Purification of human neutrophil collagenase, establishment of its monoclonal antibodies and application to gingival crevicular neutrophils

Abstract

In order to investigate the role of neutrophil collagenase in the periodontal disease, human neutrophil collagenase was purified and two monoclonal antibodies against this enzyme were obtained. This enzyme was purified by four step-affinity chromatography: heparin-aminocellurofine, gelatin Sepharose 4B, collagen-Sepharose and collagenase inhibitor column chromatographies. To produce the monoclonal antibody against the enzyme, the Balb/c mouse was immunized and its spleen cells were fused with the mouse myeloma cells. Two monoclonal antibodies to the enzyme, 2F3 (IgG1) and 3F12 (IgG1), which recognized a conformational structure of the enzyme apart from its catalytic site were obtained. Both antibodies were monospecific to leukocyte collagenase and did not cross-react with the other metalloproteinases such as leukocyte gelatinase, skin fibroblast collagenase, gelatinase and stromelysin. Using these monoclonal antibodies, collagenase was stained granularly in gingival crevicular neutrophils.