A monoclonal antibody with high affinity for a neo-antigenic site in fibrinogen degraded by polymorphonuclear leukocyte-derived elastase

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Abstract
Elastase, released by stimulated polymorphonuclear leukocytes (PMN), is thought to play an important role in the pathogenesis of chronic obstructive pulmonary disease (COPD) especially pulmonary emphysema. A test that can detect release of elastase activity from PMN would be valuable to monitor therapy or to identify patients at risk. The authors aimed to isolate and characterize monoclonal antibodies (mAb) with a high affinity for a neo-antigenic determinant in a high-molecular weight degradation product of fibrinogen (Fbg) generated by PMN-derived elastase. Using synthetic peptides, they mimicked the new amino or carboxy terminal sequences of the Aα-, Bβ-and γ-chains of Fbg that are generated by elastase. These synthetic peptides (Aα22–36, Aα350–360, Bβ44–55, and γ295–305), unidirectionally coupled to a carrier protein, were used to generate mAb specific for elastase-degraded fibrinogen (EDF). mAb that appeared to be specific for a neo-antigenic determinant (neotope) consisting of the new amino terminal amino acid(s) of the Fbg Aα-chain that is generated by elastase activity were isolated only with the Aα22–36 synthetic peptide. One mAb, designated as EF1–4, was further characterized and had an approximately 75-fold higher affinity for EDF, as compared with Fbg, in solution. Using the other peptides, no mAb specific for elastase generated fibrinogen degradation products were obtained. Incubation of immobilized fibrin(ogen) with stimulated PMN resulted in the generation of this specific neotope recognized by mAb EF1–4; the protease inhibitors α1-proteinase inhibitor (α1-PI) and antileukoprotease (ALP) decreased the generation by PMN of this neotope on Fbg. Fibrinogen degradation products generated by plasmin or by other leukocyte proteases, i.e. cathepsin G and proteinase 3, did not react with mAb EF1–4.

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