MONOCLONAL-ANTIBODIES SPECIFIC FOR NATURAL HUMAN NEUTROPHIL GELATINASE-B USED FOR AFFINITY PURIFICATION, QUANTITATION BY 2-SITE ELISA AND INHIBITION OF ENZYMATIC-ACTIVITY

Human gelatinase B was produced from peripheral blood neutrophils and purified by affinity chromatography on gelatin sepharose. This materia l was used as an antigen to prepare mouse monoclonal antibodies (mAb). The resulting hybridomas were selected on the basis of binding to bio tinylated antigen and by a sandwich ELISA using gelatinase-B-specific polyclonal rabbit antiserum and pure natural antigen, Five of these mA b were selected for further characterization. They al displayed variab le epitope specificity, binding capacity and inhibitory activity. Wher eas mAb REGA-2D9 and REGA-3G12 showed the strongest binding to biotiny lated gelatinase B and natural gelatinase B, respectively, mAb REGA-2F 9 did not bind biotinylated antigen. None of the mAb displayed cross-r eactivity to gelatinase A in a direct ELISA. The mAb REGA-1G8 was foun d to cross-react with human serum albumin. The binding capacity of the other four mAb with leukocyte gelatinase B was compared and a sensiti ve sandwich ELISA was developed with the antibodies REGA-3G12 and REGA -2D9 (detection limit 0.5 ng/ml). The mAb REGA-3G12 was unique in that it inhibited catalysis by gelatinase B, This was shown by assaying th e degradation of nasal septum type II gelatin in the presence and abse nce of each of the five mAb. Furthermore, mAb RECA-3G12 inhibited the degradation of biotinylated gelatin in a microtiterplate solution assa y. In addition to the potential use of the inhibitory mAb REGA-3G12 in the treatment of diseases with excessive gelatinase B production, sev eral of the described mAb are useful as diagnostic probes to detect ge latinase B in body fluids and tissue samples of patients with multiple sclerosis, rheumatoid arthritis and cancer.