Studies to demonstrate inhibition of functional activity of neutrophil lysosomal enzymes with ANCA.

Many autoantibodies that have enzymes as their targets bind to the catalytic sites and inhibit enzymatic activity. We have established functional assays for the targets of anti-neutrophil cytoplasm antibodies (ANCA) in order to determine whether these antibodies are directed against the corresponding catalytic sites. Anti-proteinase 3 activity was measured by the cleavage of alpha-naphthyl acetate; myeloperoxidase activity by peroxidation of monochlorodimedon or guaiacol using hydrogen peroxide; and elastase activity by a fluorimetric assay of the hydrolytic product of N-succ (ala)3 amido-methyl coumarin. The addition of immunoglobulin from patients with ANCA to these assays did not result in inhibition of functional activity of the corresponding enzyme when compared with normal immunoglobulin. Furthermore the removal of specific immunoglobulin by solid phase adsorption to the corresponding antigen did not result in an increase in enzymatic activity compared with the starting material. However preliminary results suggest that anti-neutrophil proteinase 3 binding may be inhibited in a solid-phase ELISA by preincubation with a peptide corresponding to the catalytic site.