Identification and characterization of human eosinophil cationic protein by an epitope-specific antibody.

ABSTRACT

The eosinophil cationic protein (ECP) is a basic secretion protein involved in the immune response system. ECP levels in biological fluids are an indicator of eosinophil-specific activation and degranulation and are currently used for the clinical monitoring and diagnosis of inflammatory disorders. A polyclonal epitope-specific antibody has been obtained by immunizing rabbits with a conjugated synthetic peptide. A sequence corresponding to a large exposed loop in the human ECP three-dimensional structure (D115-Y122) was selected as a putative antigenic epitope. The antibody was purified on an affinity column using recombinant ECP (rECP) as antigen. The antibody (D112-P123 Ab) specifically recognizes rECP and its native glycosylated and nonglycosylated forms in plasma, granulocytes, and sputum. The antibody detects as little as 1 ng of rECP, can be used both in reducing and nonreducing conditions, and does not cross-react with the highly homologous eosinophil-derived neurotoxin or other proteins of the pancreatic ribonuclease superfamily.