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An enzyme immunoassay for polymorphonuclear leucocyte-mediated fibrinogenolysis

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Author: Bos, R. · Leuven, C.J.M. van · Stolk, J. · Hiemstra, P.S. · Ronday, H.K. · Nieuwenhuizen, W.
Institution: TNO Preventie en Gezondheid
Source:European Journal of Clinical Investigation, 2, 27, 148-156
Identifier: 233857
Keywords: Biology · Monoclonal antibody · Polymorphonuclear leucocytes · Pulmonary emphysema · Biochemical marker · Elastase · Monoclonal antibody · Adult · Clinical article · Controlled study · Enzyme immunoassay · Female · Lung emphysema · Male · Monitoring · Protein degradation · Rheumatoid arthritis · Sepsis · Antibodies, Monoclonal · Antibody Specificity · Enzyme-Linked Immunosorbent Assay · Fibrin Fibrinogen Degradation Products · Fibrinogen · Fibrinolysis · Humans · Leukocyte Elastase · Neutrophils · Serine Endopeptidases


Upon stimulation, polymorphonuclear leucocytes (PMNs) release potent serine proteases, i.e. elastase, cathepsin C and proteinase 3, which contribute to the degradation of tissue and plasma components. Here, we describe the development of a plasma test to assess PMN-mediated fibrinogenolysis as a biochemical marker for actual PMN-derived proteolysis in vivo, useful for monitoring therapeutic efficacy, i.e. of elastase inhibitors. We generated a monoclonal antibody (MAb), designated 1-1/B3, with a high affinity for elastase-degraded fibrinogen (EDF). The epitope for 1-1/B3 becomes exposed in a time-dependent manner during digestion of fibrinogen with purified PMN-derived serine proteases and with isolated PMNs in vitro. However, 1-1/B3 does not react with plasma fibrinogen or with fibrin(ogen) degradation products generated by plasmin or by other active proteases that may occur locally, i.e. metalloproteases and lysosomal cathepsins. On the basis of MAb 1-1/B3, we developed a plasma test for the assessment of PMN-mediated fibrin(ogen) degradation products (PMN-FDP). In a panel of control plasmas, we observed concentrations of PMN-FDP of 8.2 ± 0.9 ng mL-1 (n = 18). These values were increased twofold in patients with α1-proteinase inhibitor deficiency (18.6 ± 3.3 ng mL-1; n = 12; P < 0.0001) and even more in patients with sepsis (365.7 ± 97.7 ng mL-1; n = 16; P < 0.0001). Furthermore, synovial tissue extracts from patients with rheumatoid arthritis contained increased levels of PMN-FDP, compared with synovial tissue extracts (P < 0.005) from patients with osteoarthritis. Chemicals/CAS: Antibodies, Monoclonal; Fibrin Fibrinogen Degradation Products; Fibrinogen, 9001-32-5; Leukocyte Elastase, EC; Serine Endopeptidases, EC 3.4.21.-