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. 2009 Oct;128(2):236–244. doi: 10.1111/j.1365-2567.2009.03108.x

Figure 1.

Figure 1

Determination of the structural and binding characteristics of recombinant immunoglobulin G4 PR3-anti-neutrophil cytoplasmic autoantibody (IgG4 PR3-ANCA). (a) Confirmation of the molecular weights of the recombinant IgGs by sodium dodecyl sulphate–polyacrylamide gel electrophoresis. Lane 1, IgG4 anti-4-hydroxyl-3-nitrophenylacetyl (NP); lane 2, IgG4 PR3-ANCA; lane 3, IgG1 PR3-ANCA; lanes 4 and 5, molecular weight markers; lane 6, IgG4 anti-NP; lane 7, IgG4 PR3-ANCA; lane 7, IgG1 PR3-ANCA. Lanes 1–3 contain reduced samples and lanes 6–8 contain non-reduced samples. (b) Enzyme-linked immunosorbent assay for the detection of antibodies to PR3. Antibody binding to plates coated with PR3 was detected with a specific anti-IgG4 secondary antibody. Column 1, supernatant from cells which do not contain any plasmids for IgG PR3-ANCA; column 2, supernatant form cells transfected with IgG1 PR3-ANCA; column 3, supernatant from cells transfected with IgG4 PR3-ANCA. ***P = 0·0002 (n = 3). (c) Western blot to determine recognition of antigen by IgG4 PR3-ANCA. Lane 1, recombinant PR3 (160 ng); lane 2, recombinant PR3 (320 ng); lane 3, neutrophil lysate. (d) Indirect immunofluorescence of ethanol-fixed neutrophils using either IgG4 anti-NP (left panel) or IgG4 PR3-ANCA (right panel) as detection antibody.