Production and characterization of monoclonal antibodies against the novel neutrophil activating peptide NAP/IL-8

LPS-stimulated human mononuclear cells have recently been shown to produce large amounts of a novel neutrophil-activating cytokine termed neutrophil-activating peptide NAP/IL-8. This chemotactic factor has in the meantime been biochemically and functionally well characterized. We now report on four distinct murine mAb directed against this peptide. All four mAb are different in respect to isotype and IEF pattern. The cross-reactivity with partially homologous peptides like beta-thromboglobulin and platelet factor 4 showed defined differences. With the use of these antibodies we were able to detect solid phase as well as soluble NAP/IL-8 as tested in a sandwich-ELISA. Also dose-dependent neutralization of NAP/IL-8 chemotactic activity in the Boyden chamber chemotaxis assay was observed. Immunoaffinity columns prepared with these four mAb bound NAP/IL-8 from supernatants of LPS-stimulated mononuclear cells. Furthermore, Western immunoblots showed a single protein band in the expected region of Mr of 10 kDa with all four mAb presented.