Characterization of monoclonal antibodies to proteinase-3 and application in the study of epitopes for classical anti-neutrophil cytoplasm antibodies.
Wegener's granulomatosis is associated with autoantibodies (classical antineutrophil cytoplasm antibodies, c-ANCAs) to a serine protease called proteinase-3. In this study three IgG class monoclonal antibodies, designated 4A3, 4A5 and 6A6, against proteinase-3 were generated to study the immune response of c-ANCA-positive patients. All monoclonal antibodies were tested by immunofluorescence staining of human granulocytes and gave a staining pattern identical to the pattern obtained with sera from patients with Wegener's granulomatosis. On protein transfer blots of neutrophil alpha-granule extract, all monoclonal antibodies stained a 29-kD protein band corresponding to proteinase-3. Also, in a direct binding ELISA with alpha-granule extract as antigen, binding of the monoclonal antibodies to the antigen could be completely inhibited by adding a pure preparation of proteinase-3. In the ELISA type of competition experiments, none of the monoclonal antibodies could substantially inhibit binding of any of the other antibodies to the antigen, indicating that all monoclonal antibodies recognize separate epitopes on the antigen. The same conclusion was reached from experiments by real-time competition analysis using a Pharmacia BIAcore system. The monoclonal antibodies were used to study whether some epitopes on proteinase-3 are preferred by patient autoantibodies. A total of 36 patients sera was tested by competing for autoantibody binding to proteinase-3 with the monoclonal antibodies in an ELISA. Autoantibody binding to proteinase-3 could be partially or completely inhibited by either the 4A5 (50% of the sera) or by the 6A6 antibody (11%). The 4A3 antibody could only partially inhibit 8 of the sera (22%).(ABSTRACT TRUNCATED AT 250 WORDS)
Sommarin
Y
Y
Wieslab AB, IDEON, Lund, Sweden.
Rasmussen
N
N
Wieslander
J
J
D000818:Animals; D019268:Antibodies, Antineutrophil Cytoplasmic; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D001323:Autoantibodies / Q000276:immunology*; D001667:Binding, Competitive; D003593:Cytoplasm / Q000276:immunology*; D003594:Cytoplasmic Granules / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000276:immunology*; D005455:Fluorescent Antibody Technique; D014890:Granulomatosis with Polyangiitis / Q000276:immunology*; D006801:Humans; D007074:Immunoglobulin G / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D009504:Neutrophils / Q000276:immunology* / Q000648:ultrastructure
D019268:Antibodies, Antineutrophil Cytoplasmic; D000911:Antibodies, Monoclonal; D001323:Autoantibodies; D000939:Epitopes; D007074:Immunoglobulin G