentive. [Experimental results]. 210 μL volume applications a final concentration of 40ng/ml of MPO to establish a stable enzyme reaction system, and the Michaelis constant (Km) of 0.705mM. 2.11 MPA patients, seven patients with anti-MPO antibodies to noncompetitive inhibition dose-dependent inhibition of MPO activity. The average inhibition rate was 32%, the mean inhibition constant (Ki) for 0.124mg/ml (Dixon plot mapping method). 3. Ceruloplasmin to competitive inhibition dose-dependent and time-dependent inhibition of MPO activity. The average inhibition rate of 75%, with a mean inhibition constant (Ki) of 0.547 mg / ml (Dixon plot mapping method). 4. Anti-MPO antibodies able to inhibit the binding between MPO and ceruloplasmin maximum inhibition rate reached 75.4 ± 11.6%. [Conclusion] 1. Most MPA patients with anti-MPO antibodies to a dose-dependent manner noncompetitive inhibition of the oxidation activity of MPO to suppress different ways, with the MPO physiological inhibitor ceruloplasmin. 2. Anti-MPO antibodies can inhibit the combination of the MPO and ceruloplasmin. 3. Anti-MPO antibodies of these effects may be related to the pathogenesis of primary MPA.

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